The use of deoxyfluoro-<scp>d</scp>-galactopyranoses in a study of yeast galactokinase specificity

Thomas, Peter; Bessell, E.M.; Westwood, J. H.

doi:10.1042/bj1390661

Cited by 26 publications

(13 citation statements)

References 20 publications

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“…2): the glycogen phosphorylases (16), which convert glycogen (9) into D-glucose-1-phosphate (10); fucokinases, which transfer a phosphate from ATP to the anomeric position of L-fucose (12) to provide ␤-L-fucose-1-phosphate (13) (17); and the galactokinases (GalK), which catalyze the formation of ␣-D-galactose-1-phosphate (Gal-1-P, 15) from D-galactose (14) and ATP. Previous studies have revealed that GalK from various sources have a limited substrate scope (18)(19)(20)(21), 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, to apply any of these kinases for generating a randomized sugar phosphate library, their monosaccharide substrate promiscuity must first be enhanced.…”

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confidence: 99%

Creation of the first anomeric D/L-sugar kinase by means of directed evolution

Hoffmeister

Yang²,

Liu³

et al. 2003

Proceedings of the National Academy of Sciences

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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 ...

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confidence: 99%

Creation of the first anomeric D/L-sugar kinase by means of directed evolution

Hoffmeister

Yang²,

Liu³

et al. 2003

Proceedings of the National Academy of Sciences

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“…Figure 2 illustrates the substrate profiles of wild‐type L. lactis GalK and the L. lactis Y385H GalK mutant. The L. lactis wild‐type enzyme accepted 13 sugar substrates, a striking expansion of the typically limited substrate scopes observed for the GalK family 24–27. Among these new monosaccharide substrates were three notable C‐4‐substituted sugars ( 12 , 13 , and 14 ) and two unique L ‐configured sugars ( L ‐altrose, 22 , and L ‐glucose, 23 ), all of which failed as active substrates for native GalKs from other sources 24–27.…”

Section: Representative Activities and Product Mass For Each Enzymatmentioning

confidence: 96%

“…The L. lactis wild‐type enzyme accepted 13 sugar substrates, a striking expansion of the typically limited substrate scopes observed for the GalK family 24–27. Among these new monosaccharide substrates were three notable C‐4‐substituted sugars ( 12 , 13 , and 14 ) and two unique L ‐configured sugars ( L ‐altrose, 22 , and L ‐glucose, 23 ), all of which failed as active substrates for native GalKs from other sources 24–27. As expected, the L. lactis Y385H mutant was even more promiscuous with several new structures (Figure 2, 2‐deoxy‐ D ‐glucose, 15 , 6‐amino‐6‐deoxy‐ D ‐galactose, 17 , and 6‐azido‐6‐deoxy‐ D ‐galactose, 19 ) also weakly recognized by this variant.…”

Section: Representative Activities and Product Mass For Each Enzymatmentioning

confidence: 96%

Structure‐Based Enhancement of the First Anomeric Glucokinase

2004

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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...

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“…25 In this study, they demonstrated the conversion of 6FGal to 6FGal-1P using a yeast-derived galactokinase (GalK) in the presence of ATP. Westwood et al 26 later evidenced that 2FGal, 3FGal and 4FGal could be converted by the same enzyme to 2FGal-1P, 3FGal-1P and 4FGal-1P respectively. The results of their kinetic study suggests the yeast-derived GalK has lower affinity for the four fluorinated substrates when compared with the natural Gal substrate (in the order Gal > 2FGal > 3FGal ≈ 6FGal << 4FGal).…”

Section: Fluorinated Galactose-1-phosphate and Udp-galactose Donorsmentioning

confidence: 99%