S-Adenosyl-L-Methionine Radical Enzymes: (I) Metabolism of the Product 5’-Deoxyadenosine (II) Investigation of the Mechanism of Biotin Synthase

Abstract

5’-Deoxyadenosyl radicals are formed when the S-C5’ bond of S-adenosyl-L-methionine (SAM) undergoes a one-electron reductive cleavage. The 5’-deoxyadenosyl radical then abstracts a hydrogen atom from the substrate, RH, forming a substrate-centered radical and 5’-deoxyadenosine (5’-dAH). Radical SAM enzymes are present in many biochemical pathways, and most of these enzymes generate 5’-dAH as a product. Buildup of 5’-dAH would be detrimental due to product inhibition of Radical SAM enzymes and potentially other SAM-dependent enzymes. The metabolic fate of 5’-dAH is unknown. Notably, 5’-methylthioadenosine (MTA), which is structurally similar to 5’-dAH, is also a byproduct of several SAM-dependent enzymes. The MTA pathway, commonly known as the methionine salvage pathway, has been partially biochemically characterized in Bacillus subtilis and Klebsiella pneumoniae. We propose that some of the enzymes in this pathway may be promiscuous for 5’-dAH. This thesis will first consist of work on the synthesis of 5-deoxyribose (5dr) and 5-methylthioribose (MTR), the products of the first enzyme in the bacterial MTA pathway, MTA nucleosidase (MtnN). In this work, we will show that the subsequent enzyme in the MTA pathway, methylthioribose kinase (MtnK) is also promiscuous and phosphorylates both 5dr and MTA. The product of the enzyme reaction with 5dr is 5-deoxyribose-1-phosphate (5-dR1P). A complete structural characterization of this molecule will be described. Biotin is biosynthesized by the radical SAM enzyme biotin synthase. Biotin is a water-soluble enzyme cofactor that is not biosynthesized by mammals and is therefore an essential vitamin in humans. This cofactor is involved in the biosynthesis of fats and carbohydrates and the metabolism of amino acids. A mechanistic understanding of the chemistry that biotin synthase catalyzes in its active site may be useful in the discovery of drugs that can treat infections caused by microbes like E. coli and Mycobacterium Tuberculosis. One useful tool in probing the mechanism of this enzyme is to examine the kinetic isotope effect (KIE) that occurs when deuterium is incorporated into the substrate dethiobiotin. The biotin biosynthetic pathway transforms the C3 methyl group of L-alanine into the C9 methyl group of dethiobiotin. The second part of my thesis work will focus on the synthesis of mono- or di-deuterated alanine, which would then be used to biosynthesize mono- or di-deuterated dethiobiotin. Finally, a novel method for the chemical synthesis of dethiobiotin that begins with a syn-diamination across an alkene will be described.