PMID:14612558
| Citation |
Hoffmeister, D, Yang, J, Liu, L and Thorson, JS (2003) Creation of the first anomeric D/L-sugar kinase by means of directed evolution. Proc. Natl. Acad. Sci. U.S.A. 100:13184-9 |
|---|---|
| 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. |
| Links |
PubMed PMC263743 Online version:10.1073/pnas.2235011100 |
| Keywords |
Carbohydrate Sequence; Catalysis; Deoxy Sugars/chemistry; Escherichia coli/enzymology; Galactokinase/chemistry; Galactokinase/genetics; Gene Library; Glucose/chemistry; Glycosylation; Hexosamines/chemistry; Hexoses/chemistry; Hexuronic Acids/chemistry; Lactococcus lactis/enzymology; Models, Chemical; Mutagenesis; Mutation; Phosphorylation; Protein Engineering/methods; Stereoisomerism; Substrate Specificity |
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