U.S. patent application number 12/356208 was filed with the patent office on 2009-05-28 for sugar kinases with expanded substrate specificity and their use.
Invention is credited to Jon S. Thorson.
Application Number | 20090137006 12/356208 |
Document ID | / |
Family ID | 34676549 |
Filed Date | 2009-05-28 |
United States Patent
Application |
20090137006 |
Kind Code |
A1 |
Thorson; Jon S. |
May 28, 2009 |
Sugar Kinases with Expanded Substrate Specificity and Their Use
Abstract
One preferred embodiment of the present invention provides a
GalK variant comprising a Y371H, M173L or Y371H-M173L mutation for
in vivo and in vitro glycorandomization. In another preferred
embodiment, the E. coli GalK variant is mutated at one or more
amino acids including R28, E34, D37, D174, Y223, C339, Y371, Y371H,
M173, and M173L. The GalK variants display catalytic activity
toward a variety of D or L sugars. Another preferred embodiment
provides method of phosphorylating sugars comprising the step of
incubating a nucleotide triphosphate (NTP) and a D or L sugar in
the presence of a GalK variant such that a sugar phosphate is
produced. This sugar phosphate may be further incubated with a
nucleotidylyltransferase, such that a NDP-sugar is produced. The
NDP-sugar may be further incubated with a biomolecule capable of
being glycosylated in the presence of a glycosyltransferase, such
that a glycosylated biomolecule is produced.
Inventors: |
Thorson; Jon S.; (Middleton,
WI) |
Correspondence
Address: |
QUARLES & BRADY LLP
411 E. WISCONSIN AVENUE, SUITE 2040
MILWAUKEE
WI
53202-4497
US
|
Family ID: |
34676549 |
Appl. No.: |
12/356208 |
Filed: |
January 20, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10904941 |
Dec 6, 2004 |
7479385 |
|
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12356208 |
|
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60481742 |
Dec 5, 2003 |
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Current U.S.
Class: |
435/72 ;
435/194 |
Current CPC
Class: |
C12N 9/1205 20130101;
C12P 19/18 20130101 |
Class at
Publication: |
435/72 ;
435/194 |
International
Class: |
C12P 19/00 20060101
C12P019/00; C12N 9/12 20060101 C12N009/12 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] The present invention was made by government support from
the National Institutes of Health--Grant No. AI52218. The United
States government has certain rights in this invention.
Claims
1. A method of providing a sugar phosphate comprising the step of
incubating a nucleotide triphosphate (NTP) and a D or L sugar in
the presence of a GalK variant of the wild type E. Coli GalK amino
acid sequence set forth in SEQ ID NO: 1, wherein the variant has
one or both of the following mutations: tyrosine 371 is mutated to
histidine (Y371H), and/or (b) methionine 173 is mutated to leucine
(M173L), and whereby a sugar phosphate is produced.
2. The method of claim 1, wherein the NTP is ATP.
3. The method of claim 1, wherein said method is carried out in a
host cell.
4. The method of claim 1, wherein the D or L sugar is galactose or
glucose-configured sugars having substitutions at C-2, C-3, C-4,
C-6 positions.
5. The method of claim 1, wherein the sugar is selected from the
group consisting of D-galactose, 2-deoxy D-galactose,
D-galactose-amine, D-talose, 3-deoxy-D-galactose,
6-deoxy-D-galactose, 6-amino-D-galactose, D-galacturonic acid,
L-altrose and L-glucose.
6. A method of phosphorylating sugars comprising the step of
incubating a nucleotide triphosphate (NTP) and a D or L sugar in
the presence of a GalK variant of the wild type E. coli GalK amino
acid sequence set forth in SEQ ID NO: 1, wherein the variant has
one or more mutations selected from the group consisting of R28,
E34, D37, D174, Y223, C339, Y371, and M173, whereby a sugar
phosphate is produced.
7. The method of claim 6, wherein NTP is ATP.
8. The method of claim 6, wherein said method is carried out in a
host cell.
9. The method of claim 6, wherein the D or L sugar is selected from
the group consisting of D-galactose, 2-deoxy D-galactose,
D-galactose-amine, D-talose, 3-deoxy-D-galactose,
6-deoxy-D-galactose, 6-amino-D-galactose, D-galacturonic acid,
L-altrose and L-glucose.
10. A method of synthesizing an NDP-sugar, comprising the steps of:
(a) incubating a nucleotide triphosphate (NTP) and a D or L sugar
in the presence of a GalK variant of the wild type E. coli GalK
amino acid sequence set forth in SEQ ID NO: 1, wherein the variant
has one or more mutations selected from the group consisting of
R28, E34, D37, D174, Y223, C339, Y371, and M173, whereby a sugar
phosphate is produced; and (b) incubating the sugar phosphate with
a nucleotidylyltransferase, whereby a NDP-sugar is produced.
11. The method of claim 10, wherein the D or L sugar is selected
from the group consisting of D-galactose, 2-deoxy D-galactose,
D-galactose-amine, D-talose, 3-deoxy-D-galactose,
6-deoxy-D-galactose, 6-amino-D-galactose, D-galacturonic acid,
L-altrose and L-glucose.
12. The method of claim 10, wherein the nucleotidylyltransferase is
Ep or a mutated variant thereof.
13. The method of claim 12, wherein the mutated Ep variant is
mutated at one or more amino acids selected from the group
consisting of V173, G147, W224, N112, G175, D111, E162, T201, I200,
E199, R195, L89, L89T, L109, Y146 and Y177.
14. The method of claim 10, wherein the NTP is ATP.
15. The method of claim 10, wherein the GalK variant is Y371H,
M173L or Y371H-M173L.
16. The method of claim 10, wherein the method is carried out in
vitro.
17. The method of claim 10, wherein the method is carried out in a
host cell.
18. The method of claim 17, wherein the host cell is a
bacterium.
19. The method of claim 17, wherein the host cell is selected from
the group consisting of E. coli and S. lividans.
20. A method of producing a glycosylated biomolecule containing at
least one sugar moeity, comprising the steps of: (a) incubating a
nucleotide triphosphate (NTP) and a D or L sugar in the presence of
a GalK variant of the wild type E. coli GalK amino acid sequence
set forth in SEQ ID NO: 1, wherein the variant has one or more
mutations selected from the group consisting of R28, E34, D37,
D174, Y223, C339, Y371, and M173, whereby a sugar phosphate is
produced; (b) incubating the sugar phosphate with a
nucleotidylyltransferase, whereby a NDP-sugar is produced; and (c)
incubating the NDP-sugar with a biomolecule capable of being
glycosylated in the presence of a glycosyltransferase, whereby a
glycosylated biomolecule is produced.
21. The method of claim 20, wherein the D or L sugar is selected
from the group consisting of D-galactose, 2-deoxy D-galactose,
D-galactose-amine, D-talose, 3-deoxy-D-galactose,
6-deoxy-D-galactose, 6-amino-D-galactose, D-galacturonic acid,
L-altrose and L-glucose.
22. The method of claim 20, wherein the nucleotidylyltransferase is
Ep or a mutated variant thereof.
23. The method of claim 22, wherein the mutated Ep variant is
mutated at one or more amino acids selected from the group
consisting of V173, G147, W224, N112, G175, D111, E162, T201, I200,
E199, R195, L89, L89T, L109, Y146 and Y177.
24. The method of claim 20, wherein the glycosyltransferase is
selected from the group consisting of CalB, CalE, CalN, CalU, Gra
orf14, Gra orf5, LanGT1, LanGT2, LanGT3, LanGT4, MtmGI, MtmGII,
MtmGTIII, MtmGTIV, NovM, RhlB, Rif orf 7, SnogD, SnogE, SnogZ,
UrdGT1a, UrdGT1b, UrdGT1c, UrdGT2, AknK, AknS, DesVII, DnrS, OleG1,
OleG2, TylCV, TylMII, TylN, DauH, DnrH, EryBV, EryCIII, Ngt, BgtA,
BgtB, BgtC, GftA, GftB, GftC, GftD, GftE, Gp1-1, Gp1-2, RtfA,
AveBI, BlmE, BlmF, MgtA, NysD1, OleD, OleI, SpcF, SpcG, StrH,
Ugt51B1, Ugt51C1, UGT52, UgtA, UgtB, UgtC, UgtD and homologs
thereof.
25. The method of claim 20, wherein the NTP is ATP.
26. The method of claim 20, wherein the GalK variant is Y371H,
M173L or Y371H-M173L.
27. The method of claim 20, wherein the method is carried out in
vitro.
28. The method of claim 20, wherein the method is carried out in a
host cell.
29. The method of claim 28, wherein the host cell is a
bacterium.
30. The method of claim 28, wherein the host cell is selected from
the group consisting of E. coli and S. lividans.
31. The method of claim 20, wherein the biomolecule capable of
being glycosylated is selected from the group consisting of natural
and synthetic metabolites, pyran rings, furan rings, enediynes,
anthracyclines, angucyclines, aureolic acids, orthosomycins,
macrolides, aminoglycosides, non-ribosomal peptides, polyenes,
steroids, lipids, indolocarbazoles, bleomycins, amicetins,
benzoisochromanequinones coumarins, polyketides, pluramycins,
aminoglycosides, oligosaccharides, peptides, proteins, hybrids
consisting of one or more these components, analogs and bioactive
aglycons thereof.
32. A method of claim 20, wherein the glycosylated biomolecule is
further incubated with at least one chemoselectively ligatable
moiety, whereby at least one chemoselectively ligated compound is
produced.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. application
Ser. No. 10/904,941, filed Dec. 6, 2004, which claims the benefit
of U.S. Provisional 60/481,742, filed Dec. 5, 2003, both of which
are incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0003] This invention generally relates to sugar kinases and
specifically to novel anomeric D/L sugar kinases with expanded
substrate specificity and methods of use.
[0004] Many clinically important medicines are derived from
glycosylated natural products, the D- or L-sugar substituents of
which often dictate their overall biological activity. This
paradigm is found throughout the anticancer and antiinfective
arenas with representative clinical examples (FIG. 1a) including
enediynes (calicheamicin, 1), polyketides (doxorubicin, 2;
erythromycin, 3), indolocarbazoles (staurosporine, 4),
non-ribosomal peptides (vancomycin, 5), polyenes (nystatin, 6),
coumarins (novobiocin, 7), or cardiac glycosides (digitoxin, 8).
Given the importance of the sugars attached to these and other
biologically significant metabolites, extensive effort has been
directed in recent years toward altering sugars as a means to
enhance or alter natural product-based therapeutics by both in vivo
and in vitro approaches. Among these, in vitro glycorandomization
(IVG) makes use of the inherent or engineered substrate promiscuity
of nucleotidylyltransferases and glycosyltransferases to activate
and attach chemically synthesized sugar precursors to various
natural product scaffolds. This efficiently incorporates highly
functionalized "unnatural" sugar substitutions into the
corresponding natural product scaffold (FIG. 1b).
[0005] Accordingly, the need remains for natural and/or engineered
enzymes that are promiscuous in their substrate specificity and
capable of increased catalytic activity to enhance multiplicity of
available glycosylated natural compounds.
SUMMARY OF THE INVENTION
[0006] The present invention provides sugar kinases with expanded
substrate specificity and methods of use. One embodiment of the
present invention provides a GalK variant for in vivo
glycorandomization selected from the group consisting of a Y371H,
M173L and Y371H-M173L mutations. The GalK variant displays
substrate specificity toward a D or L sugar. Preferably, the D or L
sugar may be selected from the group consisting of D-galactose,
2-deoxy D-galactose, D-galactose-amine, D-talose,
3-deoxy-D-galactose, 6-deoxy-D-galactose, 6-amino-D-galactose,
D-galacturonic acid, L-altrose and L-glucose.
[0007] Another embodiment of the present invention provides a
method of providing a sugar phosphate. The method comprises the
step of incubating a nucleotide triphosphate (NTP) and a D or L
sugar in the presence of a GalK variant as discussed in the above
paragraph, such that a sugar phosphate is produced. In this method,
the NTP is ATP. Also this method may be carried out in a host cell.
Further, in this method, the D or L sugar includes galactose or
glucose-configured sugars having substitutions at C-2, C-3, C-4,
C-6 positions. Preferably, the D or L sugar include D-galactose,
2-deoxy D-galactose, D-galactose-amine, D-talose,
3-deoxy-D-galactose, 6-deoxy-D-galactose, 6-amino-D-galactose,
D-galacturonic acid, L-altrose and L-glucose.
[0008] Yet another embodiment of the present invention provides an
E. coli GalK variant of the wild type amino acid sequence of SEQ ID
NO: 1 wherein the wild type amino acid sequence is mutated at one
or more amino acid residues. The mutations are selected from the
group consisting of R28, E34, D37, D174, Y223, C339, Y371, Y371H,
M173, M173L and C353. This variant is capable of displaying
catalytic activity toward a D or L sugar selected from the group
consisting of D-galactose, 2-deoxy D-galactose, D-galactose-amine,
D-talose, 3-deoxy-D-galactose, 6-deoxy-D-galactose,
6-amino-D-galactose, D-galacturonic acid, L-altrose and L-glucose.
In a preferred embodiment, the GalK variant is Y371H-M173L.
[0009] Another embodiment of the present invention provides a
method of phosphorylating sugars. This method comprises the step of
incubating a nucleotide triphosphate (NTP) and a D or L sugar in
the presence of a GalK variant according as discussed above, such
that a sugar phosphate is produced. In this method also, the NTP is
ATP. Further, the method is carried out in a host cell. Also the D
or L sugar in this method is selected from the group consisting of
D-galactose, 2-deoxy D-galactose, D-galactose-amine, D-talose,
3-deoxy-D-galactose, 6-deoxy-D-galactose, 6-amino-D-galactose,
D-galacturonic acid, L-altrose and L-glucose.
[0010] Yet another aspect of the present invention provides a
method of synthesizing an NDP-sugar. This method comprises the
steps of: (a) incubating a nucleotide triphosphate (NTP) and a D or
L sugar in the presence of a GalK variant as discussed, whereby a
sugar phosphate is produced; and (b) incubating the sugar phosphate
with a nucleotidylyltransferase, such that a NDP-sugar is produced.
In this method, the D or L sugar is selected from the group
consisting of D-galactose, 2-deoxy D-galactose, D-galactose-amine,
D-talose, 3-deoxy-D-galactose, 6-deoxy-D-galactose,
6-amino-D-galactose, D-galacturonic acid, L-altrose and L-glucose.
Further, the nucleotidylyltransferase is Ep or a mutated variant
thereof. Preferably, the mutated Ep variant includes an Ep mutated
at one or more amino acids selected from the group consisting of
V173, G147, W224, N112, G175, D111, E162, T201, I200, E199, R195,
L89, L89T, L109, Y146 and Y177. In this method also, the NTP is
ATP. Also in this method the GalK variant is Y371H, M173L or
Y371H-M173L. This method may be carried out in vitro or in a host
cell. When the method is carried out in a host cell, the host cell
is preferably a bacterium. More preferably, the host cell is
selected from the group consisting of E. coli and S. lividans.
