W0462

Structural Characterization of the Thiamin Regulatory Protein TenI from Bacillus subtilis. Angela V. Toms, Joo-Heon Park, Tadhg P. Begley, Steven E. Ealick, Dept. of Chemistry and Chemical Biology, Cornell University, Ithaca, NY.

Thiamin is an essential cofactor in all living systems and is a required component of the human diet. Thiamin pyrophosphate, the active form of vitamin B₁, plays an important role in carbohydrate metabolism and in branched-chain amino acid metabolism where it stabilizes acyl carbanion intermediates. Thiamin biosynthesis is not yet well understood and the reconstitution of the pyrimidine and the thiazole moieties has only recently been accomplished in a defined biochemical system. In Bacillus subtilis, thiamin pyrophosphate is synthesized from glycine, deoxy-D-xylulose 5-phosphate, cysteine and aminoimidizaole ribotide. The biosynthetic pathway is complex and uses 14 gene products. The tenI gene is part of the tenA-tenI-thiO-thiS-thiG-thiF-thiD operon. TenI is important for thiamin biosynthesis because a deletion mutant shows a thiamin requiring phenotype. Sequence analysis suggests that TenI may be a regulatory protein in the Lys R family. In addition, comparison of the TenI sequence with that of the structurally characterized thiamin phosphate synthase (TPS) sequence demonstrates that many of the thiamin binding residues at the active site of TPS are conserved in TenI, even though the overall sequence identity is only 23%. This strongly suggesting that TenI also binds thiamin phosphate. We have cloned and overexpressed TenI from B. subtilis. Crystals belong to the space group C222₁, with a = 96.97 Å, b = 104.24 Å and c = 217.35 Å, and diffract to 2.1 Å resolution. The structure of TenI was determined by selenomethionyl SAD phasing to 2.5 Å resolution. The asymmetric unit contains two TenI homodimers. Each TenI monomer is a (βα)₈ barrel and resembles the structure of TPS. The structure of TenI will be presented and compared with that of TPS.