[Para 3] Statement regarding Federally-sponsored Research and Development [Para 4] The present invention was supported in part by contribution from the National Institutes of Health grant AI52218. The government of the United States of America may have certain rights in this invention.

[Para 5] Background of Invention [Para 6] This invention generally relates to sugar kinases and specifically to novel anomeric D/L sugar kinases with expanded substrate specificity and methods of use.

[Para 7] 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. 1 a) including enediynes (calicheamicin,/), polyketides (doxorubicin, 2 ; erythromycin,. 3), indolocarbazoles (staurosporine, 4, non-ribosomal peptides (vancomycin, 5), polyenes (nystatin, 6), coumarins (novobiocin,/), or cardiac glycosides (digitoxin, $. 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. 1 b).

[Para 8] 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.

[Para 9] Summary of invention [Para 10] The present invention provides sugar kinases with expanded subtrate 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 R28, E34, D37, D1 74, Y233, C339, Y371, Y371 H, M173, M1 73L, Y371 H-M1 73L and C353. 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.

[Para 11] 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 Gall< 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.

[Para 12] 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.

[Para 1 3] 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, G1 75, D111, E162, T201, 1200, E199, R195, L89, L89T, L109, Y146 and Y177. In this method also, the NTP is ATP. Also in this method the GalK variant is Y371 H, M1 73L or Y371 H-M1 73L. 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, coliand S. lividans.

[Para 14] Another aspect of the invention provides a method of producing a glycosylated biomolecule containing at least one sugar moeity. 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, D1 11, E162, T201, 1200, E199, R195, L89, L89T, L109, Y146 and Y177. Further the glycosyltransferase is selected from the group consisting of CalB, CalE, CaIN, CalU, Gra orfl4, Gra orf5, LanGT1, LanGT2, LanGT3, LanGT4, MtmGl, MtmGll, MtmGTIII, MtmGTIV, NovM, RhIB, Rif orf 7, SnogD, SnogE, SnogZ, UrdGTI a, UrdGTI b, UrdGTI c, UrdGT2, AknK, AknS, DesVll, DnrS, OleGI, OleG2, TyICV, TyIMII, TyIN, DauH, DnrH, EryBV, EryCIII, Ngt, BgtA, BgtB, BgtC, GftA, GftB, GftC, GftD, GftE, Gpl-1, Gpl-2, RtfA, AveBI, BImE, BImF, MgtA, NysD1, OleD, Olel, SpcF, SpcG, StrH, Ugt51 B1, Ugt51C1, UGT52, UgtA, UgtB, UgtC, UgtD and homologs thereof. Also in this method, the NTP is ATP. Preferably, the GalK variant is Y371 H, M1 73L or Y371 H-M1 73L. 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. coliand 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.

[Para 15] 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.

(Para 16] Brief Description of Drawings [Para 17] 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. Ep denotes oc-D-glucopyranosyl phosphate thymidylyltransferase, GlyTn different glycosyltransferases.

[Para 18] 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 Y371 H.

[Para 19] 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.

[Para 20] FIG. 4. Provides 3JH-H coupling patterns and NOESY correlations for the Gall ( Y371 H product-L-altrose-1-phosphate.

[Para 211 FIG. 5. Glycorandomization overview and two potential scenarios for an in vivo approach. (a) In vitro glycorandomization utilizes two enhanced enzymes-E1 (a general kinase) and E2 (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 l-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 11 - feeding monosaccharides and aglycons to a non-producing host engineered to express the 'NDP-sugar factory'and an appropriate glycosyltransferase or glycosyltransferase library.

[Para 22] FIG. 6. Comparison of D-glucose docked within the active site of L. lactis GalK and homology-model of E. coliGalK. (a) Wild type L. lactis GalK bound to D-Glc. (b) L182M (the homology model for E. coliGalK active site) with D-Glc.

[Para 23] FIG. 7.'Natural'and'unnatural'substrates of wild-type GalK and GalK mutants (M1 73L, Y371 H and M1 73L-Y371 H).

