Structure-based engineering of E. coli galactokinase as a first step toward in vivo glycorandomization

Jie Yang, Xun Fu, Jianchun Liao, Lesley Liu, Jon S. Thorson

Research output: Contribution to journalArticlepeer-review

62 Scopus citations

Abstract

In vitro glycorandomization is a rapid chemoenzymatic strategy to diversify complex natural product scaffolds. The glycorandomization sugar activation pathway is dependent upon the efficient construction of diverse sugar-1-phosphate libraries. In the context of the previously evolved GalK Y371H "gatekeeper" mutation, the active site M173L mutation described herein presents a kinase with remarkably broadened substrate range to include 28 diverse natural and unnatural sugars. Among these new substrates, 6-azido-6-deoxy-galactose and 6-azido-6-deoxy-glucose present unique chemical probes to assess the utility of an E. coli Y371H/M173L-GalK-overproducing strain to generate unnatural sugar-1-phosphates in vivo. Remarkably, the in vivo conversion of both unnatural sugars rival that demonstrated in vitro. This notable in vivo success stands as the first step toward constructing short sugar-activation pathways in vivo and, ultimately, in vivo natural-product glycorandomization.

Original languageEnglish
Pages (from-to)657-664
Number of pages8
JournalChemistry and Biology
Volume12
Issue number6
DOIs
StatePublished - 2005

Bibliographical note

Funding Information:
This contribution was supported in part by the National Institutes of Health grants AI52218, CA84374, and GM70637. We are grateful to the University of Wisconsin-Madison School of Pharmacy Analytical Facility for analytical support.

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

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