[0011] Another aspect of the invention provides a method of
producing a glycosylated biomolecule containing at least one sugar
moiety. The method comprises the steps of: (a) incubating a
nucleotide triphosphate (NTP) and a D or L sugar in the presence of
a GalK variant such that a sugar phosphate is produced; (b)
incubating the sugar phosphate with a nucleotidylyltransferase,
such that a NDP-sugar is produced; and (c) incubating the NDP-sugar
with a biomolecule capable of being glycosylated in the presence of
a glycosyltransferase, whereby a glycosylated biomolecule is
produced. Preferably in this method, the D or L sugar is selected
from the group consisting of D-galactose, 2-deoxy D-galactose,
D-galactose-amine, D-talose, 3-deoxy-D-galactose,
6-deoxy-D-galactose, 6-amino-D-galactose, D-galacturonic acid,
L-altrose and L-glucose. Also, preferably, the
nucleotidylyltransferase is Ep or a mutated variant thereof.
Mutated Ep variant includes Ep that is mutated at one or more amino
acids selected from the group consisting of V173, G147, W224, N112,
G175, D111, E162, T201, I200, E199, R195, L89, L89T, L109, Y146 and
Y177. Further the glycosyltransferase is selected from the group
consisting of CalB, CalE, CalN, CalU, Gra orf14, Gra orf5, LanGT1,
LanGT2, LanGT3, LanGT4, MtmGI, MtmGII, MtmGTIII, MtmGTIV, NovM,
RhlB, Rif orf 7, SnogD, SnogE, SnogZ, UrdGT1a, UrdGT1b, UrdGT1c,
UrdGT2, AknK, AknS, DesVII, DnrS, OleG1, OleG2, TylCV, TylMII,
TylN, DauH, DnrH, EryBV, EryCIII, Ngt, BgtA, BgtB, BgtC, GftA,
GftB, GftC, GftD, GftE, Gp1-1, Gp1-2, RtfA, AveBI, BlmE, BlmF,
MgtA, NysD1, OleD, OleI, SpcF, SpcG, StrH, Ugt51B1, Ugt51C1, UGT52,
UgtA, UgtB, UgtC, UgtD and homologs thereof. Also in this method,
the NTP is ATP. Preferably, the GalK variant is Y371H, M173L or
Y371H-M173L. This method may be carried out in vitro or in a host
cell. When the method is carried out in a host cell, preferably,
the host cell is a bacterium. More preferably, the host cell is
selected from the group consisting of E. coli and S. lividans.
Also, in this method the biomolecule capable of being glycosylated
is selected from the group consisting of natural and synthetic
metabolites, pyran rings, furan rings, enediynes, anthracyclines,
angucyclines, aureolic acids, orthosomycins, macrolides,
aminoglycosides, non-ribosomal peptides, polyenes, steroids,
lipids, indolocarbazoles, bleomycins, amicetins,
benzoisochromanequinones coumarins, polyketides, pluramycins,
aminoglycosides, oligosaccharides, peptides, proteins, hybrids
consisting of one or more these components, analogs and bioactive
aglycons thereof. Furthermore, the glycosylated biomolecule is
further incubated with at least one chemoselectively ligatable
moiety, such that at least one chemoselectively ligated compound is
produced.
[0012] Various other features, objects, and advantages of the
invention will be apparent to those skilled in the art from the
following detailed description including illustrative examples
setting forth how to make and use the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1. a) Provides representative examples for natural
product glycosides used as therapeutics: calicheamicin (1),
doxorubicin (2), erythromycin (3), staurosporine (4), vancomycin
(5), nystatin (6), novobiocin (7), and digitoxin (8). The attached
sugars are highlighted in color with red indicating L-configured
sugars, and blue representing D-sugars. b) Schematic for natural
product in vitro glycorandomization. E.sub.p denotes
.alpha.-D-glucopyranosyl phosphate thymidylyltransferase,
GlyT.sub.n different glycosyltransferases.
[0014] FIG. 2. Provides reactions catalyzed by anomeric kinases. a)
Glycogen phosphorylase. b) Fucokinase. c) Galactokinase. d)
Proposed phosphorylation of L-altrose accomplished by the evolved
GalK mutant Y371H.
[0015] FIG. 3. Provides representative quantitative data for a set
of GalK variants, illustrating screen for D-galacturonic acid
(X-axis) and L-altrose (Y-axis). The higher the loss in absorption
(shown in absorption units (AU), the more active the enzyme
variant.
[0016] FIG. 4. Provides .sup.3J.sub.H-H coupling patterns and NOESY
correlations for the GalK Y371H product
.beta.-L-altrose-1-phosphate.
[0017] FIG. 5. Glycorandomization overview and two potential
scenarios for an in vivo approach. (a) In vitro glycorandomization
utilizes two enhanced enzymes--E.sub.1 (a general kinase) and
E.sub.2 (a general nucleotidylyltransferase)--to generate NDP-sugar
substrate libraries to be utilized by a flexible natural
product-associated glycosyltransferase (GlyT). (b) In vivo
glycorandomization scenario I--feeding monosaccharides to a natural
product-producing host engineered to express the `NDP-sugar
factory`. In this scenario, both the aglycon and
glycosyltransferase are provided by the bacterial host. (c) In vivo
glycorandomization scenario II--feeding monosaccharides and
aglycons to a non-producing host engineered to express the
`NDP-sugar factory` and an appropriate glycosyltransferase or
glycosyltransferase library.
[0018] FIG. 6. Comparison of D-glucose docked within the active
site of L. lactis GalK and homology-model of E. coli GalK. (a) Wild
type L. lactis GalK bound to D-Glc. (b) L182M (the homology model
for E. coli GalK active site) with D-Glc.
[0019] FIG. 7. `Natural` and `unnatural` substrates of wild-type
GalK and GalK mutants (M173L, Y371H and M173L-Y371H).
[0020] FIG. 8. FIG. 8 Percent conversion of sugar substrates by
wild-type and mutant GalKs. For each enzymatic reaction: [sugar]=8
mM, [ATP]=14 mM, [MgCl.sub.2]=3.5 mM, [enzyme]=15.0 .mu.M and
reaction time=180 min.
[0021] FIG. 9. In vivo GalK-catalyzed sugar-1-phosphate production.
(a) Experimental overview using 6-azido-6-deoxy-D-galactose (22) as
an example: 22 is fed to an E. coli host expressing the M173L-Y371H
GalK double mutant and the reactants and products subsequently
labeled using a 1,3-dicycloaddition with the fluorescent tag 50.
(b) HPLC chromatographs of bioconversions: i) 22 with Y371H-M173L
GalK; ii) 46 with Y371H-M173L GalK; iii) 22 with wild-type GalK.
MS: 50, calculated for C.sub.20H.sub.25N.sub.3O.sub.3S 387.2, found
m/z 386.1 [M-H].sup.-; 51, calculated for
C.sub.26H.sub.37N.sub.6O.sub.11PS 672.2, found m/z 671.2
[M-H].sup.-; 52, calculated for C.sub.26H.sub.36N.sub.6O.sub.8S
592.2, found m/z 591.2 [M-H].sup.-
DETAILED DESCRIPTION
[0022] Sugar phosphates, as the starting material, play a key role
in the entire IVG process. Thus, the ability to rapidly construct
sugar phosphate libraries would directly contribute to the
efficiency of IVG. Compared with the existing chemical synthetic
methods for anomeric phosphorylation, single step enzymatic
(kinase) routes bypass required multistep synthetic manipulations
and could be coupled to IVG in a single reaction vessel. Known C-1
phosphorylating enzymes are limited to mainly three types (FIG. 2):
the glycogen phosphorylases which convert glycogen (9) into
D-glucose-1-phosphate (10), fucokinases which transfer a phosphate
from ATP to the anomeric position of L-fucose (12) to provide
.beta.-L-fucose-1-phosphate (13), and the galactokinases (GalK),
which catalyze the formation of .alpha.-D-galactose-1-phosphate
(Gal-1-P, 15) from D-galactose (14) and ATP. Previous studies have
revealed GalK from various sources have a limited substrate scope
and in all C-1 kinases studied thus far, a strict adherence to
either D-sugars (GalK and glycogen phosphorylases), or L-sugars (as
in fucokinase) was observed. Thus, in order to apply any of these
kinases for generating a randomized sugar phosphate library, their
monosaccharide substrate promiscuity must first be enhanced.
[0023] Two general routes for altering enzyme substrate specificity
are currently available. Structure-based engineering relies upon
knowledge of an enzyme's three dimensional structure and an
explicit molecular-level understanding of substrate recognition. An
example of structure-based engineering as applied to IVG includes
increasing the substrate scope of nucleotidylyltransferases
employed (FIG. 1b, "Ep"). The application of Ep rational
engineering led to active site mutants capable of accepting a
variety of substrates not utilized by the wild-type enzyme. Such
techniques are also described in the U.S. Patent Publications
2003/0055235A1 and 2003/0068669A1, and International Publications
WO02079150 and WO0248331, which are incorporated herein by
reference in their entirety for all purposes.
[0024] The alternative to rational engineering is enzyme evolution,
a process primarily dependent upon the availability of a selection
or high throughput screen for the desired enhanced or altered
enzymatic properties. With respect to carbohydrate enzymology,
recent applications include the tagatose-1,6-bisphosphate aldolase
modified by in vitro evolution toward an unnatural
stereoselectivity, an evolved N-acetylneuraminic acid aldolase for
L-sialic acid synthesis, or a 2-deoxy-D-ribose-5-phosphate aldolase
with an expanded substrate range after site directed mutagenesis.
Usually, in vitro evolution strategies include error-prone PCR for
gene diversification, and/or locating critical amino acid residues
for saturation mutagenesis or, more prominently, shuffling of
fragmented diversified genes or gene families according to a number
of different protocols. Subsequently, the diversified proteins are
subjected to a screen. In a recent demonstration of IVG, >50
analogs of 5 (vancomycin) were generated, some of which displayed
enhanced and distinct antibacterial profiles from the parent
natural product.
[0025] While the first structure for a sugar C-1 kinase (GalK from
Lactococcus lactis) recently emerged, the extreme variability in
solution structures among anticipated monosaccharide library
members and the availability of a specific high throughput sugar
anomeric kinase calorimetric screen prompted an initial
evolutionary approach. As a model system, the inventors selected
the well-characterized Escherichia (E.) coli galactokinase GalK and
focused the evolutionary approach toward significant C-5 (e.g.
L-sugar variants) and C-6 alterations (e.g. deoxy, amino, uronic
acid derivatives) in an attempt to probe and elucidate the specific
enzymatic architecture responsible for restricting substrate
substitution at C-5 and C-6. All variants used herein to describe
sugar kinases are defined as unnaturally occurring variants of
naturally occurring sugar kinases.
[0026] Herein the inventors describe the application of directed
evolution and a high throughput multi-sugar calorimetric screen to
enhance the catalytic capabilities of the E. coli GalK. From this
approach, one particular GalK mutant carrying a single amino acid
exchange (Y371H) displayed a surprisingly substantial degree of
kinase activity toward sugars as diverse as D-galacturonic acid,
D-talose, L-altrose, and L-glucose, all of which failed as
wild-type GalK substrates. Furthermore, this mutant provides
enhanced turnover of the small pool of sugars converted by the
wild-type enzyme. Comparison of this mutation to the recently
solved structure of Lactococcus lactis GalK, begins to provide a
blueprint for further engineering of this vital class of enzyme. In
addition, the rapid access to such promiscuous sugar C-1 kinases
will significantly enhance accessibility to natural and unnatural
sugar-1-phosphates and thereby impact upon both in vitro and in
vivo glycosylation methodologies such as natural product
glycorandomization.
[0027] Generally the present invention provides sugar kinases with
expanded substrate specificity and methods of use. One embodiment
of the present invention provides a GalK variant for in vivo
glycorandomization selected from the group consisting of a Y371H,
M173L and Y371H-M173L mutation. The GalK variant displays substrate
specificity toward a D or L sugar. Preferably, the D or L sugar may
be selected from the group consisting of D-galactose, 2-deoxy
D-galactose, D-galactose-amine, D-talose, 3-deoxy-D-galactose,
6-deoxy-D-galactose, 6-amino-D-galactose, D-galacturonic acid,
L-altrose and L-glucose.
[0028] Another embodiment of the present invention provides a
method of providing a sugar phosphate. The method comprises the
step of incubating a nucleotide triphosphate (NTP) and a D or L
sugar in the presence of a GalK variant according to claim 1, such
that a sugar phosphate is produced. In this method, the NTP is ATP.
Also this method may be carried out in a host cell. Further, in
this method, the D or L sugar includes galactose or
glucose-configured sugars having substitutions at C-2, C-3, C-4,
C-6 positions. Preferably, the D or L sugar include D-galactose,
2-deoxy D-galactose, D-galactose-amine, D-talose,
3-deoxy-D-galactose, 6-deoxy-D-galactose, 6-amino-D-galactose,
D-galacturonic acid, L-altrose and L-glucose.
[0029] Yet another embodiment of the present invention provides an
E. coli GalK variant of the wild type amino acid sequence of SEQ ID
NO: 1 wherein the wild type amino acid sequence is mutated at one
or more amino acid residues. The mutations are selected from the
group consisting of R28, E34, D37, D174, Y223, C339, Y371, Y371H,
M173, M173L and C353. This variant is capable of displaying
catalytic activity toward a D or L sugar selected from the group
consisting of D-galactose, 2-deoxy D-galactose, D-galactose-amine,
D-talose, 3-deoxy-D-galactose, 6-deoxy-D-galactose,
6-amino-D-galactose, D-galacturonic acid, L-altrose and L-glucose.
In a preferred embodiment, the GalK variant is Y371H-M173L.
[0030] Another embodiment of the present invention provides a
method of phosphorylating sugars. This method comprises the step of
incubating a nucleotide triphosphate (NTP) and a D or L sugar in
the presence of a GalK variant according as discussed above, such
that a sugar phosphate is produced. In this method also, the NTP is
ATP. Further, the method is carried out in a host cell. Also the D
or L sugar in this method is selected from the group consisting of
D-galactose, 2-deoxy D-galactose, D-galactose-amine, D-talose,
3-deoxy-D-galactose, 6-deoxy-D-galactose, 6-amino-D-galactose,
D-galacturonic acid, L-altrose and L-glucose.
[0031] Yet another aspect of the present invention provides a
method of synthesizing an NDP-sugar. This method comprises the
steps of: (a) incubating a nucleotide triphosphate (NTP) and a D or
L sugar in the presence of a GalK variant as discussed, whereby a
sugar phosphate is produced; and (b) incubating the sugar phosphate
with a nucleotidylyltransferase, such that a NDP-sugar is produced.
In this method, the D or L sugar is selected from the group
consisting of D-galactose, 2-deoxy D-galactose, D-galactose-amine,
D-talose, 3-deoxy-D-galactose, 6-deoxy-D-galactose,
6-amino-D-galactose, D-galacturonic acid, L-altrose and L-glucose.
Further, the nucleotidylyltransferase is Ep or a mutated variant
thereof. Preferably, the mutated Ep variant includes an Ep mutated
at one or more amino acids selected from the group consisting of
V173, G147, W224, N112, G175, D111, E162, T201, I200, E199, R195,
L89, L89T, L109, Y146 and Y177. In this method also, the NTP is
ATP. Also in this method the GalK variant is Y371H, M173L or
Y371H-M173L. This method may be carried out in vitro or in a host
cell. When the method is carried out in a host cell, the host cell
is preferably a bacterium. More preferably, the host cell is
selected from the group consisting of E. coli and S. lividans.
