Joshi, Sumedh Varad (2019-11). MECHANISTIC STUDIES ON RADICAL S-ADENOSYL-L-METHIONINE ENZYMES INVOLVED IN MENAQUINONE, THIAMIN AND COBALAMIN BIOSYNTHESIS. Doctoral Dissertation.
Menaquinone (Vitamin K) is an electron transfer cofactor that is an essential component for all the domains of life. Recently discovered, futalosine-dependent menaquinone biosynthetic pathway uses radical chemistry to assemble the aromatic core of menaquinone. Aminofutalosine synthase (MqnE)- a radical S-Adenosylmethionine (SAM) enzyme, catalyzes the key C-C bond formation reaction in this pathway converting 3-((1-carboxyvinyl)oxy)benzoic acid to Aminofutalosine. We have proposed a novel reaction mechanism for the MqnE catalyzed radical rearrangement involving a radical addition to the stable benzene ring. In support of this mechanism, we have successfully trapped the captodative and aryl radical anion intermediates using intramolecular, fast and radical triggered carbon-halogen bond fragmentation reactions (b-scission and SRN1 fragmentation). We have utilized exogenous radical trapping agents such as 5,5-dimethyl-1- pyrroline-N- oxide (DMPO), and sodium dithionite to trap radical intermediates in MqnE catalyzed reaction as a DMPO-spin adduct and sulfinate adduct respectively. We also have developed a chemical derivatization strategy for these sulfinate adducts to facilitate their chromatographic detection under aerobic conditions. These radical trapping strategies are generally useful in the study of other transient enzymatic radicals. Using a mechanism-guided approach, we have identified potent inhibitors of MqnE and have demonstrated their inhibitory activity against important human pathogens - Helicobacter pylori and Campylobacter jejuni. The best inhibitor exhibits tight-binding inhibition kinetics and has comparable antibacterial to currently approved antibiotics in the treatment of H. pylori infections. Thiamin Pyrimidine Synthase (ThiC) and its paralog- hydroxybenzimidazole Synthase (BzaF) play a pivotal role in the biosynthesis of thiamin (Vitamin B1) and cobalamin (Vitamin B12) by converting 5-aminoimidazole ribonucleotide (AIR) to hydroxymethyl pyrimidine phosphate (HMP-P) and 5- hydroxybenzimidazole (5-HBI) respectively. ThiC catalyzes the most complex unresolved radical rearrangement in primary metabolism. We have used rationally designed substrate analogs to study early steps of the ThiC and BzaF catalyzed reactions and have trapped a ribose based radical intermediate in the form of a sulfinate adduct using sodium dithionite as a trapping agent. These studies have demonstrated that ThiC and BzaF reactions follow a fragmentation-recombination mechanism and have enabled us to propose credible mechanistic hypotheses for these unprecedented transformations.