[Para 24] FIG. 8 Percent conversion of sugar substrates by wild-type and mutant GalKs.

For each enzymatic reaction: [sugar] = 8 mM, [ATP] = 14 mM, [MgC121 = 3.5 mM, [enzyme] = 15.0µM and reaction time = 180 min.

[Para 25] FIG. 9. In vivo GaIK-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 M1 73L-Y371 H 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 Y371 H-M1 73L GalK ; ii) 46 with Y371 H-M1 73L GaIK ; iii) 22 with wild-type GalK. MS: 50, calculated for C2oH25N303S 387. 2, found m/z 386.1 [M-H]- ; 51, calculated for C26H37N6O11PS 672.2, found m/z 671.2 [M-H]- ; 52, calculated for C26H36N608S 592.2, found m/z 591.2 [M-H] - [Para 26] Detailed Description [Para 27] 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 (5 into D-glucose-1-phosphate (10), fucokinases which transfer a phosphate from ATP to the anomeric position of L-fucose (72) to provide P-L- fucose-1-phosphate (13), and the galactokinases (GalK), which catalyze the formation of ot-D- galactose-1-phosphate (Gal-1-P, i5) from D-galactose (i4) 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.

[Para 28] 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. 1 b,"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 W002079150 and W00248331, which are incorporated herein by reference in their entirety for all purposes.

[Para 29] 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.

[Para 30] 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 colorimetric screen prompted an initial evolutionary approach. As a model system, the inventors selected the well-characterized Escherichia (E) co/igalactokinase 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.

[Para 31] Herein the inventors describe the application of directed evolution and a high throughput multi-sugar colorimetric screen to enhance the catalytic capabilities of the E. coli GalK. From this approach, one particular Gall< mutant carrying a single amino acid exchange (Y371 H) 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 Gall< 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.

[Para 32] Generally the present invention provides sugar kinases with expanded subtrate 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 R28, E34, D37, D1 74, Y233, C339, Y371, Y371 H, M1 73, M1 73L, Y371 H-M1 73L and C353 mutation. The Gall< 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.

[Para 33] 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.

[Para 34] 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 Gall< 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.

[Para 35] 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 nucleotidylytransferase, 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, N1 12, G175, D1 1 1, E162, T201, 1200, E199, R195, L89, L89T, L109, Y146 and Y177. In this method also, the NTP is ATP. Also in this method the Gall< variant is Y371 H, Ml 73L or Y371 H-M1 73L. 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. coliand S. lividans. lPara 36] Another aspect of the invention provides a method of producing a glycosylated biomolecule containing at least one sugar moeity. 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, D11 1, E162, T201, 1200, E1 99, R1 95, L89, L89T, L109, Y146 and Y1 77. Further the glycosyltransferase is selected from the group consisting of CalB, CalE, CaIN, CalU, Gra orfl4, Gra orf5, LanGT1, LanGT2, LanGT3, LanGT4, MtmGl, MtmGll, MtmGTIII, MtmGTIV, NovM, RhlB, Rif orf 7, SnogD, SnogE, SnogZ, UrdGTI a, UrdGTI b, UrdGTI c, UrdGT2, AknK, AknS, Devil, DnrS, Ole1, OleG2, TyICV, TyIMII, TyIN, DauH, DnrH, EryBV, EryCIII, Ngt, BgtA, BgtB, BgtC, GftA, GftB, GftC, GftD, GftE, Gpl-1, Gpl-2, RtfA, AveBI, BImE, BImF, MgtA, NysD1, OleD, Olel, SpcF, SpcG, StrH, Ugt51 B1, Ugt51 C1, UGT52, UgtA, UgtB, UgtC, UgtD and homologs thereof. Also in this method, the NTP is ATP. Preferably, the GalK variant is Y371 H, M1 73L or Y371 H-M1 73L. 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. coliand 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.

[Para 37] 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.

[Para 38] Materials and Methods [Para 39] Materials. E. colistrains XL1-blue and BL21-Gold (DE3) were purchased from Stratagene (La Jolla, CA). The template plasmid pGalK has been previously described.