[0032] Another aspect of the invention provides a method of
producing a glycosylated biomolecule containing at least one sugar
moiety. The method comprises the steps of: (a) incubating a
nucleotide triphosphate (NTP) and a D or L sugar in the presence of
a GalK variant such that a sugar phosphate is produced; (b)
incubating the sugar phosphate with a nucleotidylyltransferase,
such that a NDP-sugar is produced; and (c) incubating the NDP-sugar
with a biomolecule capable of being glycosylated in the presence of
a glycosyltransferase, whereby a glycosylated biomolecule is
produced. Preferably in this method, the D or L sugar is selected
from the group consisting of D-galactose, 2-deoxy D-galactose,
D-galactose-amine, D-talose, 3-deoxy-D-galactose,
6-deoxy-D-galactose, 6-amino-D-galactose, D-galacturonic acid,
L-altrose and L-glucose. Also, preferably, the
nucleotidylyltransferase is Ep or a mutated variant thereof.
Mutated Ep variant includes Ep that is mutated at one or more amino
acids selected from the group consisting of V173, G147, W224, N112,
G175, D111, E162, T201, I200, E199, R195, L89, L89T, L109, Y146 and
Y177. Further the glycosyltransferase is selected from the group
consisting of CalB, CalE, CalN, CalU, Gra orf14, Gra orf5, LanGT1,
LanGT2, LanGT3, LanGT4, MtmGI, MtmGII, MtmGTIII, MtmGTIV, NovM,
RhlB, R1f orf 7, SnogD, SnogE, SnogZ, UrdGT1a, UrdGT1b, UrdGT1c,
UrdGT2, AknK, AknS, DesVII, DnrS, OleG1, OleG2, TylCV, TylMII,
TylN, DauH, DnrH, EryBV, EryCIII, Ngt, BgtA, BgtB, BgtC, GftA,
GftB, GftC, GftD, GftE, Gp1-1, Gp1-2, RtfA, AveBI, BlmE, BlmF,
MgtA, NysD1, OleD, OleI, SpcF, SpcG, StrH, Ugt51B1, Ugt51C1, UGT52,
UgtA, UgtB, UgtC, UgtD and homologs thereof. Also in this method,
the NTP is ATP. Preferably, the GalK variant is Y371H, M173L or
Y371H-M173L. This method may be carried out in vitro or in a host
cell. When the method is carried out in a host cell, preferably,
the host cell is a bacterium. More preferably, the host cell is
selected from the group consisting of E. coli and S. lividans.
Also, in this method the biomolecule capable of being glycosylated
is selected from the group consisting of natural and synthetic
metabolites, pyran rings, furan rings, enediynes, anthracyclines,
angucyclines, aureolic acids, orthosomycins, macrolides,
aminoglycosides, non-ribosomal peptides, polyenes, steroids,
lipids, indolocarbazoles, bleomycins, amicetins,
benzoisochromanequinones coumarins, polyketides, pluramycins,
aminoglycosides, oligosaccharides, peptides, proteins, hybrids
consisting of one or more these components, analogs and bioactive
aglycons thereof. Furthermore, the glycosylated biomolecule is
further incubated with at least one chemoselectively ligatable
moiety, such that at least one chemoselectively ligated compound is
produced.
[0033] Following examples depict preferred embodiments of the
present invention and are for illustrative purposes only. These
examples should not be deemed to narrow the scope of the present
invention.
[0034] Materials and Methods:
[0035] Materials. E. coli strains XL1-blue and BL21-Gold (DE3) were
purchased from Stratagene (La Jolla, Calif.). The template plasmid
pGalK has been previously described. Expression vector pET15b was
from Novagen (Madison, Wis.). All reagent grade chemicals and
enzymes were purchased from Promega (Madison, Wis.), Sigma (St.
Louis, Mo.), Fisher/Acros Organics (Hanover Park, Ill.), or Fluka
(Milwaukee, Wis.).
[0036] Chemical synthesis of L-idose and D-idose. The syntheses of
D- and L-idose followed literature preparations. Gene
diversification, library preparation and characterization. For the
gene library used, error-prone PCR (epPCR) was accomplished under
the following conditions: 25 mM MgCl2, 0.1 mM MnCl2, 0.2 mM (each)
dATP and dGTP, 1.0 mM (each) dCTP and dTTP, 500 pg template plasmid
pGalK, 40 pmol (each) primers 5''-CTTGGTTATGCGGGTACTGC-3'' (SEQ ID
NO: 15) and 5''-TCCCGCGAAATTAATACGAC-3'' (SEQ ID NO: 16), 5U Taq
DNA-polymerase in the buffer supplied with the enzyme, in a total
volume of 100 .mu.l using the following thermocycle parameters:
initial denaturation, 5 min, 94.degree. C.; amplification, 30
cycles, 94.degree. C. for 0.5 min, 54.degree. C. for 0.5 min,
72.degree. C. for 1.5 min; terminal hold, 5 min at 72.degree. C.
The amplification products were digested with BamHI/XbaI, purified
on an agarose gel (0.8% w/vol), eluted using the QIAquick
extraction kit (QIAGEN, Valencia, Calif.), ligated into
appropriately digested pET15b, to directly transform E. coli XL1
blue. Plasmids were isolated from randomly picked colonies
(.about.20 representatives for each library generated) and
sequenced on an ABI 310 automatic DNA sequencer (PerkinElmer,
Foster City, Calif.). Upon verifying the desired mutation rate, all
transformants were pooled, cultured overnight, the collectively
recovered plasmids used to transform E. coli BL21-Gold (DE3) and
the library processed as described below.
[0037] Bacterial fermentation. E. coli was grown in LB medium,
supplemented with ampicillin (100 .mu.L mL-1 final) under standard
conditions. For expression of GalK enzyme library members,
individual transformants were grown as 1 mL miniature cultures in
96 deep well blocks overnight as seed cultures, then replicated in
fresh medium (2%, vol/vol), afterwards added 15% (vol/vol)
glycerol, mixed, and stored at 80.degree. C. The replicated
cultures were grown to an OD600.about.0.7, protein expression was
then induced by adding 1 mM IPTG (final), for 2 hours, then
harvested by centrifugation (10 min, 3000.times.g). The cell paste
was frozen at 20.degree. C. Upon thawing the harvested expression
cultures, the biomass of each well was resuspended in 50 .mu.L
NPI-buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0) to which was then
added 70 .mu.L NPI-buffer supplemented with 1 .mu.L Lysonase
(Novagen, Madison, Wis.) for lysis. Cell debris was collected by
centrifugation (10 min, 3000.times.g), and 20 .mu.L of the clear
supernatant, containing .about.0.5 .mu.g of the expressed GalK
variant on average, was used for each kinase assay.
[0038] Library screening. Enzymatic reactions and assays were set
up and read in 96 well format on a Biomek FX automated liquid
handling workstation (Beckman Coulter, Fullerton, Calif.) fitted to
a Fluostar Optima plate reader (BMG, Durham, N.C.). The in vitro
enzymatic reactions and assays followed the protocol published
previously, slightly modified for automated liquid handling: 150
.mu.L sugar solution (8 mM final) and 12 .mu.L ATP/Mg2+ solution
(20 mM/5 mM final) were mixed, preincubated at 37.degree. C. for 5
min, then 20 .mu.L of the cleared supernatant was added, and the
reaction incubated at 37.degree. C. for an additional 2 h. To assay
the phosphorylation reactions, 50 .mu.L of the enzymatic reaction
were taken at time zero and after the 2 h incubation, mixed with
100 .mu.L of 3,5-dinitrosalicylic acid (DNS) reagent, heated at
100.degree. C. for 5 min, then immediately chilled on ice for 2
min. Assays were run as endpoint measurements following the
decrease in absorption (.lamda.=575 nm, .lamda.=758 M-1 cm-1).
[0039] Characterization of GalK Mutants. The GalK mutant Y371H was
overexpressed and purified following the procedure previously
described for wild-type GalK. The fractions containing homogenous
Y371H GalK were collected, concentrated and quantified using the
Bradford protein assay. The DNS assay was used to assess the
substrate specificity of the purified GalK mutant (Y371H) as
previously described. Standard curves for each sugar were prepared
as described. In order to determine the kinetic data for each
active monosaccharide substrate, the sugar concentration was varied
over a range of 1-16 mM, under saturating ATP (15 mM). Using the
DNS assay, change in absorbance at 575 nm as a function of time was
obtained and the initial velocity determined by the slope of the
linear phase in the progress curve. The kinetic data was analyzed
using Enzyme Kinetics Module software (SSPS, Inc., Chicago, Ill.)
as previously described.
[0040] Preparative phosphorylation of L-altrose and product
characterization. L-altrose (21.6 mg, 0.12 mmol) was dissolved in
15 mL 50 mM sodium phosphate buffer (pH 7.5). To this solution, ATP
(125 mg, 0.23 mmol), MgCl2.6H2O (15.3 mg, 0.07 mmol) were added,
the mixture incubated at 37.degree. C. for 5 min, the reaction
initiated via the addition of enzyme (Y371H) to a final
concentration of 150 .mu.g mL-1 and reaction progress monitored by
TLC. After completion, the mixture was diluted 5-fold in ddH2O and
applied to a 200 mL anion exchange column (Q-Sepharose fast flow,
Amersham, Piscataway, N.J.). The column was eluted with a gradient
of 0-400 mM NaCl and the active fraction was collected and
evaporated under reduced pressure. The crude product was desalted
on a P-2 gel filtration column (The Nest Group, Southboro, Mass.)
to give 16.1 mg of purified product (yield: 52%).
[.alpha.]D=3.5.degree. (c=1, H2O) 1H NMR (D2O): 5.48 (dd, J=8.6,
1.8 Hz, 1H), 4.14 (dd, 5.3, 3.3 Hz, 1H), 3.99 (m, 1H), 3.98 (dd,
J=4.3, 1.8 Hz, 1H), 3.91 (d, J=3.3, 1H), 3.89 (m, 1H), 3.84 (dd,
J=12.1, 7.8 Hz, 1H); 13C NMR (D2O): 94.10, 76.23, 70.70, 69.99,
65.68, 62.21; 31P NMR (D2O): 3.77; MS: calculated for C6H13O9P
260.0, found m/z 259.0 [M-H]-.
[0041] Results and Discussion:
[0042] Directed evolution of GalK with expanded specificity. The
cloned wild-type galK gene from E. coli was subjected to random
mutagenesis by epPCR performed over the entire gene. The level of
sequence alteration was adjusted to an average of 1.5 amino acid
substitutions per enzyme molecule, and verified by DNA-sequencing
of corresponding genes. However, epPCR usually results in a more or
less strong mutational bias (29, 30, 44) and is therefore not a
truly random process. Under the conditions selected, the library
contained a transition/transversion ratio of .about.3.0, the
transitions outnumbering the transversions, and the
AT.fwdarw.GC/GC.fwdarw.AT ratio was found to be 2.4. A population
of 3,500 GalK variants from this library were evaluated for their
ability to accommodate an expanded spectrum of sugar substrates.
The inventors' goals were kinase activity toward L-configured
sugars (C-5 alteration) or to those with altered substituents at
C-6. Unlike most assays in high-throughput campaigns which screen
or select toward a single substrate or substrate mimic, the
inventors' recently developed DNS assay is universally applicable
to all reducing sugars. Consequently, simultaneous screens for a
relaxed enzyme specificity were carried out with a set of
appropriately selected sugar substrates rather than a
one-dimensional single substrate screen of each library member. To
first focus upon C-5 and C-6, the set included a single C-5
challenge--L-altrose (Table 1, 16, the C-5 epimer of
D-galactose)--and three C-6 challenges-6-deoxy-D-galactose (22,
D-fucose), 6-amino-6-deoxy-D-galactose (23) and D-galacturonic acid
(24). Of this set, only D-fucose (22) showed any turnover with
wild-type GalK, albeit 2.7% that observed for D-galactose (14).
Table 1 describes the kinetic data for the ten active substrates of
the GalK variant (Y371H).
TABLE-US-00001 wild type Y371H sugar substrate K.sub.m*
k.sub.cat.sup..dagger. k.sub.catK.sub.m.sup..dagger-dbl. K.sub.m*
k.sub.cat.sup..dagger. k.sub.catK.sub.m.sup..dagger-dbl.
##STR00001## D-Gal (14) 2.1 108 51.4 5.6 220 39.3 ##STR00002##
2-deoxy- D-Gal (18) 3.6 30 8.3 4.7 200 42.6 ##STR00003##
D-GalNH.sub.2 (19) 2.9 11.7 4.0 8.8 260 29.5 ##STR00004## D-Tal
(20) --.sup..sctn. --.sup..sctn. --.sup..sctn. 2.9 45.5 15.7
##STR00005## 3-deoxy- D-Gal (21) 6.4 5.1 0.8 10.1 64.6 6.4
##STR00006## 6-deoxy- D-Gal (22) 4.9 2.9 0.59 8.0 101 12.6
##STR00007## 6-amino- D-Gal (23) --.sup..sctn. --.sup..sctn.
--.sup..sctn. 21.3 149 7.0 ##STR00008## D- Galacturonicacid (24)
--.sup..sctn. --.sup..sctn. --.sup..sctn. 3.2 58 18.1 ##STR00009##
L-Alt (16) --.sup..sctn. --.sup..sctn. --.sup..sctn. 5.2 80 15.4
##STR00010## L-Glc (25) --.sup..sctn. --.sup..sctn. --.sup..sctn.
2.7 65 24.1 *mM .sup..dagger.min.sup.-1 .sup..dagger-dbl.mM.sup.-1
min.sup.-1 .sup..sctn.no conversion
[0043] The graphical display of a typical screening result is given
in FIG. 3. Surprisingly, after only one round of evolution two GalK
variants appeared independently which phosphorylated the C-5/C-6
set of sugars with roughly similar efficiency. Subsequent
DNA-sequencing of both GalK variant genes revealed that they were
identical in their sequence and carried a single forward mutation,
a C.fwdarw.T transition at position 1111 of the wild-type reading
frame, which translates into a tyrosine 371 to histidine
replacement.
[0044] The structure of the Lactococcus (L.) lactis galactokinase
was published. Despite a rather low sequence homology to the E.
coli GalK (36% identity, 53% similarity) these two kinases clearly
share three characteristic footprint motifs, and all amino acid
residues found within the catalytic center of the L. lactis
galactokinase (R36, E42, D45, D183, and Y233) are invariably
present in its E. coli homolog (R28, E34, D37, D1174, Y223,
respectively), embedded in highly conserved sequence environments.
For this reason, the inventors speculate that the equivalent
residues also form the catalytic apparatus in the E. coli enzyme.
Surprisingly, residue 371 in the E. coli wild-type enzyme, found to
be essential for the widened substrate specificity and the activity
toward L-configured sugars, does not appear as part of its deduced
active site. In the L. lactis galactokinase the C.alpha. of the
equivalent amino acid Y385 located within the C-terminal domain
.beta.-strand K is .about.20 .ANG. from the anomeric carbon of the
substrate when bound in the active site. In the L. lactis
galactokinase crystal structure Y385 is located in close proximity
to C353 (C339 in E. coli GalK). The tyrosine phenolic oxygen is
located .about.5.5 .ANG. from the cysteine sulfur atom.
