Functional elucidation of TfuA in peptide backbone thioamidation

Andi Liu, Yuanyuan Si, Shi Hui Dong, Nilkamal Mahanta, Haley N. Penkala, Satish K. Nair, Douglas A. Mitchell

Research output: Contribution to journalArticlepeer-review


YcaO enzymes catalyze several post-translational modifications on peptide substrates, including thioamidation, which substitutes an amide oxygen with sulfur. Most predicted thioamide-forming YcaO enzymes are encoded adjacent to TfuA, which when present, is required for thioamidation. While activation of the peptide amide backbone is well established for YcaO enzymes, the function of TfuA has remained enigmatic. Here we characterize the TfuA protein involved in methyl-coenzyme M reductase thioamidation and demonstrate that TfuA catalyzes the hydrolysis of thiocarboxylated ThiS (ThiS-COSH), a proteinaceous sulfur donor, and enhances the affinity of YcaO toward the thioamidation substrate. We also report a crystal structure of a TfuA, which displays a new protein fold. Our structural and mutational analyses of TfuA have uncovered conserved binding interfaces with YcaO and ThiS in addition to revealing a hydrolase-like active site featuring a Ser–Lys catalytic pair. [Figure not available: see fulltext.]

Original languageEnglish (US)
Pages (from-to)585-592
Number of pages8
JournalNature chemical biology
Issue number5
StatePublished - May 2021

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology


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