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 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 Km for thiamin of 3.7 +/- 0.6 microM and a kcat 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 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.
Costello
C A
CA
Department of Chemistry, Cornell University, Ithaca, New York 14853, USA.
Kelleher
N L
NL
Abe
M
M
McLafferty
F W
FW
Begley
T P
TP
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005798:Genes, Bacterial; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014166:Transferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C022737:thiamin pyridinylase