Exploring the intermediate state of biotin synthase: covalent attachment of substrate to iron sulfur cluster

Abstract

Biotin synthase (BioB) is an iron-sulfur protein that catalyzes the final step in the biosynthesis of biotin. Specifically, it substitutes sulfur for hydrogen at the C9 and C6 positions of dethiobiotin, completing a thiophane ring. It is one of an ever growing group of AdoMet-dependent enzymes, which undergo multiple one-electron transfers and carbon-centered radical transition states. For the protein to be active, it requires two FeS clusters--one oxygen-sensitive [4Fe4S]2+ cluster that lies near the surface and one reductant-sensitive [2Fe2S]2+ cluster that is buried deep within the (αβ)8 barrel. Study of this enzyme has been fueled by the theory of using inhibitors as antibiotics. Biotin synthase appears to only be found in bacteria and some plants, not in any member of the animal kingdom. It has been suggested that dethiobiotin derivatives could be targets of resistant bacteria--specifically, tubercles bacillus. In order to further promote this research, more information is needed about the mechanism of biotin synthase. In this dissertation, the intermediate state of this enzyme will be examined by three different methods. I will show that 9-mercaptodethiobiotin is the catalytic intermediate, that single-electron redox changes are occurring in the [2Fe2S] cluster during turnover that parallel 9-MDTB production, and that the [2Fe2S] is the source of sulfur for biotin.