Thiaminase I (42 kDa) heterogeneity, sequence refinement, and active site location from high-resolution tandem mass spectrometry

Neil L. Kelleher, Colleen A. Costello, Tadhg P. Begley, Fred W. McLafferty*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

54 Scopus citations

Abstract

Thiaminase I (E.C. 2.5. 1.2) from Bacillus thiaminolyticus catalyzes the degradation of thiamin (vitamin B1). Unexpected mass heterogeneity (MW 42,127, 42,197, and 42,254; 1:2:1) in recombinant thiaminase I from Escherichia coli was detected by electrospray ionization Fourier-transform mass spectrometry, resolving power 7×104. Nozzle-skimmer fragmentation data reveal an extra Ala (+71.02; 71.04=theory) and GlyAla (+128.04; 128.06=theory) on the N-terminus, in addition to the fully processed enzyme. However, the fragment ion masses were consistent only with this sequence through 330 N-terminal residues; resequencing of the last 150 bps of the thiaminase I gene yields a sequence consistent with the molecular weight values and all 61 fragment ion masses. Covalently labeling the active site with a 108-Da pyrimidine moiety via mechanism-based inhibition produces a corresponding molecular weight increase in all three thiaminase I components, which indicates that they are all enzymatically active. Inspection of the fragment ions that do and do not increase by 108 Da indicates that the active site nucleophile is located between Pro79 and Thr177 in the 379 amino acid enzyme.

Original languageEnglish (US)
Pages (from-to)981-984
Number of pages4
JournalJournal of the American Society for Mass Spectrometry
Volume6
Issue number10
DOIs
StatePublished - Oct 1995

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy

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