Biotin is an essential vitamin in the human diet. The adequate daily intake for adult humans is in the range of 30-100 micrograms. The biotin market is divided between agricultural and human use with 90% and 10% going to animal and human nutrition respectively. Currently biotin is produced by chemical synthesis at the level of >20 tons/year. Biotin biosynthesis is well-understood and efforts to produce it by fermentation are currently under intense investigation in several biotechnology companies. Biotin functions as a cofactor in all biochemical carboxylation reactions involving bicarbonate as the CO2 donor. The mechanism of biotin mediated carboxylation is now well-understood. Biotin has also found extensive applications in chemical biology where the strong biotin avidin interaction has been extensively used for macromolecular tagging. In contrast to biotin biosynthesis, little is known about biotin catabolism. A few products of biotin catabolism in a soil Pseudomonad grown on biotin as the sole carbon source have been isolated but no genes or enzymes associated with biotin catabolism have yet been identified. This proposal will focus on the isolation of a biotin catabolic strain, the identification of the catabolic operon and the reconstitution of all enzymes involved in biotin catabolism. Mechanistic and structural studies will be carried out on the key enzymes involved in the degradation of the biotin heterocyclic core.