Interestingly, during the screen, a mutant on position 339 was also
discovered with a similarly expanded substrate profile yet low
catalytic activity regardless of the sugar (data not shown). This
implicates a potential Y385-C353 (Y371-C339 in E. coli GalK) side
chain interaction may play a role in stabilizing this structure and
thereby dictate substrate specificity. While an induced fit model
has not been previously put forth for this class of enzyme, such
"gate-keeping" interactions are known to occur in other enzymes
devoted to carbohydrate metabolism, such as hexokinase.
[0045] Characterization of the GalK variant (Y371H). To determine
the substrate specificity of the Y371H GalK variant, a sugar
library of twenty putative substrates was tested with the purified
enzyme. For each sugar, both the DNS assay and thin layer
chromatography were used to monitor the reaction progress and
control assays in the absence of enzyme or sugar were performed in
parallel. The mutant GalK demonstrated the ability to turn over
compounds 14, 16, 18-25 (Table 1), strikingly expanding the overall
substrate scope compared with wild-type E. coli GalK. The kinetic
parameters of the mutant enzyme with all active substrates (14, 16,
18-25) were determined using the DNS assay and compared with
wild-type GalK activity. These kinetic studies also revealed, as
expected, the evolved enzyme remains an efficient catalyst with
D-galactose (kcat=220 min-1, Km=5.6 mM) and displays remarkably
enhanced kcat values for all the previously known substrates for
wild-type GalK (14, 18, 19, 21, 22), the affinity for which is
slightly reduced in all cases.
[0046] While most in vitro evolution projects require repeated
rounds of random mutation and/or recombination to generate the
desired activity, a leap in GalK catalytic activity and substrate
selectivity was accomplished in the initial round of random
mutagenesis. Other recent similar examples of single forward
mutations leading to a catalytic shift include, for example, the
Arabidopsis thaliana cycloartenol synthase or yeast lanosterol
synthase, or the adipyl acylase evolved from a Pseudomonas glutaryl
acylase. From an analysis of the GalK substrate specificity
profiles, one can begin to construct a loose structure-activity
requirement for both wild-type enzyme and the corresponding Y371H
mutant. Specifically, wild-type GalK displays a stringent
requirement for the substrate galactose architecture from C-3
through C-6 and is capable of limited flexibility toward
substitution at C-2. Yet, it is interesting to note these stringent
requirements, with the exception of the extensive contacts at C-4,
are not readily apparent in the L. lactis GalK active site
structure. In contrast to wild-type GalK, the Y371H mutation
retains primarily only the stringent requirement for the C-4
galactose architecture with an enhanced substrate specificity
flexibility at all other positions of the sugar. Remarkably, with
essentially all substrates accepted, an enhancement of catalytic
efficiency was observed in the Y371H mutant, the enhancement
ranging from 5- to 22-fold. The only exception was the wild-type
substrate galactose for which the catalytic efficiency was
decreased slightly in comparison to wild-type GalK, albeit kcat in
this case was also increased 2-fold.
[0047] Confirmation of L-sugar conversion. The substrate
specificity studies have demonstrated GalK variant Y371H to be a
D/L-unspecific sugar kinase. To confirm the evolved enzyme retains
regio- and stereoselectivity with L-sugar substrates, a
representative L-sugar reaction product was further characterized.
Specifically, a small-scale preparative phosphorylation reaction
was performed with L-altrose (21.6 mg, 0.12 mmol). The DNS assay
indicated 91% of L-altrose conversion within four hours. Product
isolation was readily achieved by anion exchange chromatography,
and the yield of purified product was .about.52%. The purified
product was characterized by 1H and 13C NMR from which H--H
coupling and NOESY data confirmed the product to
be.beta.-L-altrose-1-phosphatein a 1C4 conformation (FIG. 4). In
particular, 3JH-H coupling data showed two typical axial-equatorial
couplings (H1-H2, H3-H4) and one equatorial-equatorial coupling
(H2-H3). NOESY data also revealed the anticipated correlations
consistent with this structure (H1-H2, H1-H5, H3-H4, H4-H6 and
H5-H6). Based upon this data the inventors propose the Y371H mutant
must bind and phosphorylate L-altrose in the same 4C1 conformation
as D-galactose (FIG. 2c and 2d) (38) the product of which
subsequently rapidly equilibrates to the more stable 1C4
conformation upon release from the enzyme.
[0048] Implications for In Vitro Glycorandomization:
[0049] Apart from total synthesis, current approaches to alter
glycosidic structures include, for example, combinatorial
biosynthesis or in vitro biocatalysis. Combinatorial biosynthesis
primarily relies on in vivo diversification via genetic engineering
of involved sugar biosynthetic pathways. Mendez, C., & Salas,
J. (2001) Trends Biotechnol. 11, 449-456. However, combinatorial
biosynthesis is significantly limited by enzyme specificity which
substantially biases the ultimate extent of diversity accessible.
In contrast, IVG presents a significant advantage by providing a
truly unbiased library of activated sugars to utilize for drug lead
glycosylation. The present advent of kinase-enhanced IVG not only
simplifies the upstream availability of sugar-1-phosphates for IVG
but also potentially opens the door to in vivo applications of
glycorandomization. Specifically, the expression of a tandem
promiscuous sugar-1-kinase (GalK) and nucleotidylyltransferase (Ep)
in a given organism, presents the prospect of generating a library
of NDP-sugars in situ. As such, the present invention provides the
foundation for eventually glycorandomizing a variety of clinically
important secondary metabolites in vivo to rapidly enhance drug
discovery efforts. Such techniques are also described in the U.S.
Patent Publications 2003/0055235A1 and 2003/0068669A1, and
International Publications WO02079150 and WO0248331, which are
incorporated herein by reference in their entirety for all
purposes.
[0050] In one embodiment of the present invention, promiscuous
sugar-1-kinase (GalK) may be used for synthesizing NDP-sugars. The
method of synthesizing comprises the steps of incubating a
nucleotide triphosphate (NTP) and a D or L sugar in the presence of
a GalK variant such that a sugar phosphate is produced. The sugar
phosphate is further incubated with a nucleotidylyltransferase,
such that a NDP-sugar is produced. Various anomeric sugars may be
used to form the sugar phosphate, including the D or L sugars such
as D-galactose, 2-deoxy D-galactose, D-galactose-amine, D-talose,
3-deoxy-D-galactose, 6-deoxy-D-galactose, 6-amino-D-galactose,
D-galacturonic acid, L-altrose and L-glucose.
[0051] In a preferred embodiment, the nucleotidylyltransferase is
Ep or a mutated variant thereof. The mutated Ep variant includes Ep
that is mutated at one or more amino acids V173, G147, W224, N112,
G175, D111, E162, T201, I200, E199, R195, L89, L89T, L109, Y146 and
Y177. In one preferred embodiment the method may be carried out in
vitro. In another preferred embodiment, the method is carried out
in a host cell. The host cell may be a bacterium. Further, the host
cell may be selected from the group consisting of E. coli and S.
lividans.
[0052] Another preferred embodiment of the present invention
provides a method of producing a glycosylated biomolecule
containing at least one sugar moiety. The method comprises the
steps of incubating a nucleotide triphosphate (NTP) and a D or L
sugar in the presence of a GalK variant, whereby a sugar phosphate
is produced; incubating the sugar phosphate with a
nucleotidylyltransferase, whereby a NDP-sugar is produced; and
incubating the NDP-sugar with a biomolecule capable of being
glycosylated in the presence of a glycosyltransferase, such that a
glycosylated biomolecule is produced. In a preferred embodiment,
the glycosyltransferase is selected from the group consisting of
CalB, CalE, CalN, CalU, Gra orf14, Gra orf5, LanGT1, LanGT2,
LanGT3, LanGT4, MtmGI, MtmGII, MtmGTIII, MtmGTIV, NovM, RhlB, Rif
orf 7, SnogD, SnogE, SnogZ, UrdGT1a, UrdGT1b, UrdGT1c, UrdGT2,
AknK, AknS, DesVII, DnrS, OleG1, OleG2, TylCV, TylMII, TylN, DauH,
DnrH, EryBV, EryCIII, Ngt, BgtA, BgtB, BgtC, GftA, GftB, GftC,
GftD, GftE, Gp1-1, Gp1-2, RtfA, AveBI, BlmE, BlmF, MgtA, NysD1,
OleD, OleI, SpcF, SpcG, StrH, Ugt51B1, Ugt51C1, UGT52, UgtA, UgtB,
UgtC, UgtD and homologs thereof. In another preferred embodiment,
the biomolecule capable of being glycosylated is selected from the
group consisting of natural and synthetic metabolites, pyran rings,
furan rings, enediynes, anthracyclines, angucyclines, aureolic
acids, orthosomycins, macrolides, aminoglycosides, non-ribosomal
peptides, polyenes, steroids, lipids, indolocarbazoles, bleomycins,
amicetins, benzoisochromanequinones coumarins, polyketides,
pluramycins, aminoglycosides, oligosaccharides, peptides, proteins,
hybrids consisting of one or more these components, analogs and
bioactive aglycons thereof. In yet another preferred embodiment,
the glycosylated biomolecule is further incubated with at least one
chemoselectively ligatable moiety, such that at least one
chemoselectively ligated compound is produced. Exempletive
chemoligation techniques are described in the United States Patent
Application No. (NOT ASSIGNED) "Glycorandomization and Production
of Novel Vancomycin Analogs", filed on Sep. 24, 2003, which is
incorporated in its entirety by reference for all purposes.
TABLE-US-00002 TABLE 2 D-Gal (29) D-Gal (29) D-Glc (27) D-Glc (27)
L-Alt (21) L-Alt (21) Km Vmax Km Vmax Km Vmax mM mM min - 1 Mm mM
min - 1 mM mM min - 1 WT 2.1 (.+-.0.4) 1.5 (.+-.0.5) -- -- -- --
GalK M173L 5.9 (.+-.0.8) 1.9 (.+-.0.6) 2.6 (.+-.0.5) 0.02
(.+-.0.01) -- -- Y371H 5.6 (.+-.0.3) 2.2 (.+-.0.6) -- -- 6.0
(.+-.0.9) 0.16 (.+-.0.04) M173L- 4.6 (.+-.0.7) 2.3 (.+-.0.4) 4.0
(.+-.0.6) 0.02 (.+-.0.01) 6.3 (.+-.1.1) 0.07 (.+-.0.03) Y371H
EXAMPLE II
[0053] Glycorandomization (FIG. 5a), a process centered upon the
inherent promiscuity of secondary metabolite-associated
glycosyltransferases, is one of the latest promising developments
toward this important goal. Critical to the success of
glycorandomization has been the ability to engineer and/or evolve
two additional promiscuous enzymes--anomeric kinases and
nucleotidylyltransferases. Taken together with the many elegant
methods to synthesize monosaccharide libraries and the intrinsic
substrate flexibility of many secondary metabolite-associated
glycosyltransferases, this two-enzyme short activation pathway
allows one to rapidly diversify the sugars attached to complex
natural products. The glycorandomization process is further
enhanced via a final diversification step which relies upon the use
of downstream chemoselective ligation.
[0054] Cumulatively, the successful demonstration of in vitro
glycorandomization, the observations that functional NDP-sugar
pathways can be reassembled in prokaryotes, and the fact that
natural and `unnatural` endogenous sugars are processed in vivo by
both prokaryotes and eukaryotes, present the foundation from which
to approach in vivo glycorandomization. For example, the expression
of a tandem promiscuous sugar-1-kinase (GalK) and
nucleotidylyltransferase (Ep)--essentially an unnatural NDP-sugar
factory--in a natural aglycon-producing host (e.g. the
erythromycin-producing Saccharopolyspora) should present the
prospect of generating a glycorandomized library in situ, the
glycorandomized metabolite output of which is controlled by
monosaccharides being fed to the strain (FIG. 5b). Alternatively,
expression of the tandem two gene `NDP-sugar factory` genes in a
non-producing host (e.g. S. lividans or E. coli) which expresses a
given glycosyltransferase (or glycosyltransferase library), should
also provide a vehicle to accomplish glycorandomization via feeding
the host with appropriate aglycon acceptors and unnatural sugar
donors (FIG. 5c). The key to either in vivo scenario is the ability
of unnatural sugars to enter the host and serve as efficient
substrates of the first enzyme of the short activation pathway (the
flexible anomeric kinase). Toward this goal, a kinase able to
process sugars bearing unique mass signatures and/or reactive
handles would, in addition to further enhancing library
diversification, greatly simplify the final analysis of in vivo
access and activity.
[0055] The inventor has applied directed enzyme evolution and
relied upon a high throughput galactokinase (GalK) assay (DNS
assay) for the screening of diverse E. coli GalK variant libraries
generated via error-prone PCR. From this approach, one particular
GalK mutant (Y371H) demonstrated remarkably widened substrate
flexibility toward C-2, C-3 and C-5 substitutions of D-galactose.
Yet, the mutant retained a stringent requirement for the axial C-4
galactose architecture. The recently solved L. lactis GalK crystal
structure suggested two highly conserved active-site residues
(Asp-37 and Tyr-223 in E. coli GalK) are responsible for
hydrogen-bonding with this C-4 axial hydroxyl group of the
substrate. Yet, saturation mutagenesis at these two critical
positions in the E. coli enzyme failed to provide mutants with
enhanced C-4 sugar flexibility while a parallel study revealed the
L. lactis wild-type GalK and Y385H orthologs to surprisingly
display weak activity toward the C-4 epimer, glucose.
[0056] Herein the inventor reports a structure-activity model,
based upon the L. lactis active site and its ability to weakly
utilize glucose, led to a specific engineered E. coli M 173L mutant
GalK with enhanced C-4 and C-6 promiscuity. Moreover, a combination
of the favorable structure-based (M173L) mutation with the
beneficial mutation previously discovered via directed evolution
(Y371H) drastically exceeds an additive enhancement for both C-4
and C-6 substitutions. Most importantly, the additional unnatural
sugar substrates accessed by this unique double mutant allowed the
unique opportunity to assess whether unnatural sugars can enter a
bacterial host and serve as efficient substrates of the first
enzyme of the glycorandomization pathway (the flexible anomeric
kinase). Specifically, feeding of the unique substrate
6-azido-6-deoxy-galactose (22) or 6-azido-6-deoxy-glucose (46) to
an E. coli host engineered to express M173L-Y371H-GalK followed by
the rapid fluorescent labeling of substrates and products via
Huisgen 1,3-dipolar cycloaddition revealed the desired efficient
sugar-1-phosphate production in vivo. This result stands as a key
first step in demonstrating the concept of in vivo
glycorandomization.
