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, 100 (23), 13184-9

Creation of the First Anomeric D/L-sugar Kinase by Means of Directed Evolution

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Creation of the First Anomeric D/L-sugar Kinase by Means of Directed Evolution

Dirk Hoffmeister et al. Proc Natl Acad Sci U S A.

Abstract

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.

Figures

Fig. 1.
Fig. 1.
(a) 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 IVG. Ep, α-d-glucopyranosyl phosphate thymidylyltransferase; GlyTn different glycosyltransferases.
Fig. 2.
Fig. 2.
Reactions catalyzed by anomeric kinases. (a) Glycogen phosphorylase. (b) Fucokinase. (c) Galactokinase. (d) Proposed phosphorylation of l-altrose accomplished by the evolved GalK mutant Y371H.
Fig. 3.
Fig. 3.
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.
Fig. 4.
Fig. 4.
3JH–H coupling patterns and NOESY correlations for the GalK Y371H product β-l-altrose-1-phosphate.

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