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. (end of abstract)

Sugar kinases with expanded substrate specificity and their use description/claims

Brief Patent Description

Full Patent Description

Patent Application Claims

CROSS-REFERENCE TO RELATED APPLICATIONS

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.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

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.

BACKGROUND OF THE INVENTION

This invention generally relates to sugar kinases and specifically to novel anomeric D/L sugar kinases with expanded substrate specificity and methods of use.

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).

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

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.

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.

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.

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.

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.

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.

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.

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