[0057] Materials and Methods:
[0058] Materials. The syntheses of 4-azido-4-deoxy-D-galactose
(21), 6-azido-6-deoxy-D-galactose (22),
6-chloro-6-deoxy-D-galactose (23), 6-bromo-6-deoxy-D-galactose
(24), 4-deoxy-D-galactose (25), 6-hydroxymethylene-D-galactose
(32), 3-deoxy-D-galactose (34), 6-amino-6-deoxy-D-galactose (35),
6-deoxy-6,6-difluoro-D-galactose (40), were reported previously
while other monosaccharide compounds 26-31, 33, 36-39, 41, 42, 45,
47 and 48 were purchased from Sigma (St. Louis, Mo.), Fisher/Acros
Organics (Hanover Park, Ill.), or Fluka (Milwaukee, Wis.). E. coli
strains XL1-blue and BL21 (DE3) were purchased from Stratagene (La
Jolla, Calif.). Expression vector pET15b was purchased from Novagen
(Madison, Wis.). Enzymes were purchased from Promega (Madison,
Wis.).
[0059] Chemical Synthesis. For chemical synthesis of
6-thio-6-deoxy-D-galactose 43, 6-thio-6-deoxy-D-glucose 44,
6-azido-6-deoxy-D-glucose 46, and
5-dimethylaminonaphthalene-1-(N-(5-propargylamidepentyl))-sulfonamide
50, see supporting methods, which is published as supporting
information on the PNAS web site.
[0060] Structure Modeling. The PDB file for the crystal structure
of L. lactis wild type GalK was obtained from the Protein Data
Bank, Research Collaboratory for Structural Bioinformatics, Rutgers
University, New Brunswick, N.J. (available on the Rutgers
University Website). The structure modeling was accomplished using
Swiss-PdbViewer software (Version 3.7).
[0061] Site-specific Mutagenesis. The GalK M173L single mutant and
M173L-Y371H double mutant were generated using the QuikChange II
Site-Directed Mutagenesis Kit from either wild-type or Y371H
template, respectively (Stratagene). The corresponding mutated
plasmids pGalKM173L and pGalKMLYH were constructed by using
PfuUltra.TM. DNA polymerase for mutagenic primer-directed
replication from pGalK or pGalKY371H template, respectively, using
a pair of mutagenic primers
(5'-GTAACTGCGGGATCCTGGATCAGCTAATTTCCG-3' (SEQ ID NO: 17) and
5'-CGGAAATTAGCTGATCCAGGATCCCGCAGTTAC-3' (SEQ ID NO: 18)).
Amplification was accomplished under the following conditions: 5
.mu.L of 10.times. reaction buffer, 40 ng template DNA, 120 ng of
each oligonucleotide primer, 1 .mu.L dNTPs mixture (2.5 mM), 2.5 U
of PfuUltra.TM. high-fidelity DNA polymerase, in a total volume of
50 .mu.L ddH2O (thermocycler parameters: initial denaturation, 2
min at 95.degree. C.; amplification, 12 cycles, 0.5 min at
95.degree. C., 1 min at 55.degree. C., 6.5 min at 68.degree. C.;
terminal hold, 5 min at 68.degree. C.). The amplified plasmids were
treated with DpnI to digest the parental DNA template and the
mutated prodigy plasmid subsequently used to transform E. coli
XL1-blue. The desired point mutation was verified by
sequencing.
[0062] Characterization of GalK Mutants. The GalK mutants Y371H was
overexpressed following the procedure previously described for
wild-type E. coli GalK, while the overexpression of mutants M173L
and M173L-Y371H were accomplished at 16.degree. C. as described for
L. lactis GalK. The mutant enzymes were purified by using metal
affinity chromatography on Ni-NTA Spin Columns (QIAGEN, Valencia,
Calif.) and fractions containing homogenous protein were collected,
concentrated and quantified using the Bradford protein assay. The
DNS assay was used to assess the substrate specificity of the
purified GalK mutants as previously described. A library of 45
different sugars was screened with each mutant (M173L, Y371H and
M173L-Y371H). For each sugar, the DNS assay was used to monitor the
reaction progress and control assays in the absence of enzyme or
sugar were performed in parallel. Standard curves for each sugar
were prepared as described. To assess general percent conversion,
each reaction contained 15.0 (M enzyme, 8 mM sugar, 14 mM ATP and
3.5 mM MgCl.sub.2. The reactions were incubated at 37.degree. C.
for 3 hrs after which the reactions were quenched with MeOH,
centrifuged (10 min, 12,000 rpm) and then the supernatant (diluted
20-fold) submitted for LC-MS and MS/MS analysis. For monosaccharide
kinetic data determination, the sugar concentration was varied over
a range of 1-8 mM, under saturating ATP (14 mM). Reaction progress
was assessed via the DNS assay, wherein a change in absorbance at
575 nm as a function of time was obtained and the initial velocity
determined by the slope of the linear phase in the progress curve.
The kinetic data was analyzed using Enzyme Kinetics Module software
(SSPS, Inc., Chicago, Ill.) as previously described.
[0063] In Vivo Conversion Analysis. The GalK double mutant
pGalKMLYH-E. coli was overexpressed at 16.degree. C. via induction
of a 40 mL of culture at an OD600.about.0.7 with IPTG (1 mM). The
induced and the cultures were incubated with shaking (140 rpm) for
1 hr and 100 mM 22 or 46 was added to the culture to a final
concentration of 4 mM. The cultures were further incubated at
16.degree. C. with shaking (140 rpm) for 16 hr. To assess
bioconversion, the cells were harvested by centrifugation (15 min,
12,000 rpm) and the recovered cell pellet (380 mg) washed twice
with sodium phosphate buffer (20 mL), frozen, thawed and
resuspended in H2O:MeOH (1:1). The resuspended solution was heated
at 100.degree. C. for 15 min and then sonicated 5.times.45 sec on
ice. Cell debris was collected by centrifugation (15 min, 12,000
rpm) and lyophilized to give the pale white solid (18 mg). To a
solution containing all the crude product in 160 .mu.L of H2O:MeOH
(1:1) was added 10 .mu.mol 50 and 3.2 .mu.mol of CuI, followed by
heating to 50.degree. C. for 24 hours. The reaction mixture was
subsequently centrifuged to remove CuI and the supernatant (diluted
2-fold) directly analyzed by HPLC and LC-MS calculated for
C26H37N6O11PS 672.20, found m/z [M+H]-671.20.
[0064] Results and Discussion:
[0065] Structure-basis for Engineering GalKs with Expanded
Specificity. Prior to any available GalK structural information,
the directed evolution of E. coli GalK presented a general sugar
anomeric kinase with widened flexibility primarily at C-5 and C-6
of the sugar substrate. Interestingly, all C-4-modified derivatives
tested in this study failed as substrates for the evolved catalyst.
In contrast, the recent analysis of the L. lactis wild-type GalK
revealed in vitro conversion of various C-4-modified analogs,
including 4-azido-4-deoxy-D-galactose (21), 4-deoxy-D-galactose
(25) and D-glucose (27). The recent structure elucidation of
Lactococcus lactis GalK potentially allows for a molecular level
assessment of this surprising C-4 specificity distinction between
the E. coli and L. lactis enzymes. With the L. lactis structure as
a template, the sequence alignment of the E. coli and L. lactis
GalKs revealed one clear difference among the sugar-binding
pockets. Specifically, Leu-182 in L. lactis GalK is near to the C-4
carbon atom of galactose (3.85 .ANG.) and, based upon sequence
alignment, this residue is replaced by Met-173 in E. coli GalK. A
model of the C-4 epimer of galactose (D-glucose) within the L.
lactis active site predicts the Glc-C-4 equatorial hydroxyl to be
3.79 .ANG. from the .gamma. methyl of Leu 182 (FIG. 6a). However,
the identical model in which Leu-182 has been replaced by Met (to
mimic the E. coli GalK active site) revealed the same Glc-C-4
equatorial hydroxyl to be 1.72 .ANG. from Met sulfur (FIG. 6b).
Thus, this model clearly suggests Met 173 in E. coli GalK may
exclude glucose and thereby limit sugar C-4 specificity to
galacto-configured substrates. Moreover, given the close proximity
of the sugar C-6 hydroxyl to Met in this structural model (2.85
.ANG.) (FIG. 6b), the inventors believe that M-173 in E. coli GalK
may also limit C-6 variation.
[0066] Characterization of Engineered E. coli GalK Mutants. To test
the above hypothesis, a single E. coli GalK M173L mutant was
generated and screened against a panel 45 potential sugar
substrates. As predicted, the E. coli M173L engineered mutant
displayed moderate D-glucose 27 activity (20% conversion in 3 hr).
Moreover, three additional D-gluco-configured structures 28, 37, 38
(FIGS. 7 and 8), which were not substrates of wild-type E. coli
GalK (or the evolved E. coli Y371H mutant), were also substrates of
the new structure-based variant. While these studies clearly
revealed the structure-based M173L mutant to accept a substrate set
distinct to that of the previously evolved Y371H mutant, in
contrast to the structural model described above, both mutants
failed in the presence of substrates presenting even moderate C-6
bulk such as 6-azido-6-deoxy-galactose 22 or
6-azido-6-deoxy-D-glucose 46. In an attempt to further generalize
the sugar kinase activity, an E. coli double M173L-Y371H mutant was
examined. Remarkably, not only did this double mutant retain the
activity of both corresponding single mutants, but this prodigy
demonstrated a substantial degree of kinase activity toward a
variety of new structures (21-26, 43-48). Most of the new
substrates share modifications at C-4 and/or C-6, with many of
D-gluco origin. It is also noteworthy that three among this new
substrate set are azidosugars (21, 22 and 46), thereby setting the
stage for rapid analysis of in vivo bioconversion via
post-bioconversion labeling of substrates and products with a
fluorescent tag using Huisgen 1,3-cycloaddition. FIGS. 7 and 8
illustrate the complete substrate profiles for wild-type E. coli
GalK, the E. coli GalK mutant M173L, Y371H and M173L-Y371H.
[0067] To better understand the distinct role of the two particular
amino acid residues (Met-173 and Tyr-371) in determining the
substrate specificity, the inventor chose the native substrate
D-galactose 29, the unique M173L substrate D-glucose 27 and the
unique Y371H substrate L-altrose 41 for complete comparative
steady-state kinetic profiling. In comparison to wild-type E. coli
GalK, a slight (around 2-fold) D-galactose Km increase was observed
in all three variants (M173L, Y371H and M173L-Y371H). Moreover, a
comparison of the D-glucose kinetic parameters for M173L and the
L-altrose kinetic values for Y371H to those of the double mutant
revealed very little change. Thus, in contrast to the notable gain
of function (in terms of the shear number of new M173L-Y371H
substrates) illustrated in FIG. 7, the kinetic analysis only
predicts the gain of function to be additive at best. In other
words, the kinetic analysis predicts the double mutant should
accept only known M173L and Y371H substrates but would not predict
an expansion beyond this dual substrate set. Yet, while it is
difficult to explain this remarkable gain of function in the
M173L-Y371H variant, the unique ability of this double mutant to
accept compounds 21, 22 and 46 sets the stage to assess the first
step of in vivo glycorandomization as described below.
[0068] In Vivo Bioconversion of Unnatural Sugars Using an
Engineered GalK. In the context of assessing in vivo bioconversion,
the specific M173L-Y371H-22 relationship is advantageous for two
reasons. First, as described above, 21, 22 and 46 are not a
substrates for wild-type E. coli GalK and therefore, the use of a
standard E. coli host strain (which contains the inherent wild-type
E. coli GalK) should not interfere. Second, as previously
mentioned, 21, 22 and 46 each offer a unique functional handle to
provide for the rapid installation of a fluorescent label to
simplify the chromatographic analysis. In this context, 22 and 46
are equally reactive to the required fluorescent-labeling via
Husigen 1,3-cycloaddition while 21 is poorly reactive (<10% X.
Fu, unpublished). Thus, for the current in vivo analysis, 21 was
excluded. The set selected (22 and 46) still offer the opportunity
to test a range of substrates with distinctly unique in vitro
profiles. Specifically, 22 is known to have >50% in vitro in 2
hrs while 46 shows .about.15% conversion under the same
conditions.
[0069] To assess the Y371H-M173L GalK-catalyzed in vivo production
of unnatural sugar-1-phosphates (FIG. 9a), the unnatural sugars (22
or 46, 4 mM final concentration) were fed to an E. coli host (40 mL
culture) which overexpressed the promiscuous GalK. After a
designated time, the extracts were analyzed via the specific
attachment of a fluorescent tag (50), to both starting material (22
or 46) and desired sugar-1-phosphate products, using 1,3-dipolar
cycloaddition. Two controls were run in parallel. The first
utilized a strain containing an empty expression vector
(pET-15b--the vector used for overexpression of the GalK mutants)
while the second employed a wild-type GalK overexpression strain.
The crude products from each bioconversion were isolated, labelled
via 1,3-dipolar cycloaddition and analyzed by fluorescence HPLC and
LC-MS. As illustrated in FIG. 9b, .about.69% conversion of
6-azido-6-deoxy-D-galactose (22) was observed, a slight improvement
over the in vitro yield (.about.50% conversion). In a similar
manner, 15% conversion of 6-azido-6-deoxy-D-glucose (46) was
observed, consistent with the in vitro yield (.about.50%
conversion). Notably, this success illustrates that unnatural
sugars are able to enter the heterologous E. coli host and access
the engineered promiscuous sugar kinase. Given this key result, it
is likely that the addition of a flexible nucleotidylytransferase
(E2) and glycosyltransferase to this host will allow for in vivo
glycorandomization.
[0070] Implications for In Vivo Glycorandomization:
[0071] The recent developed chemoenzymatic approach in vitro
glycorandomization significantly contributes to the diversity of
novel therapeutics via altering glycosylation patterns on secondary
metabolites. However, the general application of this approach is
significantly limited by the two primary issues. First, expensive
substrates/cofactors significantly hamper the scaling up the
process, although the alternative solutions for regenerating these
reagents are available. Second, the application of IVG to most, or
all, classes of glycosylated natural products is heavily dependent
upon the expression of appropriate glycosyltransferases and
establishing in vitro conditions for an active enzyme which, in
some cases, can be severely dictated by the solubility of the
aglycon acceptor. The present advent of kinase-enhanced IVG
potentially opens the door to in vivo applications of
glycorandomization, and the inventor believes the in vivo process
would be able to overcome these limitations. The current result
illustrates the entry of unnatural sugars and their subsequent
utilization by the engineered GalK. This result clearly stands as
strong evidence supporting the overall feasibility of in vivo
glycorandomization. As such, this work provides the foundation for
the eventual glycorandomization a variety of clinically important
secondary metabolites in vivo to rapidly enhance drug discovery
efforts.
[0072] The sugar kinase with expanded substrate specificity useful
for glycorandomization of the present invention has many other
applications aside from those described in the preferred embodiment
and examples. Thus, although the invention has been herein shown
and described in what is perceived to be the most certain
embodiments, it is to be understood that the invention is not
intended to be limited to the specific embodiments set forth above.
Rather, it is recognized that certain modifications, substitutions,
alterations, omissions may be made by one of skill in the art of
the invention without departing from the spirit or intent of the
invention. Accordingly, the invention is to be taken as including
all reasonable equivalents to the subject matter of the appended
claims and the foregoing description is meant to be exemplary only
and should not limit the scope of the invention set forth in the
following claims.
[0073] All references are incorporated by reference herein for all
purposes.