Expression vector pET1 5b was from Novagen (Madison, WI). All reagent grade chemicals and enzymes were purchased from Promega (Madison, WI), Sigma (St. Louis, MO), Fisher/Acros Organics (Hanover Park, IL), or Fluka (Milwaukee, WI).

[Para 40] 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 MgC12, 0.1 mM MnCI2, 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"and 5"-TCCCGCGAAATTAATACGAC-3", 5U Taq DNA- polymerase in the buffer supplied with the enzyme, in a total volume of 100 ul using the following thermocycle parameters: initial denaturation, 5 min, 94 °C ; amplification, 30 cycles, 94 °C for 0.5 min, 54 °C for 0.5 min, 72 °C for 1.5 min; terminal hold, 5 min at 72 °C. The amplification products were digested with BamHl/Xbal, purified on an agarose gel (0. 8% w/vol), eluted using the QlAquick extraction kit (QIAGEN, Valencia, CA), ligated into appropriately digested pETI 5b, to directly transform E. coliXLl blue. Plasmids were isolated from randomly picked colonies (-20 representatives for each library generated) and sequenced on an ABI 310 automatic DNA sequencer (PerkinElmer, Foster City, CA). Upon verifying the desired mutation rate, all transformants were pooled, cultured overnight, the collectively recovered plasmids used to transform E. co/iBL21-Gold (DE3) and the library processed as described below.

[Para 41] Bacterial fermentation. E. coliwas grown in LB medium, supplemented with ampicillin (100 uL mL-1 final) under standard conditions. For expression of Gall< 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 °C. The replicated cultures were grown to an OD600 # 0. 7, protein expression was then induced by adding 1 mM IPTG (final), for 2 hours, then harvested by centrifugation (10 min, 3000 x g). The cell paste was frozen at 20°C. Upon thawing the harvested expression cultures, the biomass of each well was resuspended in 50 uL NPI-buffer (50 mM NaH2PO4, 300 mM NaCI, pH 8.0) to which was then added 70 pL NPI-buffer supplemented with 1 uL Lysonase (Novagen, Madison, WI) for lysis. Cell debris was collected by centrifugation (1 0 min, 3000 x g), and 20 uL of the clear supernatant, containing-0. 5 ug of the expressed Gall< variant on average, was used for each kinase assay.

[Para 42] 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, CA) fitted to a Fluostar Optima plate reader (BMG, Durham, NC). The in vitro enzymatic reactions and assays followed the protocol published previously, slightly modified for automated liquid handling: 1 50 uL sugar solution (8 mM final) and 12, uL ATP/Mg2+ soiution (20 mM/5 mM final) were mixed, preincubated at 37 °C for 5 min, then 20 uL of the cleared supernatant was added, and the reaction incubated at 37 °C for an additional 2 h. To assay the phosphorylation reactions, 50 uL of the enzymatic reaction were taken at time zero and after the 2 h incubation, mixed with 100 uL of 3, 5-dinitrosalicylic acid (DNS) reagent, heated at 100 °C for 5 min, then immediately chilled on ice for 2 min. Assays were run as endpoint measurements following the decrease in absorption (X=575 nm, c=758 M-1 cm-1) [Para 43] Characterization of GalK Mutants. The Gall< mutant Y371 H was overexpressed and purified following the procedure previously described for wild-type Gall<. The fractions containing homogenous Y371 H Gall< were collected, concentrated and quantified using the Bradford protein assay. The DNS assay was used to assess the substrate specificity ofthe purified GalK mutant (Y371 H) 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, IL) as previously described.

[Para 44] Preparative phosphorylation of L-altrose and product characterization. L- altrose (21.6 mg, 0.12 mmol) was dissolved in 1 5 mL 50 mM sodium phosphate buffer (pH 7.5).

