MECHANISTIC STUDIES ON THIAMINASE-I - OVEREXPRESSION AND IDENTIFICATION OF THE ACTIVE-SITE NUCLEOPHILE

Thiaminase I (EC 2.5.1.2) catalyzes the replacement of the thiazole mo iety of thiamin with a wide variety of nucleophiles. Here we report th e sequencing of a thiaminase I clone from Bacillus thiaminolyticus, th e overexpression of the cloned gene in Escherichia coli, and the purif ication and characterization of the enzyme. Recombinant thiaminase I f unctions as a monomer with a K-m for thiamin of 3.7 +/- 0.6 mu M and a k(cat) of 34 s(-1). Electrospray ionization Fourier-transform mass sp ectrometry identified a single sequencing error and demonstrated heter ogeneity, finding molecular weights of 42, 127, 42, 198, and 42,255 du e to added Ala and Gly-Ala at the amino terminus. Similar analysis of the 4-amino-2-methyl-6-chloropyrimidine (8) inactivated enzyme indicat ed that the active site nucleophile involved in catalysis of the subst itution reaction is located between Pro(79) and Thr(177). Subsequent c ysteine-specific labeling and site-directed mutagenesis identified Cys (113) as the active site nucleophile.