Twelve genes involved in thiamin biosynthesis in prokaryotes have been identified and overexpressed. Of these, six are required for the thiazole biosynthesis (thiFSGH, thil, and dxs), one is involved in the pyrimidine biosynthesis (thiC), one is required for the linking of the thiazole and the pyrimidine (thiE), and four are kinase genes (thiD, thiM, thiL, and pdxK). The specific reactions catalyzed by ThiEF, Dxs, ThiDM, ThiL, and PdxK have been reconstituted in vitro and ThiS thiocarboxylate has been identified as the sulfur source. The X-ray structures of thiamin phosphate synthase and 5-hydroxyethyl-4-methylthiazole kinase have been completed. The genes coding for the thiamin transport system (thiBPQ) have also been identified. Remaining problems include the cloning and characterization of thiK (thiamin kinase) and the gene(s) involved in the regulation of thiamin biosynthesis. The specific reactions catalyzed by ThiC (pyrimidine formation), and ThiGH and ThiI (thiazole formation) have not yet been identified.

Oxalate decarboxylase is a manganese-dependent enzyme that catalyzes the conversion of oxalate to formate and carbon dioxide. We have determined the structure of oxalate decarboxylase from Bacillus subtilis at 1.75 A resolution in the presence of formate. The structure reveals a hexamer with 32-point symmetry in which each monomer belongs to the cupin family of proteins. Oxalate decarboxylase is further classified as a bicupin because it contains two cupin folds, possibly resulting from gene duplication. Each oxalate decarboxylase cupin domain contains one manganese binding site. Each of the oxalate decarboxylase domains is structurally similar to oxalate oxidase, which catalyzes the manganese-dependent oxidative decarboxylation of oxalate to carbon dioxide and hydrogen peroxide. Amino acid side chains in the two metal binding sites of oxalate decarboxylase and the metal binding site of oxalate oxidase are very similar. Four manganese binding residues (three histidines and one glutamate) are conserved as well as a number of hydrophobic residues. The most notable difference is the presence of Glu333 in the metal binding site of the second cupin domain of oxalate decarboxylase. We postulate that this domain is responsible for the decarboxylase activity and that Glu333 serves as a proton donor in the production of formate. Mutation of Glu333 to alanine reduces the catalytic activity by a factor of 25. The function of the other domain in oxalate decarboxylase is not yet known.

The crystal structure of Bacillus subtilis orotidine 5-monophosphate (OMP) decarboxylase with bound uridine 5-monophosphate has been determined by multiple wavelength anomalous diffraction phasing techniques and refined to an R-factor of 19.3% at 2.4 Å resolution. OMP decarboxylase is a dimer of two identical subunits. Each monomer consists of a triosephosphate isomerase barrel and contains an active site that is located across one end of the barrel and near the dimer interface. For each active site, most of the residues are contributed by one monomer with a few residues contributed from the adjacent monomer. The most highly conserved residues are located in the active site and suggest a novel catalytic mechanism for decarboxylation that is different from any previously proposed OMP decarboxylase mechanism. The uridine 5-monophosphate molecule is bound to the active site such that the phosphate group is most exposed and the C5-C6 edge of the pyrimidine base is most buried. In the proposed catalytic mechanism, the ground state of the substrate is destabilized by electrostatic repulsion between the carboxylate of the substrate and the carboxylate of Asp60. This repulsion is reduced in the transition state by shifting negative charge from the carboxylate to C6 of the pyrimidine, which is close to the protonated amine of Lys62. We propose that the decarboxylation of OMP proceeds by an electrophilic substitution mechanism in which decarboxylation and carbon-carbon bond protonation by Lys62 occur in a concerted reaction. O rotidine monophosphate (OMP, 1) decarboxylase catalyzes the final step in the de novo biosynthesis of uridine monophosphate (UMP, 2) (Eq. 1).In most prokaryotes, OMP decarboxylase is a dimer of identical subunits whereas in higher organisms, it is part of a bifunctional enzyme that also catalyzes the formation of OMP. Amino acid sequence comparisons suggest that monomeric and bifunctional OMP decarboxylases are structurally homologous with about a dozen residues conserved throughout all species. The enzyme accelerates this decarboxylation reaction by 10 17 and is the most proficient enzyme identified so far (1). OMP decarboxylase does not use any cofactors (2). Its mechanism is novel because the carbanion generated by carbon dioxide loss is localized in an sp 2 orbital perpendicular to the system of the pyrimidine. In all other decarboxylases, the carbanion is delocalized either into an adjacent carbonyl or into a covalently bound thiamin, pyridoxal, or pyruvoyl cofactor (3). Although several hypotheses have been advanced to explain how the enzyme stabilizes the carbanion intermediate, the mechanistic details of this reaction are currently unclear.Three mechanisms have been proposed for OMP decarboxylase (Scheme 1). In the first mechanism (zwitterion mechanism), protonation of the C2 carbonyl group would generate the zwitterion 3, in which the positive charge at N1 could stabilize the negative charge accumulating at C6 during the decarboxylation. This proposal was supported by a model study that dem...

The substrates for Bacillus subtilis PLP synthase (YaaD and YaaE) are identified, and the first reconstitution of PLP biosynthesis using this pathway is described. Three partial reactions catalyzed by YaaD are also identified.

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