A protein-derived oxygen is the source of the amide oxygen of nitrile hydratases

Micah T. Nelp, Yang Song, Vicki H. Wysocki, Vahe Bandarian

Research output: Contribution to journalArticle

4 Scopus citations

Abstract

Nitrile hydratase metalloenzymes are unique and important biocatalysts that are used industrially to produce high value amides from their corresponding nitriles. After more than three decades since their discovery, the mechanism of this class of enzymes is becoming clear with evidence from multiple recent studies that the cysteine-derived sulfenato ligand of the active site metal serves as the nucleophile that initially attacks the nitrile. Herein we describe the first direct evidence from solution phase catalysis that the source of the product carboxamido oxygen is the protein. Using 18O-labeled water under single turnover conditions and native high resolution protein mass spectrometry, we show that the incorporation of labeled oxygen into both product and protein is turnover-dependent and that only a single oxygen is exchanged into the protein even under multiple turnover conditions, lending significant support to proposals that the post-translationally modified sulfenato group serves as the nucleophile to initiate hydration of nitriles.

Original languageEnglish (US)
Pages (from-to)7822-7829
Number of pages8
JournalJournal of Biological Chemistry
Volume291
Issue number15
DOIs
StatePublished - Apr 8 2016
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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