Converting Pasteurella multocida α2-3-sialyltransferase 1 (PmST1) to a regioselective α2-6-sialyltransferase by saturation mutagenesis and regioselective screening

John B. McArthur, Hai Yu, Jie Zeng, Xi Chen

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

A microtiter plate-based screening assay capable of determining the activity and regioselectivity of sialyltransferases was developed. This assay was used to screen two single-site saturation libraries of Pasteurella multocida α2-3-sialyltransferase 1 (PmST1) for α2-6-sialyltransferase activity and total sialyltransferase activity. PmST1 double mutant P34H/M144L was found to be the most effective α2-6-sialyltransferase and displayed 50% reduced donor hydrolysis and 50-fold reduced sialidase activity compared to the wild-type PmST1. It retained the donor substrate promiscuity of the wild-type enzyme and was used in an efficient one-pot multienzyme (OPME) system to selectively catalyze the sialylation of the terminal galactose residue in a multigalactose-containing tetrasaccharide lacto-N-neotetraoside.

Original languageEnglish (US)
Pages (from-to)1700-1709
Number of pages10
JournalOrganic and Biomolecular Chemistry
Volume15
Issue number7
DOIs
StatePublished - 2017

ASJC Scopus subject areas

  • Biochemistry
  • Physical and Theoretical Chemistry
  • Organic Chemistry

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