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Volume 271, Number 7, Issue of February 16, 1996 pp. 3445-3452
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Mechanistic Studies on Thiaminase I
OVEREXPRESSION AND IDENTIFICATION OF THE ACTIVE SITE NUCLEOPHILE

(Received for publication, September 12, 1995; and in revised form, November 8, 1995)

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

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 for thiamin of 3.7 ± 0.6 µM and a k of 34 s. 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 Pro and Thr. Subsequent cysteine-specific labeling and site-directed mutagenesis identified Cys as the active site nucleophile.



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