Mechanistic Studies of the Adenosylmethionine Radical Enzyme Biotin Synthase

University Of Hawaii, Honolulu

Investigators

Abstract

Biotin is a small molecular cofactor used in several important enzymes that carry out reactions that involve the transfer of carboxylate groups; for example, the addition of a carboxylate group to acetyl CoA by the biotin-containing enzyme acetyl CoA carboxylase is the first step in fatty acid biosynthesis in humans as well as most other organisms. Biotin is not made in humans (it is an essential vitamin), but is made in bacteria, yeast, and plants by a conserved pathway that concludes with the substitution of a sulfur atom in place of two hydrogen atoms, a reaction that is catalyzed by the enzyme biotin synthase. This biochemical reaction is unique and unprecedented: it requires S-adenosyl-L-methionine (AdoMet or SAM) to remove the hydrogen atoms from the precursor dethiobiotin and it appears to use an iron-sulfur cluster cofactor as the source of sulfur for constructing biotin. The involvement of SAM in removing hydrogen atoms suggests that biotin synthase belongs to an emerging family of enzymes that utilize SAM as a substrate or cofactor to facilitate the controlled generation of substrate or protein radicals. In the present research, the formation and decay of this intermediate will be examined using several techniques. First, mass spectrometry and heavy atom labeling will be used to determine the chemical nature of the intermediate, probe the kinetics of formation and decay of the intermediate, and optimize conditions for maximizing the amount of enzyme in this state. Second, vibrational spectroscopy and electron paramagnetic resonance spectroscopy with isotopically-labeled substrates will be employed to probe whether 9-mercaptodethiobiotin is covalently attached to the iron-sulfur cluster during the catalytic reaction. A detailed description of the factors that control SAM radical generation, substrate activation, and carbon-sulfur bond formation will contribute to our understanding of mechanistic and structural features common to all radical enzymes. BROADER IMPACTS. The research projects described provide an excellent forum for teaching the basic techniques involved in characterizing enzyme reaction intermediates to undergraduate and graduate students and postdoctoral researchers. The University of Hawaii serves a diverse student population that includes a significant percentage of students from the Hawaiian and Pacific Islands, and a number of undergraduates will be trained in protein and enzyme characterization through participation in a "Directed Research" course. More specifically, this research project will support the scientific work of 2-3 graduate students and 3-4 undergraduate students over three years. In addition, our improved knowledge of the mechanism of biotin synthase will contribute to the development of organisms engineered to overproduce biotin. Biotin is an expensive but essential vitamin that is incorporated into both human nutritional supplements and, more significantly, into animal feedstock. The current research could contribute to a low-cost biological method for the production of biotin (as well as other vitamins) which would benefit society by contributing to less expensive and more efficient food production. This award was co-funded by the Division of Molecular and Cellular Biosciences and the Organic and Macromolecular Chemistry Program of the Chemistry Division.