Creation of the first anomeric D/L-sugar kinase by means of directed evolution.
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.
Hoffmeister
Dirk
D
Laboratory for Biosynthetic Chemistry, Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705, USA. jsthorson@pharmacy.wisc.edu
Yang
Jie
J
Liu
Lesley
L
Thorson
Jon S
JS
D002240:Carbohydrate Sequence; D002384:Catalysis; D003837:Deoxy Sugars / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D005686:Galactokinase / Q000737:chemistry* / Q000235:genetics*; D015723:Gene Library; D005947:Glucose / Q000737:chemistry; D006031:Glycosylation; D006595:Hexosamines / Q000737:chemistry; D006601:Hexoses / Q000737:chemistry; D006603:Hexuronic Acids / Q000737:chemistry; D013294:Lactococcus lactis / Q000201:enzymology; D008956:Models, Chemical; D016296:Mutagenesis; D009154:Mutation; D010766:Phosphorylation; D015202:Protein Engineering / Q000379:methods; D013237:Stereoisomerism; D013379:Substrate Specificity
D003837:Deoxy Sugars; D006595:Hexosamines; D006601:Hexoses; D006603:Hexuronic Acids; C007819:galacturonic acid; D005686:Galactokinase; D005947:Glucose