Crystal structure of thiamin phosphate synthase from Bacillus subtilis at 1.25 angstrom resolution

The crystal Structure of Bacillus subtilis thiamin phosphate synthase compl exed with the reaction products thiamin phosphate and pyrophosphate has bee n determined by multiwavelength anomalous diffraction phasing techniques an d refined to 1.25 Angstrom resolution. Thiamin phosphate synthase is an alp ha/beta protein with a triosephosphate isomerase fold. The active site is i n a pocket formed primarily by the loop regions, residues 59-67 (A loop, jo ining alpha 3 and beta 2), residues 109-114 (B loop, joining alpha 5 and be ta 4), and residues 151-168 (C loop, joining alpha 7 and beta 6). The high- resolution structure of thiamin phosphate synthase complexed with its react ion products described here provides a detailed picture of the catalyticall y important interactions between the enzyme and the substrates. The structu re and other mechanistic studies are consistent with a reaction mechanism i nvolving the ionization of 4-amino-2-methyl-5-hydroxymethylpyrimidine pyrop hosphate at the active site to give the pyrimidine carbocation. Trapping of the carbocation by the thiazole followed by product dissociation completes the reaction. The ionization step is catalyzed by orienting the C-O bond p erpendicular to the plane of the pyrimidine, by hydrogen bonding between th e C4' amino group and one of the terminal oxygen atoms of the pyrophosphate , and by extensive hydrogen bonding and electrostatic interactions between the pyrophosphate and the enzyme.