In vitro glycorandomization is a rapid chemoenzymatic strategy to diversify complex natural product scaffolds. The glycorandomization sugar activation pathway is dependent upon the efficient construction of diverse sugar-1-phosphate libraries. In the context of the previously evolved GalK Y371H "gatekeeper" mutation, the active site M173L mutation described herein presents a kinase with remarkably broadened substrate range to include 28 diverse natural and unnatural sugars. Among these new substrates, 6-azido-6-deoxy-galactose and 6-azido-6-deoxy-glucose present unique chemical probes to assess the utility of an E. coli Y371H/M173L-GalK-overproducing strain to generate unnatural sugar-1-phosphates in vivo. Remarkably, the in vivo conversion of both unnatural sugars rival that demonstrated in vitro. This notable in vivo success stands as the first step toward constructing short sugar-activation pathways in vivo and, ultimately, in vivo natural-product glycorandomization.

No abstract

Many pharmaceutically important compounds derive from carbohydrate-containing natural products, and sugar moieties of these molecules have been proven to play an important role in drug targeting, biological activity, and pharmacology. [1±7] Thus, altering the glycosylation of natural products would significantly contribute to the diversity of novel therapeutics. Among a number of routes for altering glycosylation, naturalproduct glycorandomization is one of the most efficient approaches for complex secondary metabolites.[8±14] In vitro glycorandomization (IVG) technology establishes a promiscuous chemoenzymatic system to quickly build diverse glycorandomized libraries based on natural-product structures (Figure 1 a). This process utilizes chemical synthesis to provide a repertoire of unique sugar precursors, and three promiscuous enzymes to activate (anomeric sugar kinases and nucleotidylyltransferases) and attach (glycosyltransferases) these carbohydrate libraries to various complex natural-product aglycons. Anomeric sugar kinases, as key components of glycorandomization, directly determine the availability of sugar phosphates for chemoenzymatic routes toward complex glycoconjugates. Thus, generation of a flexible sugar kinase capable of accepting a wide array of monosaccharide substrates would directly enhance the efficiency of IVG. Of particular interest are d-glucoconfigured scaffolds given their prevalence in glycosylated natural products (Figure 1 b), [1,3,4,6] glycoproteins, [15±18] as well as many bacterial and eukaryotic cell-surface glycosylation patterns.[18±20]To date, an enzyme capable of d-glucose (Figure 2, 13) anomeric phosphorylation (a glucokinase, or GlcK) remains elusive. Toward this goal, directed evolution of E. coli galactokinase (GalK) led to the Y371H variant with remarkably widened substrate flexibility at C-2, C-3, and C-5 of the sugar. Yet, all sugars tested containing alternative C-4 substitutions (e.g. 4-deoxy-d-galactose, 12 or 13, Figure 2) were not accepted as substrates for the evolved catalyst.[21] As an alternative approach, the recently elucidated structure of Lactococcus lactis GalK provides a template for rational sugar kinase engineering.[22] The L. lactis GalK structure reveals that the strongly conserved active-site residues Asp45 and Tyr233 hydrogen bond with the critical galactose C-4 axial hydroxyl.[22] While a recent saturation mutagenesis of the equivalent residues within the E. coli GalK revealed the role of the active-site tyrosine to be hydrophobic stacking and expanded the substrate range to include 12, [23] a GlcK has still not been found. To continue our quest for an anomeric GlcK and further explore the relevance of the L. lactis structural model to other GalKs, we report the generation and characterization of the Y385H L. lactis GalK mutant-the promiscuous E. coli equivalent of which was discovered through directed evolution.[21] Remarkably, we reveal that this L. lactis variant displays a substantial degree of kinase activity toward several C-4-substitut...

Chemoenzymatic routes toward complex glycoconjugates often depend on the availability of sugar-1-phosphates. Yet the chemical synthesis of these vital components is often tedious, whereas natural enzymes capable of anomeric phosphorylation are known to be specific for one or only a few monosaccharides. Herein we describe the application of directed evolution and a high-throughput multisugar colorimetric screen to enhance the catalytic capabilities of the Escherichia coli galactokinase 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 both in vitro and in vivo glycosylation methodologies, such as natural product glycorandomization.galactokinase ͉ glycorandomization ͉ in vitro evolution ͉ enzyme M any 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), nonribosomal peptides (vancomycin, 5), polyenes (nystatin, 6), coumarins (novobiocin, 7), and cardiac glycosides (digitoxin, 8) (1-7). 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 (ref. 8 and references therein). 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 (6,7,(9)(10)(11)(12)(13)(14)(15), the advantage of which is the ability to efficiently incorporate highly functionalized ''unnatural'' sugar substitutions into the corresponding natural product scaffold (Fig. 1b). In a recent demonstration of IVG, Ͼ50 analogs of 5 were generated, some of which displayed enhanced and distinct antibacterial profiles from the parent natural product (13).As starting materials, sugar phosphates play a key role in the entire IVG process. Thus, the ability to rapidly construct sugar phosphate ...

Chemoenzymatic routes toward complex glycoconjugates often depend on the availability of sugar-1-phosphates. Yet the chemical synthesis of these vital components is often tedious, whereas natural enzymes capable of anomeric phosphorylation are known to be specific for one or only a few monosaccharides. Herein we describe the application of directed evolution and a high-throughput multisugar colorimetric screen to enhance the catalytic capabilities of the Escherichia coli galactokinase 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 both in vitro and in vivo glycosylation methodologies, such as natural product glycorandomization.galactokinase ͉ glycorandomization ͉ in vitro evolution ͉ enzyme M any 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), nonribosomal peptides (vancomycin, 5), polyenes (nystatin, 6), coumarins (novobiocin, 7), and cardiac glycosides (digitoxin, 8) (1-7). 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 (ref. 8 and references therein). 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 (6,7,(9)(10)(11)(12)(13)(14)(15), the advantage of which is the ability to efficiently incorporate highly functionalized ''unnatural'' sugar substitutions into the corresponding natural product scaffold (Fig. 1b). In a recent demonstration of IVG, Ͼ50 analogs of 5 were generated, some of which displayed enhanced and distinct antibacterial profiles from the parent natural product (13).As starting materials, sugar phosphates play a key role in the entire IVG process. Thus, the ability to rapidly construct sugar phosphate ...