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.

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.