To this solution, ATP (1 25 mg, 0. 23 mmol), MgCI2. 6H20 (15. 3 mg, 0.07 mmol) were added, the mixture incubated at 37 oC for 5 min, the reaction initiated via the addition of enzyme (Y371 H) to a final concentration of 150, ug mL-1 and reaction progress monitoredby TLC. After completion, the mixture was diluted 5-fold in ddH20 and applied to a 200 mL anion exchange column (Q-Sepharose fast flow, Amersham, Piscataway, NJ). The column was eluted with a gradient of 0-400 mM NaCI 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, MA) to give 16.1 mg of purified product (yield: 52%). Mo= 3. 5° (c= 1, H20) H NMR (D20) : 5.48 (dd,. J = 8.6, 1.8 Hz, 1 H), 4.14 (dd, 5.3, 3.3 Hz, 1 H), 3.99 (m, 1 H), 3.98 (dd, J = 4.3, 1.8 Hz, 1 H), 3.91 (d, J= 3.3, 1H), 3.89 (m, 1H), 3.84 (dd, J= 12.1, 7.8 Hz, 1H) ; 13C NMR (D20): 94.10, 76.23, 70.70, 69.99, 65. 68, 62. 21 ; 31p NMR (D20) : 3.77 ; MS: calculated for C6H1309P 260.0, found m/z 259.0 [M-H] -.

[Para 45] Resu/ts and Discussion.

[Para 46] Directed evolution of GalK with expanded specificity. The cloned wild-type galK gene from E. coliwas 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 mutation bias (29,30, 44) and is therefore not a truly random process. Under the conditions selected, the library contained a transition/transversion ratio of # 3. 0, the transitions outnumbering the transversions, and the AT-GC/GC-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, 76, 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 (1). Table 1 describes the kinetic data for the ten active substrates of the GalK variant (Y371 H). wild type Y371H sugar substrate Km* kcat# kcat/Km# Km* kcat# kcat/Km# HoOH HO D-Gal (14) 2. 1 108 51. 4 5. 6 220 39. 3 HoOH 2-doxy-3. 6 30 8. 3 4. 7 200 42. 6 . 6H D-Gal (18) HO D-GAINH, (19) 2. 9 11. 7 4. 0 8. 8 260 29. 5 tOH HOU D-Tal (20) 2. 9 45. 5 15. 7 HoOH OH Ho cxi \\ 3-doxy-6. 4 5. 1 0. 8 10. 1 64. 6 6. 4 °"-aa D-Gal (21) Ho 6-dwxy-4. 9 2. 9 0. 59 8. 0 101 12. 6 o"on D-Gal (22) HO 6-mmo-21. 3 149 7. 0 °"cH D-Gal (23) . D-3. 2 58 18. 1 a Galacturonicac OH in 24 c _o L. tt (1 5. 2 80 15. 4 ai ai Hot r (c (253 ~§ ~§ 2. 7 65 24. 1 Ho api HO *mM <BR> <BR> t nan ~<BR> #mM-1 min-1 § no conversion [Para 47] 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~T transition at position 1111 of the wild-type reading frame, which translates into a tyrosine 371 to histidine replacement.

[Para 48] The structure of the Lactococcus (L.) lactis galactokinase was published.

Despite a rather low sequence homology to the E co/iGalK (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./actisgalactokinase (R36, E42, D45, D183, and Y233) are invariably present in its E. co/ihomolog (R28, E34, D37, D1 74, 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. colienzyme.

Surprisingly, residue 371 in the E. co/iwild-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./actisgalactokinase the Ca of the equivalent amino acid Y385 located within the C-terminal domain 0-strand K is-20 A 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. coliGaIK). The tyrosine phenolic oxygen is located-5. 5 A 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 EcoliGaIK) 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.

[Para 49] Characterization of the Gall variant (Y371 H). To determine the substrate specificity of the Y371 H Gall 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, 76, IS-25 (Table 1), strikingly expanding the overall substrate scope compared with wild-type E. coliGalK. The kinetic parameters of the mutant enzyme with all active substrates (74, 76, 78-25) 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 (kyat = 220 min-', Km = 5.6 mM) and displays remarkably enhanced kcat values for all the previously known substrates for wild-type GalK (i4, i8, i9, 2i, 2Z), the affinity for which is slightly reduced in all cases.

