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

Dirk Hoffmeister, Jie Yang, Lesley Liu, Jon S. Thorson

Research output: Contribution to journalArticlepeer-review

87 Scopus citations

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.

Original languageEnglish
Pages (from-to)13184-13189
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume100
Issue number23
DOIs
StatePublished - Nov 11 2003

Keywords

  • Enzyme
  • Galactokinase
  • Glycorandomization
  • In vitro evolution

ASJC Scopus subject areas

  • General

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