National Science Foundation Pre-doctoral Fellowship
Biotin is an essential vitamin for humans; it must be included in infant formulas and in fluids for intravenous nutrition. Even though it is a necessary component of our diet, we do not yet know the mechanism by which biotin, also known as vitamin H, is synthesized in the body from dethiobiotin. The enzyme that accomplishes this conversion has been termed biotin synthase. This enzyme catalyzes the insertion of sulfur at nonactivated positions of dethiobiotin, between carbons C1 and C4, to generate biotin. This mechanism is of interest because it appears to involve uncommon chemistry and because of the commercial value of the product. Biotin is sold as a pharmaceutical and as a food and cosmetic additive. This water soluble vitamin is essential for the growth and well-being of animals and humans, and it is used in the formation of fats the utilization of carbon dioxide.
Prof. Richard H. Holm's bioinorganic chemistry class sparked my interest in the understanding of enzyme mechanisms as model systems for the design of more selective drugs and better synthetic catalysts that allow us to run reactions with good yield, little waste, and inexpensively. Having worked for two years with Prof. JoAnne Stubbe on the mechanism of nucleotide reduction in E. coli by ribonucleoside diphosphate reductase, I have come to appreciate the positive effects of an understanding of this mechanism in the synthesis of more efficient drugs for the treatment of cancer and viral diseases. Increased knowledge of the biotin biosynthase mechanism should allow us to produce biotin more efficiently, and the knowledge gained from this mechanism could be applied to similar enzymes.
Despite recent advances in the elucidation of the biotin biosynthetic pathway, the final step of sulfur insertion has not been characterized. It is this final step in which dethiobiotin is converted into biotin that I would like to study in detail. As an organic chemist I would be very interested in the elucidation of the mechanism needed to effect this transformation. Some mechanistic work involving radioactive labeling has been done by Parry and coworkers showing that the incorporation of sulfur is stereospecific, and that only a single hydrogen was lost at each of the carbons bridging the sulfur.5,6,7,8 Biotin synthase has been a difficult enzyme to study due to, among other things, the complexity of the assays which has made it difficult to assess the enzyme's activity. Most of the work that has been done so far in this step of the pathway has been related to improving the in vitro activity of the enzyme and identifying the sulfur donor.
Recently Sanyal et al.1 isolated the purified enzyme from E. coli. This red protein appears to be a homodimer and electron paramagnetic resonance studies have shown the existence of a [2Fe-2S] cluster per monomer.1 The discovery that S-adenosylmethionine and NADPH were necessary cofactors for the...