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Sequence CWU 1
1
181382PRTEscherichia coli 1Met Ser Leu Lys Glu Lys Thr Gln Ser Leu
Phe Ala Asn Ala Phe Gly1 5 10 15Tyr Pro Ala Thr His Thr Ile Gln Ala
Pro Gly Arg Val Asn Leu Ile 20 25 30Gly Glu His Thr Asp Tyr Asn Asp
Gly Phe Val Leu Pro Cys Ala Ile 35 40 45Asp Tyr Gln Thr Val Ile Ser
Cys Ala Pro Arg Asp Asp Arg Lys Val 50 55 60Arg Val Met Ala Ala Asp
Tyr Glu Asn Gln Leu Asp Glu Phe Ser Leu65 70 75 80Asp Ala Pro Ile
Val Ala His Glu Asn Tyr Gln Trp Ala Asn Tyr Val 85 90 95Arg Gly Val
Val Lys His Leu Gln Leu Arg Asn Asn Ser Phe Gly Gly 100 105 110Val
Asp Met Val Ile Ser Gly Asn Val Pro Gln Gly Ala Gly Leu Ser 115 120
125Ser Ser Ala Ser Leu Glu Val Ala Val Gly Thr Val Leu Gln Gln Gly
130 135 140Tyr His Leu Pro Leu Asp Gly Ala Gln Ile Ala Leu Asn Gly
Gln Glu145 150 155 160Ala Glu Asn Gln Phe Val Gly Cys Asn Cys Gly
Ile Met Asp Gln Leu 165 170 175Ile Ser Ala Leu Gly Lys Lys Asp His
Ala Leu Leu Ile Asp Cys Arg 180 185 190Ser Leu Gly Thr Lys Ala Val
Ser Met Pro Lys Gly Val Ala Val Val 195 200 205Ile Ile Asn Ser Asn
Phe Lys Arg Thr Leu Val Gly Ser Glu Tyr Asn 210 215 220Thr Arg Arg
Glu Gln Cys Glu Thr Gly Ala Arg Phe Phe Gln Gln Pro225 230 235
240Ala Leu Arg Asp Val Thr Ile Glu Glu Phe Asn Ala Val Ala His Glu
245 250 255Leu Asp Pro Ile Val Ala Lys Arg Val Arg His Ile Leu Thr
Glu Asn 260 265 270Ala Arg Thr Val Glu Ala Ala Ser Ala Leu Glu Gln
Gly Asp Leu Lys 275 280 285Arg Met Gly Glu Leu Met Ala Glu Ser His
Ala Ser Met Arg Asp Asp 290 295 300Phe Glu Ile Thr Val Pro Gln Ile
Asp Thr Leu Val Glu Ile Val Lys305 310 315 320Ala Val Ile Gly Asp
Lys Gly Gly Val Arg Met Thr Gly Gly Gly Phe 325 330 335Gly Gly Cys
Ile Val Ala Leu Ile Pro Glu Glu Leu Val Pro Ala Val 340 345 350Gln
Gln Ala Val Ala Glu Gln Tyr Glu Ala Lys Thr Gly Ile Lys Glu 355 360
365Thr Phe Tyr Val Cys Lys Pro Ser Gln Gly Ala Gly Gln Cys 370 375
3802382PRTEscherichia coliMISC_FEATURE(28)..(28)Xaa can be any
naturally occurring amino acid. 2Met Ser Leu Lys Glu Lys Thr Gln
Ser Leu Phe Ala Asn Ala Phe Gly1 5 10 15Tyr Pro Ala Thr His Thr Ile
Gln Ala Pro Gly Xaa Val Asn Leu Ile 20 25 30Gly Glu His Thr Asp Tyr
Asn Asp Gly Phe Val Leu Pro Cys Ala Ile 35 40 45Asp Tyr Gln Thr Val
Ile Ser Cys Ala Pro Arg Asp Asp Arg Lys Val 50 55 60Arg Val Met Ala
Ala Asp Tyr Glu Asn Gln Leu Asp Glu Phe Ser Leu65 70 75 80Asp Ala
Pro Ile Val Ala His Glu Asn Tyr Gln Trp Ala Asn Tyr Val 85 90 95Arg
Gly Val Val Lys His Leu Gln Leu Arg Asn Asn Ser Phe Gly Gly 100 105
110Val Asp Met Val Ile Ser Gly Asn Val Pro Gln Gly Ala Gly Leu Ser
115 120 125Ser Ser Ala Ser Leu Glu Val Ala Val Gly Thr Val Leu Gln
Gln Leu 130 135 140Tyr His Leu Pro Leu Asp Gly Ala Gln Ile Ala Leu
Asn Gly Gln Glu145 150 155 160Ala Glu Asn Gln Phe Val Gly Cys Asn
Cys Gly Ile Met Asp Gln Leu 165 170 175Ile Ser Ala Leu Gly Lys Lys
Asp His Ala Leu Leu Ile Asp Cys Arg 180 185 190Ser Leu Gly Thr Lys
Ala Val Ser Met Pro Lys Gly Val Ala Val Val 195 200 205Ile Ile Asn
Ser Asn Phe Lys Arg Thr Leu Val Gly Ser Glu Tyr Asn 210 215 220Thr
Arg Arg Glu Gln Cys Glu Thr Gly Ala Arg Phe Phe Gln Gln Pro225 230
235 240Ala Leu Arg Asp Val Thr Ile Glu Glu Phe Asn Ala Val Ala His
Glu 245 250 255Leu Asp Pro Ile Val Ala Lys Arg Val Arg His Ile Leu
Thr Glu Asn 260 265 270Ala Arg Thr Val Glu Ala Ala Ser Ala Leu Glu
Gln Gly Asp Leu Lys 275 280 285Arg Met Gly Glu Leu Met Ala Glu Ser
His Ala Ser Met Arg Asp Asp 290 295 300Phe Glu Ile Thr Val Pro Gln
Ile Asp Thr Leu Val Glu Ile Val Lys305 310 315 320Ala Val Ile Gly
Asp Lys Gly Gly Val Ala Met Thr Gly Gly Gly Phe 325 330 335Gly Gly
Cys Ile Val Ala Leu Ile Pro Glu Glu Leu Val Pro Ala Val 340 345
350Gln Gln Ala Val Ala Glu Gln Tyr Glu Ala Lys Thr Gly Ile Lys Glu
355 360 365Thr Phe Tyr Val Cys Lys Pro Ser Gln Gly Ala Gly Gln Cys
370 375 3803382PRTEscherichia coliMISC_FEATURE(34)..(34)Xaa can be
any naturally occurring amino acid. 3Met Ser Leu Lys Glu Lys Thr
Gln Ser Leu Phe Ala Asn Ala Phe Gly1 5 10 15Tyr Pro Ala Thr His Thr
Ile Gln Ala Pro Gly Arg Val Asn Leu Ile 20 25 30Gly Xaa His Thr Asp
Tyr Asn Asp Gly Phe Val Leu Pro Cys Ala Ile 35 40 45Asp Tyr Gln Thr
Val Ile Ser Cys Ala Arg Arg Asp Asp Arg Lys Val 50 55 60Arg Val Met
Ala Ala Asp Tyr Glu Asn Gln Leu Asp Glu Phe Ser Leu65 70 75 80Asp
Ala Pro Ile Val Ala His Glu Asn Tyr Gln Trp Ala Asn Tyr Val 85 90
95Arg Gly Val Val Lys His Leu Gln Leu Arg Asn Asn Ser Phe Gly Gly
100 105 110Val Asp Met Val Ile Ser Gly Asn Val Pro Gln Gly Ala Gly
Leu Ser 115 120 125Ser Ser Ala Ser Leu Glu Val Ala Val Gly Thr Val
Leu Gln Gln Leu 130 135 140Tyr His Leu Pro Leu Asp Gly Ala Gln Ile
Ala Leu Asn Gly Gln Glu145 150 155 160Ala Glu Asn Gln Phe Val Gly
Cys Asn Cys Gly Ile Met Asp Gln Leu 165 170 175Ile Ser Ala Leu Gly
Lys Lys Asp His Ala Leu Leu Ile Asp Cys Arg 180 185 190Ser Leu Gly
Thr Lys Ala Val Ser Met Pro Lys Gly Val Ala Val Val 195 200 205Ile
Ile Asn Ser Asn Phe Lys Arg Thr Leu Val Gly Ser Glu Tyr Asn 210 215
220Thr Arg Arg Glu Gln Cys Glu Thr Gly Ala Arg Phe Phe Gln Gln
Pro225 230 235 240Ala Leu Arg Asp Val Thr Ile Glu Glu Phe Asn Ala
Val Ala His Glu 245 250 255Leu Asp Pro Ile Val Ala Lys Arg Val Arg
His Ile Leu Thr Glu Asn 260 265 270Ala Arg Thr Val Glu Ala Ala Ser
Ala Leu Glu Gln Gly Asp Leu Lys 275 280 285Arg Met Gly Glu Leu Met
Ala Glu Ser His Ala Ser Met Arg Asp Asp 290 295 300Phe Glu Ile Thr
Val Pro Gln Ile Asp Thr Leu Val Glu Ile Val Lys305 310 315 320Ala
Val Ile Gly Asp Lys Gly Gly Val Arg Met Thr Gly Gly Gly Phe 325 330
335Gly Gly Cys Ile Val Ala Leu Ile Pro Glu Glu Leu Val Pro Ala Val
340 345 350Gln Gln Ala Val Ala Glu Gln Tyr Glu Ala Lys Thr Gly Ile
Lys Glu 355 360 365Thr Phe Tyr Val Cys Lys Pro Ser Gln Gly Ala Gly
Gln Cys 370 375 3804382PRTEscherichia coliMISC_FEATURE(37)..(37)Xaa
can be any naturally occurring amino acid. 4Met Ser Leu Lys Glu Lys
Thr Gln Ser Leu Phe Ala Asn Ala Phe Gly1 5 10 15Tyr Pro Ala Thr His
Thr Ile Gln Ala Pro Gly Arg Val Asn Leu Ile 20 25 30Gly Glu His Thr
Xaa Tyr Asn Asp Gly Phe Val Leu Pro Cys Ala Ile 35 40 45Asp Tyr Gln
Thr Val Ile Ser Cys Ala Pro Arg Asp Asp Arg Lys Val 50 55 60Arg Val
Met Ala Ala Asp Tyr Glu Asn Gln Leu Asp Glu Phe Ser Leu65 70 75
80Asp Ala Pro Ile Val Ala His Glu Asn Tyr Gln Trp Ala Asn Tyr Val
85 90 95Arg Gly Val Val Lys His Leu Gln Leu Arg Asn Asn Ser Phe Gly
Gly 100 105 110Val Asp Met Val Ile Ser Gly Asn Val Pro Gln Gly Ala
Gly Leu Ser 115 120 125Ser Ser Ala Ser Leu Glu Val Ala Val Gly Thr
Val Leu Gln Gln Leu 130 135 140Tyr His Leu Pro Leu Asp Gly Ala Gln
Ile Ala Leu Asn Gly Gln Glu145 150 155 160Ala Glu Asn Gln Phe Val
Gly Cys Asn Cys Gly Ile Met Asp Gln Leu 165 170 175Ile Ser Ala Leu
Gly Lys Lys Asp His Ala Leu Leu Ile Asp Cys Arg 180 185 190Ser Leu
Gly Thr Lys Ala Val Ser Met Pro Lys Gly Val Ala Val Val 195 200
205Ile Ile Asn Ser Asn Phe Lys Arg Thr Leu Val Gly Ser Glu Tyr Asn
210 215 220Thr Arg Arg Glu Gln Cys Glu Thr Phe Ala Arg Phe Phe Gln
Gln Pro225 230 235 240Ala Leu Arg Asp Val Thr Ile Glu Glu Phe Asn
Ala Val Ala His Glu 245 250 255Leu Asp Pro Ile Val Ala Lys Arg Val
Arg His Ile Leu Thr Glu Asn 260 265 270Ala Arg Thr Val Gly Ala Ala
Ser Ala Leu Glu Gln Gly Asp Leu Lys 275 280 285Arg Met Gly Glu Leu
Met Ala Glu Ser His Ala Ser Met Arg Asp Asp 290 295 300Phe Glu Ile
Thr Val Pro Gln Ile Asp Thr Leu Val Glu Ile Val Lys305 310 315
320Ala Val Ile Gly Asp Lys Gly Gly Val Arg Met Thr Gly Gly Gly Phe
325 330 335Gly Gly Cys Ile Val Ala Leu Ile Pro Glu Glu Leu Val Pro
Ala Val 340 345 350Gln Gln Ala Val Ala Glu Gln Tyr Glu Ala Lys Thr
Gly Ile Lys Glu 355 360 365Thr Phe Tyr Val Cys Lys Pro Ser Gln Gly
Ala Gly Gln Cys 370 375 3805382PRTEscherichia
coliMISC_FEATURE(174)..(174)Xaa can be any naturally occurring
amino acid. 5Met Ser Leu Lys Glu Lys Thr Gln Ser Leu Phe Ala Asn
Ala Phe Gly1 5 10 15Tyr Pro Ala Thr His Thr Ile Gln Ala Pro Gly Arg
Val Asn Leu Ile 20 25 30Gly Glu His Thr Asp Tyr Asn Asp Gly Phe Val
Leu Pro Cys Ala Ile 35 40 45Asp Tyr Glu Thr Val Ile Ser Cys Ala Pro
Arg Asp Asp Arg Lys Val 50 55 60Arg Val Met Ala Ala Asp Tyr Glu Asn
Gln Leu Asp Glu Phe Ser Leu65 70 75 80Asp Ala Pro Ile Val Ala His
Glu Asn Tyr Gln Trp Ala Asn Tyr Val 85 90 95Arg Gly Val Val Lys His
Leu Gln Leu Arg Asn Asn Ser Phe Gly Gly 100 105 110Val Asp Met Val
Ile Ser Gly Asn Val Pro Gln Gly Ala Gly Leu Ser 115 120 125Ser Ser
Ala Ser Leu Glu Val Ala Val Gly Thr Val Leu Gln Gln Leu 130 135
140Tyr His Leu Pro Leu Asp Gly Ala Gln Ile Ala Leu Asn Gly Gln
Glu145 150 155 160Ala Glu Asn Gln Phe Val Gly Cys Asn Cys Gly Ile
Met Xaa Gln Leu 165 170 175Ile Ser Ala Leu Gly Lys Lys Asp His Ala
Leu Leu Ile Asp Cys Arg 180 185 190Ser Leu Gly Thr Lys Ala Val Ser
Met Pro Lys Gly Val Ala Val Val 195 200 205Ile Ile Asn Ser Asn Phe
Lys Arg Thr Leu Val Gly Ser Glu Tyr Asn 210 215 220Thr Arg Arg Glu
Gln Cys Glu Thr Gly Ala Arg Phe Phe Gln Gln Pro225 230 235 240Ala
Leu Arg Asp Val Thr Ile Glu Glu Phe Asn Ala Val Ala His Glu 245 250
255Leu Asp Pro Ile Val Ala Lys Arg Val Arg His Ile Leu Thr Glu Asn
