Mechanistic studies on thiaminase I. Overexpression and identification of the active site nucleophile

C. A. Costello, N. L. Kelleher, M. Abe, F. W. McLafferty, T. P. Begley*

*Corresponding author for this work

Research output: Contribution to journalArticle

50 Scopus citations

Abstract

Thiaminase I (EC 2.5.1.2) catalyzes the replacement of the thiazole moiety of thiamin with a wide variety of nucleophiles. Here we report the sequencing of a thiaminase I clone from Bacillus thiaminolyticus, the overexpression of the cloned gene in Escherichia coli, and the purification and characterization of the enzyme. Recombinant thiaminase I functions as a monomer with a K(m) for thiamin of 3.7 ± 0.6 μM and a k(cat) of 34 s-1. Electrospray ionization Fourier-transform mass spectrometry identified a single sequencing error and demonstrated heterogeneity, finding molecular weights of 42,127, 42,198, and 42,255 due to added Ala and Gly-Ala at the amino terminus. Similar analysis of the 4-amino-2-methyl-6-chloropyrimidine (8) inactivated enzyme indicated that the active site nucleophile involved in catalysis of the substitution reaction is located between Pro79 and Thr177. Subsequent cysteine-specific labeling and site-directed mutagenesis identified Cys113 as the active site nucleophile.

Original languageEnglish (US)
Pages (from-to)3445-3452
Number of pages8
JournalJournal of Biological Chemistry
Volume271
Issue number7
DOIs
StatePublished - 1996

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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