[Para 50] 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 Pseudomonasglutaryl 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 Y371 H 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 Y371 H 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 Y371 H 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 kcatin this case was also increased 2- fold.

[Para 51] Confirmation of L-sugar conversion. The substrate specificity studies have demonstrated GalK variant Y371 H 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 mol). 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-52%. The purified product was characterized by'H and 13C NMR from which H-H coupling and NOESY data confirmed the product to beß-L-altrose-1-phosphatein a I C4 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 Y371 H mutant must bind and phosphorylate L-altrose in the same 4C conformation as D-galactose (Fig. 2cand 2d) (38) the product of which subsequently rapidly equilibrates to the more stable'C4 conformation upon release from the enzyme.

[Para 52] Implications for in vitroglycorandomization [Para 53] 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. I i, 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 (Gall<) 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 W0020791 50 and W00248331, which are incorporated herein by reference in their entirety for all purposes.

[Para 54] In one embodiment of the present invention, promiscuous sugar-1-kinase (GaIK) 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.

[Para 5 5] 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, N11 2, G1 75, D111, E162, T201, 1200, El 99, R1 95, 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.

[Para 56] 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 Gall 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 orfl4, Gra orf5, LanGTI, LanGT2, LanGT3, LanGT4, MtmGl, MtmGll, MtmGTlll, MtmGTIV, NovM, RhlB, Rif orf 7, SnogD, SnogE, SnogZ, UrdGTla, UrdGTlb, UrdGTlc, UrdGT2, AknK, AknS, DesVll, DnrS, OleG1, OleG2, TyICV, TyIMII, TyIN, DauH, DnrH, EryBV, EryClll, Ngt, BgtA, BgtB, BgtC, GftA, GftB, GftC, GftD, GftE, Gpl-1, Gpl-2, RtfA, AveBI, BImE, BImF, MgtA, NysD1, OleD, Olel, SpcF, SpcG, StrH, Ugt51 B1, Ugt51 C1, 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. (NOTASSIGNED)"Glycorandomization and Production of Novel Vancomycin Analogs", filed on September 24,2003, which is incorporated in its entirety by reference for all purposes.

Table D-Gal (29) D-Gal (29) D-Glc (27) D-Glc (27) L-Alt (21) L-Alt (21) 2.

Km Vmax Km Vmax Km Vmax mM mM min1 Mm mM min 1 mM mM min WT 2. 1 (t 0. 4) 1.5 (~ 0.5) - - - - GaIK M173L 5.9 (t 0. 8) 1.9 (t 0. 6) 2.6 (t 0. 5) 0.02 0. 01)- Y371H 5.6 (t 0. 3) 2.2 0. 6) 6. 0 (t 0. 9) 0. 16 0.04) M173L-4.6 (t 0. 7) 2.3 (t 0. 4) 4.0 (~ 0. 6) 0.02 (t 0. 01) 6.3 1. 1) 0.07 ( Y371H 0.03) [Para 5 7] Example 11 [Para 58] 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.

[Para 59] 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 (GaIK) and nucleotidylyltransferase (Ep) -essentially an unnatural NDP-sugar factory-in a natural aglycon-producing host (e. g. the erythromycin-producing Saccharopo/yspora) 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. coll) 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.

[Para 60] The inventor has applied directed enzyme evolution and relied upon a high throughput galactokinase (GaIK) assay (DNS assay) for the screening of diverse E. co/iGalK variant libraries generated via error-prone PCR. From this approach, one particular GalK mutant (Y371 H) 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. co/iGaIK) 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. co/ienzyme failed to provide mutants with enhanced C-4 sugar flexibility while a parallel study revealed the L./actiswild-type GalK and Y385H orthologs to surprisingly display weak activity toward the C-4 epimer, glucose.