260 265 270Ala Arg Thr Val Glu Ala Ala Ser Ala Leu Glu Gln Gly Asp
Leu Lys 275 280 285Arg Met Gly Glu Leu Met Ala Glu Ser His Ala Ser
Met Arg Asp Asp 290 295 300Phe Glu Ile Thr Val Pro Gln Ile Asp Thr
Leu Val Glu Ile Val Lys305 310 315 320Ala Val Ile Gly Asp Lys Gly
Gly Val Arg Met Thr Gly Gly Gly Phe 325 330 335Gly Gly Cys Ile Val
Ala Leu Ile Pro Glu Glu Leu Val Pro Ala Val 340 345 350Gln Gln Ala
Val Ala Glu Gln Tyr Glu Ala Lys Thr Gly Ile Lys Glu 355 360 365Thr
Phe Tyr Val Cys Lys Pro Ser Gln Gly Ala Gly Gln Cys 370 375
3806399PRTLactococcus lactisMISC_FEATURE(233)..(233)Xaa can be any
naturally occurring amino acid. 6Met Ser Ile Val Val Glu Asn Ser
Thr Val Leu Ser Ala Leu Thr Glu1 5 10 15Lys Phe Ala Glu Val Phe Gly
Asp Thr Lys Glu Val Glu Tyr Phe Phe 20 25 30Ser Pro Gly Arg Ile Asn
Leu Ile Gly Glu His Thr Asp Tyr Asn Gly 35 40 45Gly Tyr Val Phe Pro
Ala Ser Ile Ile Ile Gly Thr Thr Gly Leu Ala 50 55 60Arg Leu Arg Glu
Asp Lys Lys Val Lys Leu Tyr Ser Glu Asn Phe Pro65 70 75 80Lys Leu
Gly Val Ile Glu Phe Asp Leu Asp Glu Val Glu Lys Lys Asp 85 90 95Gly
Glu Leu Trp Ser Asn Tyr Val Lys Gly Met Ile Val Met Leu Lys 100 105
110Gly Ala Gly Tyr Glu Ile Asp Lys Gly Phe Glu Leu Leu Ile Lys Gly
115 120 125Glu Ile Pro Thr Ala Ser Gly Leu Ser Ser Ser Ala Ser Leu
Glu Leu 130 135 140Leu Val Gly Val Val Leu Asp Asp Leu Phe Asn Asp
Asn Val Pro Arg145 150 155 160Leu Glu Leu Val Gln Leu Gly Gln Lys
Thr Glu Asn Asp Tyr Ile Gly 165 170 175Val Asn Ser Gly Ile Leu Asp
Gln Phe Ala Ile Gly Phe Gly Glu Val 180 185 190Lys Lys Ala Ile Leu
Leu Asp Cys Asn Thr Leu Lys Tyr Glu Met Val 195 200 205Pro Val Glu
Leu Arg Asp Tyr Asp Ile Val Ile Met Asn Thr Asn Lys 210 215 220Pro
Arg Ala Leu Thr Glu Ser Lys Xaa Asn Glu Arg Phe Ala Glu Thr225 230
235 240Arg Glu Ala Leu Lys Arg Met Gln Thr Arg Leu Asp Ile Gln Ser
Leu 245 250 255Gly Glu Leu Ser Asn Glu Glu Phe Asp Ala Asn Thr Asp
Leu Ile Gly 260 265 270Asp Glu Thr Leu Ile Lys Arg Ala Arg His Ala
Val Tyr Glu Asn Asn 275 280 285Arg Thr Lys Ile Ala Gln Lys Ala Phe
Val Ala Gly Asn Leu Thr Lys 290 295 300Phe Gly Glu Leu Leu Asn Ala
Ser His Ala Ser Leu Lys Asp Asp Tyr305 310 315 320Glu Val Thr Gly
Leu Glu Leu Asp Thr Leu Ala Glu Thr Ala Gln Lys 325 330 335Gln Ala
Gly Val Leu Gly Ala Arg Met Thr Gly Ala Gly Phe Gly Gly 340 345
350Cys Ala Ile Ala Leu Val Ala His Asp Asn Val Ser Ala Phe Glu Lys
355 360 365Ala Val Gly Gln Val Tyr Glu Glu Val Val Gly Tyr Pro Ala
Ser Phe 370 375 380Tyr Val Ala Gln Ile Gly Ser Gly Ser Thr Lys Leu
Asp Val Glu385 390 3957382PRTEscherichia
coliMISC_FEATURE(339)..(339)Xaa can be any naturally occurring
amino acid. 7Met Ser Leu Lys Glu Lys Thr Gln Ser Leu Phe Ala Asn
Ala Phe Gly1 5 10 15Tyr Pro Ala Thr His Thr Ile Gln Ala Pro Gly Arg
Val Asn Leu Ile 20 25 30Gly Glu His Thr Asp Tyr Asn Asp Gly Phe Val
Leu Pro Cys Ala Ile 35 40 45Asp Tyr Gln Thr Val Ile Ser Cys Ala Pro
Arg Asp Asp Arg Lys Val 50 55 60Arg Val Met Ala Ala Asp Tyr Glu Asn
Gln Leu Asp Glu Phe Ser Leu65 70 75 80Asp Ala Pro Ile Val Ala His
Glu Asn Tyr Gln Arg Ala Asn Tyr Val
85 90 95Arg Gly Val Val Lys His Leu Gln Leu Arg Asn Asn Ser Phe Gly
Gly 100 105 110Val Asp Met Val Ile Ser Gly Asn Val Pro Gln Gly Ala
Gly Leu Ser 115 120 125Ser Ser Ala Ser Leu Glu Val Ala Val Gly Thr
Val Leu Gln Gln Leu 130 135 140Tyr Gly Leu Pro Leu Asp Gly Ala Gln
Ile Ala Leu Asn Gly Gln Glu145 150 155 160Ala Glu Asn Gln Phe Val
Gly Cys Asn Cys Gly Ile Met Asp Gln Leu 165 170 175Ile Ser Ala Leu
Gly Lys Lys Asp His Ala Leu Leu Ile Asp Cys Arg 180 185 190Ser Leu
Gly Thr Lys Ala Val Ser Met Pro Lys Gly Val Ala Val Val 195 200
205Ile Ile Asn Ser Asn Phe Lys Arg Thr Leu Val Gly Ser Glu Tyr Asn
210 215 220Thr Arg Arg Glu Gln Cys Glu Thr Gly Ala Arg Phe Phe Gln
Gln Pro225 230 235 240Ala Leu Arg Asp Val Thr Ile Glu Glu Phe Asn
Ala Val Ala His Glu 245 250 255Leu Asp Pro Ile Val Ala Lys Arg Val
Arg His Ile Leu Thr Glu Asn 260 265 270Ala Arg Thr Val Glu Ala Ala
Ser Ala Leu Glu Gln Gly Asp Leu Lys 275 280 285Arg Met Gly Glu Leu
Met Ala Glu Ser His Ala Ser Met Arg Asp Asp 290 295 300Phe Glu Ile
Thr Val Pro Gln Ile Asp Thr Leu Val Glu Ile Val Lys305 310 315
320Ala Val Ile Gly Asp Lys Gly Gly Val Arg Met Thr Gly Gly Gly Phe
325 330 335Gly Gly Xaa Ile Val Ala Leu Ile Pro Glu Glu Leu Val Pro
Ala Val 340 345 350Gln Gln Ala Val Ala Glu Gln Tyr Glu Ala Lys Thr
Gly Ile Lys Glu 355 360 365Thr Phe His Val Cys Lys Pro Ser Gln Gly
Ala Gly Gln Cys 370 375 3808382PRTEscherichia coli 8Met Ser Leu Lys
Glu Lys Thr Gln Ser Leu Phe Ala Asn Ala Phe Gly1 5 10 15Tyr Pro Ala
Thr His Thr Ile Gln Ala Pro Gly Arg Val Asn Leu Ile 20 25 30Gly Glu
His Thr Asp Tyr Asn Asp Gly Phe Val Leu Pro Cys Ala Ile 35 40 45Asp
Tyr Gln Thr Val Ile Ser Cys Ala Pro Arg Asp Asp Arg Lys Val 50 55
60Arg Val Met Ala Ala Asp Tyr Glu Asn Gln Leu Asp Glu Phe Ser Leu65
70 75 80Asp Ala Pro Ile Val Ala His Glu Asn Tyr Gln Arg Ala Asn Tyr
Val 85 90 95Arg Gly Val Val Lys His Leu Gln Leu Arg Asn Asn Ser Phe
Gly Gly 100 105 110Val Asp Met Val Ile Ser Gly Asn Val Pro Gln Gly
Ala Gly Leu Ser 115 120 125Ser Ser Ala Ser Leu Glu Val Ala Val Gly
Thr Val Leu Gln Gln Leu 130 135 140Tyr Gly Leu Pro Leu Asp Gly Ala
Gln Ile Ala Leu Asn Gly Gln Glu145 150 155 160Ala Glu Asn Gln Phe
Val Gly Cys Asn Cys Gly Ile Met Asp Gln Leu 165 170 175Ile Ser Ala
Leu Gly Lys Lys Asp His Ala Leu Leu Ile Asp Cys Arg 180 185 190Ser
Leu Gly Thr Lys Ala Val Ser Met Pro Lys Gly Val Ala Val Val 195 200
205Ile Ile Asn Ser Asn Phe Lys Arg Thr Leu Val Gly Ser Glu Tyr Asn
210 215 220Thr Arg Arg Glu Gln Cys Glu Thr Gly Ala Arg Phe Phe Gln
Gln Pro225 230 235 240Ala Leu Arg Asp Val Thr Ile Glu Glu Phe Asn
Ala Val Ala His Glu 245 250 255Leu Asp Pro Ile Val Ala Lys Arg Val
Arg His Ile Leu Thr Glu Asn 260 265 270Ala Arg Thr Val Glu Ala Ala
Ser Ala Leu Glu Gln Gly Asp Leu Lys 275 280 285Arg Met Gly Glu Leu
Met Ala Glu Ser His Ala Ser Met Arg Asp Asp 290 295 300Phe Glu Ile
Thr Val Pro Gln Ile Asp Thr Leu Val Glu Ile Val Lys305 310 315
320Ala Val Ile Gly Asp Lys Gly Gly Val Arg Met Thr Gly Gly Gly Phe
325 330 335Gly Gly Cys Ile Val Ala Leu Ile Pro Glu Glu Leu Val Pro
Ala Val 340 345 350Gln Gln Ala Val Ala Glu Gln Tyr Glu Ala Lys Thr
Gly Ile Lys Glu 355 360 365Thr Phe His Val Cys Lys Pro Ser Gln Gly
Ala Gly Gln Cys 370 375 3809382PRTEscherichia
coliMISC_FEATURE(371)..(371)Xaa can be any naturally occurring
amino acid. 9Met Ser Leu Lys Glu Lys Thr Gln Ser Leu Phe Ala Asn
Ala Phe Gly1 5 10 15Tyr Pro Ala Thr His Thr Ile Gln Ala Pro Gly Arg
Val Asn Leu Ile 20 25 30Gly Glu His Thr Asp Tyr Asn Asp Gly Phe Val
Leu Pro Cys Ala Ile 35 40 45Asp Tyr Gln Thr Val Ile Ser Cys Ala Pro
Arg Asp Asp Arg Lys Val 50 55 60Arg Val Met Ala Ala Asp Tyr Glu Asn
Gln Leu Asp Glu Phe Ser Leu65 70 75 80Asp Ala Pro Ile Val Ala His
Glu Asn Tyr Gln Arg Ala Asn Tyr Val 85 90 95Arg Gly Val Val Lys His
Leu Gln Leu Arg Asn Asn Ser Phe Gly Gly 100 105 110Val Asp Met Val
Ile Ser Gly Asn Val Pro Gln Gly Ala Gly Leu Ser 115 120 125Ser Ser
Ala Ser Leu Glu Val Ala Val Gly Thr Val Leu Gln Gln Leu 130 135
140Tyr Gly Leu Pro Leu Asp Gly Ala Gln Ile Ala Leu Asn Gly Gln
Glu145 150 155 160Ala Glu Asn Gln Phe Val Gly Cys Asn Cys Gly Ile
Met Asp Gln Leu 165 170 175Ile Ser Ala Leu Gly Lys Lys Asp His Ala
Leu Leu Ile Asp Cys Arg 180 185 190Ser Leu Gly Thr Lys Ala Val Ser
Met Pro Lys Gly Val Ala Val Val 195 200 205Ile Ile Asn Ser Asn Phe
Lys Arg Thr Leu Val Gly Ser Glu Tyr Asn 210 215 220Thr Arg Arg Glu
Gln Cys Glu Thr Gly Ala Arg Phe Phe Gln Gln Pro225 230 235 240Ala
Leu Arg Asp Val Thr Ile Glu Glu Phe Asn Ala Val Ala His Glu 245 250
255Leu Asp Pro Ile Val Ala Lys Arg Val Arg His Ile Leu Thr Glu Asn
260 265 270Ala Arg Thr Val Glu Ala Ala Ser Ala Leu Glu Gln Gly Asp
Leu Lys 275 280 285Arg Met Gly Glu Leu Met Ala Glu Ser His Ala Ser
Met Arg Asp Asp 290 295 300Phe Glu Ile Thr Val Pro Gln Ile Asp Thr
Leu Val Glu Ile Val Lys305 310 315 320Ala Val Ile Gly Asp Lys Gly
Gly Val Arg Met Thr Gly Gly Gly Phe 325 330 335Gly Gly Cys Ile Val
Ala Leu Ile Pro Glu Glu Leu Val Pro Ala Val 340 345 350Gln Gln Ala
Val Ala Glu Gln Tyr Glu Ala Lys Thr Gly Ile Lys Glu 355 360 365Thr
Phe Xaa Val Cys Lys Pro Ser Gln Gly Ala Gly Gln Cys 370 375
38010382PRTEscherichia coli 10Met Ser Leu Lys Glu Lys Thr Gln Ser
Leu Phe Ala Asn Ala Phe Gly1 5 10 15Tyr Pro Ala Thr His Thr Ile Gln
Ala Pro Gly Arg Val Asn Leu Ile 20 25 30Gly Glu His Thr Asp Tyr Asn
Asp Gly Phe Val Leu Pro Cys Ala Ile 35 40 45Asp Tyr Gln Thr Val Ile
Ser Cys Ala Pro Arg Asp Asp Arg Lys Val 50 55 60Arg Val Met Ala Ala
Asp Tyr Glu Asn Gln Leu Asp Glu Phe Ser Leu65 70 75 80Asp Ala Pro
Ile Val Ala His Glu Asn Tyr Gln Arg Ala Asn Tyr Val 85 90 95Arg Gly
Val Val Lys His Leu Gln Leu Arg Asn Asn Ser Phe Gly Gly 100 105
110Val Asp Met Val Ile Ser Gly Asn Val Pro Gln Gly Ala Gly Leu Ser
115 120 125Ser Ser Ala Ser Leu Glu Val Ala Val Gly Thr Val Leu Gln
Gln Leu 130 135 140Tyr Gly Leu Pro Leu Asp Gly Ala Gln Ile Ala Leu
Asn Gly Gln Glu145 150 155 160Ala Glu Asn Gln Phe Val Gly Cys Asn
Cys Gly Ile Leu Asp Gln Leu 165 170 175Ile Ser Ala Leu Gly Lys Lys
