CRYSTAL-STRUCTURE OF THIAMINASE-I FROM BACILLUS-THIAMINOLYTICUS AT 2.0 ANGSTROM RESOLUTION

Thiaminase-I catalyzes the replacement of the thiazole moiety of thiam in with a wide variety of nucleophiles, such as pyridine, aniline, cat echols, quinoline, and cysteine. The crystal structure of the enzyme f rom Bacillus thiaminolyticus was determined at 2.5 Angstrom resolution by multiple isomorphous replacement and refined to an R factor of 0.1 95 (R-free = 0.272). Two other structures, one native and one containi ng a covalently bound inhibitor, were determined at 2.0 Angstrom resol ution by molecular replacement from a second crystal form and were ref ined to R factors of 0.205 and 0.217 (R-free = 0.255 and 0.263), respe ctively. The overall structure contains two alpha/beta-type domains se parated by a large cleft. At the base of the cleft lies Cys113, previo usly identified as a key active site nucleophile. The structure with a covalently bound thiamin analogue, which functions as a mechanism-bas ed inactivating agent, confirms the location of the active site. Glu24 1 appears to function as an active site base to increase the nucleophi licity of Cys113. The mutant Glu241Gln was made and shows no activity. Thiaminase-I shows no sequence identity to other proteins in the sequ ence databases, but the three-dimensional structure shows very high st ructural homology to the periplasmic binding proteins and the transfer rins.