[Para 61] 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. colt 73L mutant GalK with enhanced C-4 and C-6 promiscuity. Moreover, a combination of the favorable structure-based (M1 73L) mutation with the beneficial mutation previously discovered via directed evolution (Y371 H) 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. colihost engineered to express M1 73L-Y371 H-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.

[Para 62] Materials and Methods [Para 63] 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, IL), or Fluka (Milwaukee, WI). E. colistrains XL1-blue and BL21 (DE3) were purchased from Stratagene (Lajolla, CA). Expression vector pET1 5b was purchased from Novagen (Madison, WI). Enzymes were purchased from Promega (Madison, WI).

[Para 64] 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-I- (N- (5-propargylamidepentyl))-sulfonamide 50, see supporting methods, which is published as supporting information on the PNAS web site.

[Para 65] Structure Modeling. The PDB file for the crystal structure of L./actiswild type GalK was obtained from the Protein Data Bank, Research Collaboratoryfor Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www. rcsb. org/). The structure modeling was accomplished using Swiss-PdbViewer software (Version 3.7).

[Para 66] Site-specific Mutagenesis. The Gall< M1 73L single mutant and M1 73L-Y371 H double mutant were generated using the QuikChange II Site-Directed Mutagenesis Kit from either wild-type or Y371 H template, respectively (Stratagene). The corresponding mutated plasmids pGaIKMI 73L and pGaIKMLYH were constructed by using PfuUltraTM DNA polymerase for mutagenic primer-directed replication from pGalK or pGalKY371 H template, respectively, using a pair of mutagenic primers (5'-GTAACTGCGGGATCCTGGATCAGCTAATTTCCG-3'and 5'-CGGAAATTAGCTGATCCAGGATCCCGCAGTTAC-3'). Amplification was accomplished under the following conditions: 5 uL of 1 Ox reaction buffer, 40 ng template DNA, 120 ng of each oligonucleotide primer, 1 uL dNTPs mixture (2.5 mM), 2.5 U of PfuUltraTM high-fidelity DNA polymerase, in a total volume of 50, uL ddH20 (thermocycler parameters: initial denaturation, 2 min at 95°C ; amplification, 12 cycles, 0.5 min at 95°C, 1 min at 55°C, 6.5 min at 68°C ; terminal hold, 5 min at 68°C). The amplified plasmids were treated with Dpnl to digest the parental DNA template and the mutated prodigy plasmid subsequently used to transform E. co/iXLI-blue.

The desired point mutation was verified by sequencing.

[Para 67] Characterization of GalK Mutants. The GalK mutants Y371 H was overexpressed following the procedure previously described for wild-type E. co/iGalK, while the overexpression of mutants M1 73L and M1 73L-Y371 H were accomplished at 16OC as described for L. lactis GalK. The mutant enzymes were purified by using metal affinity chromatography on Ni-NTA Spin Columns (QIAGEN, Valencia, CA) 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 (M1 73L, Y371 H and M1 73L-Y371 H). 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 MgCI2. The reactions were incubated at 37 oC 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, IL) as previously described.

[Para 68] In VivoConversion Analysis. The GalK double mutant pGaIKMLYH-E. coliwas overexpressed at 16 oC via induction of a 40 mL of culture at an ODeoo-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 oC with shaking (140 rpm) for 16 hr. To assess bioconversion, the cells were harvested by centrifugation (1 5 min, 12,000 rpm) and the recovered cell pellet (380 mg) washed twice with sodium phosphate buffer (20 mL), frozen, thawed and resuspended in HzO : MeOH (1: 1). The resuspended solution was heated at 100 oC for 15 min and then sonicated 5 x 45 sec on ice. Cell debris was collected by centrifugation (1 5 min, 12,000 rpm) and lyophilized to give the pale white solid (18 mg). To a solution containing all the crude product in 160 uL of H2O : MeOH (1: 1) was added 10 pmol 50 and 3.2 pmol of Cul, followed by heating to 50 oc for 24 hours. The reaction mixture was subsequently centrifuged to remove Cul and the supernatant (diluted 2-fold) directly analyzed by HPLC and LC-MS calculated for C26H37Nr, OiiPS 672. 20, found m/z [M+H]-671. 20.

