Enzymes can take advantage of the high reactivity of radicals to carry out otherwise difficult chemical transformations. Such enzymes are found in all branches of life, and are particularly ubiquitous in the diverse classes of microbes. A large emerging superfamily of enzymes found in virtually all organisms utilize S-adenosyl-L-methionine (AdoMet or SAM) as a substrate or cofactor to generate substrate or protein radicals. An important subset of the Radical SAM superfamily catalyzes the radical-mediated addition of sulfur to biomolecules. Biotin synthase catalyzes the oxidative addition of sulfur to saturated carbon atoms on dethiobiotin, generating the biotin thiophane ring. Biotin synthase is an iron-sulfur enzyme, and isotopic labeling suggests the sulfur atom incorporated into biotin derives from an iron-sulfur cluster. Our research focuses on understanding the chemical details of radical generation, substrate activation, and sulfur incorporation.
In the working hypothesis for the biotin synthase reaction sequence, in a manner identical with all Radical SAM enzymes, catalysis is initiated by reductive cleavage of SAM to generate methionine and a 5´-deoxyadenosyl radical. Hydrogen atom transfer from dethiobiotin to this radical results in formation of 5´-deoxyadenosine and a dethiobiotinyl substrate radical, which is then quenched through formation of a new C-S bond with sulfide from a nearby [2Fe-2S]2+ cluster. A similar reaction sequence using a second equivalent of AdoMet leads to completion of the thiophane ring. There is previously demonstrated formation and decay of an isolable chemical intermediate, 9-mercaptodethiobiotin (MDTB), and the parallel formation and decay of an unusual signal observable by electron paramagnetic resonance (EPR) spectroscopy assigned to a [2Fe-2S]+ cluster. This project will use pulsed EPR methods with isotopically labeled substrates and enzyme to examine the structure and electronic state of this paramagnetic intermediate. Mössbauer spectroscopy and mass spectrometry will be used to determine whether the [2Fe-2S]2+ cluster is regenerated following each turnover. In addition, site-directed mutagenesis experiments will determine whether the [2Fe-2S]2+ cluster is truly essential for activity. The exceptionally slow kinetics observed for the formation of both 9-mercaptodethiobiotin and biotin, both in vitro and in vivo will be investigated by deuterium labeling and isotope effect analysis to determine the extent to which the specific hydrogen trans,fer steps are rate limiting. A detailed description of the factors that control substrate activation and C-S bond formation will contribute to the understanding of mechanistic and structural features common to all Radical SAM 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. The PI is the organizer and director of a new undergraduate biochemistry degree program, through which talented undergraduate students will become involved in laboratory research through participation in a "Directed Research" course. In particular, the University of Hawaii serves a diverse student population that includes a significant percentage of students from underrepresented groups in science. In addition, an 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 feedstocks. A low-cost biological method for the production of biotin (as well as other vitamins) would benefit society by contributing to less expensive and more efficient food production.
