Mechanistic studies of biotin synthase using isotopically-labeled dethiobiotin

Abstract

Biotin synthase is a S-adenosylmethionine radical enzyme that catalyzes the conversion of dethiobiotin to biotin by replacing two hydrogen atoms in dethiobiotin with a sulfur atom to form the thiophane ring of biotin. Key questions remain unanswered in the mechanism of conversion including the role of the [2Fe-2S] coordinating cysteine residues, the structure of the intermediate state, and the rate-determining step of the reaction. The [2Fe-2S] coordinating cysteine residues have been found to be essential for conversion of DTB to biotin. Mutation of any of the coordinating cysteine residues to aspartate abolished activity. Mutation of the coordinating cysteine residues to aspartate causes a significant shift in the redox potential of the [2Fe-2S] cluster. This shift in redox potential is likely the cause of inactivity as the [2Fe-2S] cluster is likely destroyed once reduced by an external electron donor. It is necessary to produce isotopically labeled DTB in order to probe various aspects of the mechanism of biotin synthase. The biotin biosynthetic pathway has successfully been co-opted to generate various isotopically labeled compounds. d3-9-dethiobiotin was used to determine if hydrogen atom abstraction from the C9 position of DTB is rate limiting. A KIE of 7.95 was measured that confirms that hydrogen atom abstraction is in fact rate limiting. The intermediate state of catalysis is proposed to exist as DTB covalently bound to an intact reduced [2Fe-2S] cluster. HYSCORE spectroscopy was used to examine the intermediate state generated using 13C-(9-methyl)-dethiobiotin and provided the first direct evidence for the existence of the proposed intermediate. In summation, this body of would has provided many insights into the mechanism of sulfur insertion catalyzed by biotin synthase.