Asp His Ala Leu Leu Ile Asp Cys Arg 180 185 190Ser Leu Gly Thr Lys
Ala Val Ser Met Pro Lys Gly Val Ala Val Val 195 200 205Ile Ile Asn
Ser Asn Phe Lys Arg Thr Leu Val Gly Ser Glu Tyr Asn 210 215 220Thr
Arg Arg Glu Gln Cys Glu Thr Gly Ala Arg Phe Phe Gln Gln Pro225 230
235 240Ala Leu Arg Asp Val Thr Ile Glu Glu Phe Asn Ala Val Ala His
Glu 245 250 255Leu Asp Pro Ile Val Ala Lys Arg Val Arg His Ile Leu
Thr Glu Asn 260 265 270Ala Arg Thr Val Glu Ala Ala Ser Ala Leu Glu
Gln Gly Asp Leu Lys 275 280 285Arg Met Gly Glu Leu Met Ala Glu Ser
His Ala Ser Met Arg Asp Asp 290 295 300Phe Glu Ile Thr Val Pro Gln
Ile Asp Thr Leu Val Glu Ile Val Lys305 310 315 320Ala Val Ile Gly
Asp Lys Gly Gly Val Arg Met Thr Gly Gly Gly Phe 325 330 335Gly Gly
Cys Ile Val Ala Leu Ile Pro Glu Glu Leu Val Pro Ala Val 340 345
350Gln Gln Ala Val Ala Glu Gln Tyr Glu Ala Lys Thr Gly Ile Lys Glu
355 360 365Thr Phe Tyr Val Cys Lys Pro Ser Gln Gly Ala Gly Gln Cys
370 375 38011382PRTEscherichia coliMISC_FEATURE(171)..(171)Xaa can
be any naturally occurring amino acid. 11Met Ser Leu Lys Glu Lys
Thr Gln Ser Leu Phe Ala Asn Ala Phe Gly1 5 10 15Tyr Pro Ala Thr His
Thr Ile Gln Ala Pro Gly Arg Val Asn Leu Ile 20 25 30Gly Glu His Thr
Asp Tyr Asn Asp Gly Phe Val Leu Pro Cys Ala Ile 35 40 45Asp Tyr Gln
Thr Val Ile Ser Cys Ala Pro Arg Asp Asp Arg Lys Val 50 55 60Arg Val
Met Ala Ala Asp Tyr Glu Asn Gln Leu Asp Glu Phe Ser Leu65 70 75
80Asp Ala Pro Ile Val Ala His Glu Asn Tyr Gln Arg Ala Asn Tyr Val
85 90 95Arg Gly Val Val Lys His Leu Gln Leu Arg Asn Asn Ser Phe Gly
Gly 100 105 110Val Asp Met Val Ile Ser Gly Asn Val Pro Gln Gly Ala
Gly Leu Ser 115 120 125Ser Ser Ala Ser Leu Glu Val Ala Val Gly Thr
Val Leu Gln Gln Leu 130 135 140Tyr Gly Leu Pro Leu Asp Gly Ala Gln
Ile Ala Leu Asn Gly Gln Glu145 150 155 160Ala Glu Asn Gln Phe Val
Gly Cys Asn Cys Gly Ile Xaa Asp Gln Leu 165 170 175Ile Ser Ala Leu
Gly Lys Lys Asp His Ala Leu Leu Ile Asp Cys Arg 180 185 190Ser Leu
Gly Thr Lys Ala Val Ser Met Pro Lys Gly Val Ala Val Val 195 200
205Ile Ile Asn Ser Asn Phe Lys Arg Thr Leu Val Gly Ser Glu Tyr Asn
210 215 220Thr Arg Arg Glu Gln Cys Glu Thr Gly Ala Arg Phe Phe Gln
Gln Pro225 230 235 240Ala Leu Arg Asp Val Thr Ile Glu Glu Phe Asn
Ala Val Ala His Glu 245 250 255Leu Asp Pro Ile Val Ala Lys Arg Val
Arg His Ile Leu Thr Glu Asn 260 265 270Ala Arg Thr Val Glu Ala Ala
Ser Ala Leu Glu Gln Gly Asp Leu Lys 275 280 285Arg Met Gly Glu Leu
Met Ala Glu Ser His Ala Ser Met Arg Asp Asp 290 295 300Phe Glu Ile
Thr Val Pro Gln Ile Asp Thr Leu Val Glu Ile Val Lys305 310 315
320Ala Val Ile Gly Asp Lys Gly Gly Val Arg Met Thr Gly Gly Gly Phe
325 330 335Gly Gly Cys Ile Val Ala Leu Ile Pro Glu Glu Leu Val Pro
Ala Val 340 345 350Gln Gln Ala Val Ala Glu Gln Tyr Glu Ala Lys Thr
Gly Ile Lys Glu 355 360 365Thr Phe Tyr Val Cys Lys Pro Ser Gln Gly
Ala Gly Gln Cys 370 375 38012382PRTEscherichia coli 12Met Ser Leu
Lys Glu Lys Thr Gln Ser Leu Phe Ala Asn Ala Phe Gly1 5 10 15Tyr Pro
Ala Thr His Thr Ile Gln Ala Pro Gly Arg Val Asn Leu Ile 20 25 30Gly
Glu His Thr Asp Tyr Asn Asp Gly Phe Val Leu Pro Cys Ala Ile 35 40
45Asp Tyr Gln Thr Val Ile Ser Cys Ala Pro Arg Asp Asp Arg Lys Val
50 55 60Arg Val Met Ala Ala Asp Tyr Glu Asn Gln Leu Asp Glu Phe Ser
Leu65 70 75 80Asp Ala Pro Ile Val Ala His Glu Asn Tyr Gln Arg Ala
Asn Tyr Val 85 90 95Arg Gly Val Val Lys His Leu Gln Leu Arg Asn Asn
Ser Phe Gly Gly 100 105 110Val Asp Met Val Ile Ser Gly Asn Val Pro
Gln Gly Ala Gly Leu Ser 115 120 125Ser Ser Ala Ser Leu Glu Val Ala
Val Gly Thr Val Leu Gln Gln Leu 130 135 140Tyr Gly Leu Pro Leu Asp
Gly Ala Gln Ile Ala Leu Asn Gly Gln Glu145 150 155 160Ala Glu Asn
Gln Phe Val Gly Cys Asn Cys Gly Ile Leu Asp Gln Leu 165 170 175Ile
Ser Ala Leu Gly Lys Lys Asp His Ala Leu Leu Ile Asp Cys Arg 180 185
190Ser Leu Gly Thr Lys Ala Val Ser Met Pro Lys Gly Val Ala Val Val
195 200 205Ile Ile Asn Ser Asn Phe Lys Arg Thr Leu Val Gly Ser Glu
Tyr Asn 210 215 220Thr Arg Arg Glu Gln Cys Glu Thr Gly Ala Arg Phe
Phe Gln Gln Pro225 230 235 240Ala Leu Arg Asp Val Thr Ile Glu Glu
Phe Asn Ala Val Ala His Glu 245 250 255Leu Asp Pro Ile Val Ala Lys
Arg Val Arg His Ile Leu Thr Glu Asn 260 265 270Ala Arg Thr Val Glu
Ala Ala Ser Ala Leu Glu Gln Gly Asp Leu Lys 275 280 285Arg Met Gly
Glu Leu Met Ala Glu Ser His Ala Ser Met Arg Asp Asp 290 295 300Phe
Glu Ile Thr Val Pro Gln Ile Asp Thr Leu Val Glu Ile Val Lys305 310
315 320Ala Val Ile Gly Asp Lys Gly Gly Val Arg Met Thr Gly Gly Gly
Phe 325 330 335Gly Gly Cys Ile Val Ala Leu Ile Pro Glu Glu Leu Val
Pro Ala Val 340 345 350Gln Gln Ala Val Ala Glu Gln Tyr Glu Ala Lys
Thr Gly Ile Lys Glu 355 360 365Thr Phe His Val Cys Lys Pro Ser Gln
Gly Ala Gly Gln Cys 370 375 38013399PRTLactococcus
lactisMISC_FEATURE(353)..(353)Xaa can be any naturally occurring
amino acid. 13Met Ser Ile Val Val Glu Asn Ser Thr Val Leu Ser Ala
Leu Thr Glu1 5 10 15Lys Phe Ala Glu Val Phe Gly Asp Thr Lys Glu Val
Glu Tyr Phe Phe 20 25 30Ser Pro Gly Arg Ile Asn Leu Ile Gly Glu His
Thr Asp Tyr Asn Gly 35 40 45Gly Tyr Val Phe Pro Ala Ser Ile Ile Ile
Gly Thr Thr Gly Leu Ala 50 55 60Arg Leu Arg Glu Asp Lys Lys Val Lys
Leu Tyr Ser Glu Asn Phe Pro65 70 75 80Lys Leu Gly Val Ile Glu Phe
Asp Leu Asp Glu Val Glu Lys Lys Asp 85 90 95Gly Glu Leu Trp Ser Asn
Tyr Val Lys Gly Met Ile Val Met Leu Lys 100 105 110Gly Ala Gly Tyr
Glu Ile Asp Lys Gly Phe Glu Leu Leu Ile Lys Gly 115 120 125Glu Ile
Pro Thr Ala Ser Gly Leu Ser Ser Ser Ala Ser Leu Glu Leu 130 135
140Leu Val Gly Val Val Leu Asp Asp Leu Phe Asn Leu Asn Val Pro
Arg145 150 155 160Leu Glu Leu Val Gln Leu Gly Gln Lys Thr Glu Asn
Asp Tyr Ile Gly 165 170 175Val Asn Ser Gly Ile Leu Asp Gln Phe Ala
Ile Gly Phe Gly Glu Val 180 185 190Lys Lys Ala Ile Leu Leu Asp Cys
Asn Thr Leu Lys Tyr Glu Met Val 195 200 205Pro Val Glu Leu Arg Asp
Tyr Asp Ile Val Ile Met Asn Thr Asn Lys 210 215
220Pro Arg Ala Leu Thr Glu Ser Lys Tyr Asn Glu Arg Phe Ala Glu
Thr225 230 235 240Arg Glu Ala Leu Lys Arg Met Gln Thr Arg Leu Asp
Ile Gln Ser Leu 245 250 255Gly Glu Leu Ser Asn Glu Glu Phe Asp Ala
Asn Thr Asp Leu Ile Gly 260 265 270Asp Glu Thr Leu Ile Lys Arg Ala
Arg His Ala Val Tyr Glu Asn Asn 275 280 285Arg Thr Lys Ile Ala Gln
Lys Ala Phe Val Ala Gly Asn Leu Thr Lys 290 295 300Phe Gly Glu Leu
Leu Asn Ala Ser His Ala Ser Leu Lys Asp Asp Tyr305 310 315 320Glu
Val Thr Gly Leu Glu Leu Asp Thr Leu Ala Glu Thr Ala Gln Lys 325 330
335Gln Ala Gly Val Leu Gly Ala Arg Met Thr Gly Ala Gly Phe Gly Gly
340 345 350Xaa Ala Ile Ala Leu Val Ala His Asp Asn Val Ser Ala Phe
Glu Lys 355 360 365Ala Val Gly Gln Val Tyr Glu Glu Val Val Gly Tyr
Pro Ala Ser Phe 370 375 380Tyr Val Ala Gln Ile Gly Ser Gly Ser Thr
Lys Leu Asp Val Glu385 390 39514399PRTLactococcus lactis 14Met Ser
Ile Val Val Glu Asn Ser Thr Val Leu Ser Ala Leu Thr Glu1 5 10 15Lys
Phe Ala Glu Val Phe Gly Asp Thr Lys Glu Val Glu Tyr Phe Phe 20 25
30Ser Pro Gly Arg Ile Asn Leu Ile Gly Glu His Thr Asp Tyr Asn Gly
35 40 45Gly Tyr Val Phe Pro Ala Ser Ile Ile Ile Gly Thr Thr Gly Leu
Ala 50 55 60Arg Leu Arg Glu Asp Lys Lys Val Lys Leu Tyr Ser Glu Asn
Phe Pro65 70 75 80Lys Leu Gly Val Ile Glu Phe Asp Leu Asp Glu Val
Glu Lys Lys Asp 85 90 95Gly Glu Leu Trp Ser Asn Tyr Val Lys Gly Met
Ile Val Met Leu Lys 100 105 110Gly Ala Gly Tyr Glu Ile Asp Lys Gly
Phe Glu Leu Leu Ile Lys Gly 115 120 125Glu Ile Pro Thr Ala Ser Gly
Leu Ser Ser Ser Ala Ser Leu Glu Leu 130 135 140Leu Val Gly Val Val
Leu Asp Asp Leu Phe Asn Leu Asn Val Pro Arg145 150 155 160Leu Glu
Leu Val Gln Leu Gly Gln Lys Thr Glu Asn Asp Tyr Ile Gly 165 170
175Val Asn Ser Gly Ile Leu Asp Gln Phe Ala Ile Gly Phe Gly Glu Val
180 185 190Lys Lys Ala Ile Leu Leu Asp Cys Asn Thr Leu Lys Tyr Glu
Met Val 195 200 205Pro Val Glu Leu Arg Asp Tyr Asp Ile Val Ile Met
Asn Thr Asn Lys 210 215 220Pro Arg Ala Leu Thr Glu Ser Lys Tyr Asn
Glu Arg Phe Ala Glu Thr225 230 235 240Arg Glu Ala Leu Lys Arg Met
Gln Thr Arg Leu Asp Ile Gln Ser Leu 245 250 255Gly Glu Leu Ser Asn
Glu Glu Phe Asp Ala Ser Thr Asp Leu Ile Gly 260 265 270Asp Glu Thr
Leu Ile Lys Arg Ala Arg His Ala Val Tyr Glu Asn Asn 275 280 285Arg
Thr Lys Ile Ala Gln Lys Ala Phe Val Ala Gly Asn Leu Thr Lys 290 295
300Phe Gly Glu Leu Leu Asn Ala Ser His Ala Ser Leu Lys Asp Asp
Tyr305 310 315 320Glu Val Thr Gly Leu Glu Leu Asp Thr Leu Ala Glu
Thr Ala Gln Lys 325 330 335Gln Ala Gly Val Leu Gly Ala Arg Met Thr
Gly Ala Gly Phe Gly Gly 340 345 350Cys Tyr Ile Ala Leu Val Ala His
Asp Asn Val Ser Ala Phe Glu Lys 355 360 365Ala Val Gly Gln Val Tyr
Glu Glu Val Val Gly Tyr Pro Ala Ser Phe 370 375 380Tyr Val Ala Gln
Ile Gly Ser Gly Ser Thr Lys Leu Asp Val Glu385 390
3951520DNAARTIFICIALSYNTHETIC NUCLEOTIDE-PCR PRIMER 15cttggttatg
cgggtactgc 201619DNAARTIFICIALSYNTHETIC NUCLEOTIDE-PCR PRIMER
16tcccgcgaaa ttmtacgac 191732DNAARTIFICIALSYNTHETIC NUCLEOTIDE-PCR
PRIMER 17gtmctgcggg atcctggatc agctaatttc cg
321832DNAARTIFICIALSYNTHETIC NUCLEOTIDE-PCR PRIMER 18cggmattagc
tgatccagga tcccgcagtt ac 32
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