MECHANISTIC IMPLICATIONS AND FAMILY RELATIONSHIPS FROM THE STRUCTURE OF DETHIOBIOTIN SYNTHETASE

Background: Biotin is the vitamin essential for many biological carbox ylation reactions, such as the conversion of acetyl-coenzyme A (CoA) t o malonyl-CoA in fatty acid biosynthesis. Dethiobiotin synthetase (DTB S) facilitates the penultimate, ureido ring closure in biotin synthesi s, which is a non-biotin-dependent carboxylation. DTBS displays no seq uence similarity to any other protein in the database. Structural stud ies provide a molecular insight into the reaction mechanism of DTBS. R esults: We present the structure of DTBS refined to 1.80 Angstrom reso lution with an R-factor of 17.2% for all terms plus unrefined data on the binding of the substrate, 7,8-diaminopelargonic acid and the produ ce, dethiobiotin. These studies confirm that the protein forms a homod imer with each subunit folded as a single globular alpha/beta domain. The presence of sulphate ions in the crystals and comparisons with the related Ha-ras-p21 oncogene product are used to infer the ATP-binding site, corroborated by the difference electron density for the ATP ana logue AMP-PNP. Conclusions: This study establishes that the enzyme act ive site is situated at the dimer interface, with the substrate bindin g to one monomer and ATP to the other. The overall fold of DTBS closel y resembles that of three other enzymes, adenylosuccinate synthetase ( purA), Ha-rar-p21, and nitrogenase iron protein, that are unrelated by sequence or function, indicating that DTBS is a member of a diverse f amily of enzymes.