[Para 69] Results and Discussion [Para 70] Structure-basis for Engineering GalKs with Expanded Specificity. Prior to any available GalK structural information, the directed evolution of E. co/iGalK 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./actiswild-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. coliand L. enzymes. With the L./acti5structure as a template, the sequence alignment of the E. coliand L. lactis GalKs revealed one clear difference among the sugar-binding pockets. Specifically, Leu-I 82 in L. lactl5 GalK is near to the C-4 carbon atom of galactose (3.85 A) and, based upon sequence alignment, this residue is replaced by Met-173 in E. coliGal K. 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 A from the y methyl of Leu 182 (Fig 6a). However, the identical model in which Leu-182 has been replaced by Met (to mimic the E coliGalK active site) revealed the same Glc-C-4 equatorial hydroxyl to be 1.72 A from Met sulfur (Fig 6b). Thus, this model clearly suggests Met 173 in E. co/iGalK may exclude glucose and thereby limit sugar C-4 specificity to ga/acto-configured substrates. Moreover, given the close proximity of the sugar C-6 hydroxyl to Met in this structural model (2.85 A) (Fig 6b), the inventors believe that M-1 73 in E. co/iGalK may also limit C-6 variation.

[Para 71] Characterization of Engineered E. coliGalK Mutants. To test the above hypothesis, a single E. co/iGalK M1 73L mutant was generated and screened against a panel 45 potential sugar substrates. As predicted, the E. colt 73L engineered mutant displayed moderate D-glucose 27 activity (20% conversion in 3 hr). Moreover, three additional D-gluco- configured structures 28,37, 38 (Fig 7 and 8), which were not substrates of wild-type E. coli GalK (or the evolved E. coli371 H 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 Y371 H 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. co/idouble Ml 73L-Y371 H 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. Figures 7 and 8 illustrate the complete substrate profiles forwild-type E. co/iGalK, the E. coliGalK mutant M1 73L, Y371 H and M1 73L-Y371 H.

[Para 72] To better understand the distinct role of the two particular amino acid residues (Met-1 73 and Tyr-371) in determining the substrate specificity, the inventor chose the native substrate D-galactose 29, the unique M1 73L substrate D-glucose 27 and the unique Y371 H substrate L-altrose 41 for complete comparative steady-state kinetic profiling. In comparison to wild-type E. co/iGalK, a slight (around 2-fold) D-galactose Km increase was observed in all three variants (M1 73L, Y371 H and M1 73L-Y371 H). Moreover, a comparison of the D-glucose kinetic parameters for M1 73L and the L-altrose kinetic values for Y371 H 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 M1 73L-Y371 H 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 M1 73L and Y371 H 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 M1 73L-Y371 H 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.

[Para 73] In Vivo Bioconversion of Unnatural Sugars Using an Enginee, red GalK. In the context of assessing in vivo bioconversion, the specific M1 73L-Y371 H-22 relationship is advantageous for two reasons. First, as described above, 21,22 and 46 are not a substrates forwild-type E. coliGalK and therefore, the use of a standard E. colihost strain (which contains the inherent wild-type E. co/iGaIK) 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-cycloaddtion 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-1 5% conversion under the same conditions.

[Para 74] To assess the Y371 H-M1 73L GaIK-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. colihost (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-1 5b-the vector used for overexpression of the Gallo mutants) while the second employed a wild-type GaIK 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,-69% conversion of 6- azido-6-deoxy-D-galactose (22) was observed, a slight improvement over the in vitro yield (~ 50% conversion). In a similar manner, ~ 15% conversion of 6-azido-6-deoxy-D-glucose (46) was observed, consistent with the in vitroyield ( 50% conversion). Notably, this success illustrates that unnatural sugars are able to enter the heterologous E. colihost 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.

[Para 75] Implications for In Vivo Glycorandomization [Para 76] 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.

[Para 77] 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 sl<ill 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.

(Para 78] All references are incorporated herein for all purposes.

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