Mechanistic Studies on the Radical S-Adenosylmethionine Enzymes Involved in Molybdopterin, Thiamin and Vitamin B12 Biosynthesis

Abstract

This dissertation focuses on radical S-adenosylmethionine enzymes involved in cofactor biosynthesis. Mechanistic studies discussed here include: (i) molybdenum cofactor biosynthetic enzyme - MoaA, (ii) thiamin pyrimidine synthase – ThiC (iii) hydroxybenzimidazole synthase, HBI synthase, involved in anaerobic vitamin B12 biosynthesis.

MoaA catalyzes the first step in molydopterin biosynthesis where GTP is converted to pterin. This catalysis involves a remarkable rearrangement reaction where the C8 of guanosine-5’-triphosphate (GTP) is inserted between the C2’ and C3’ carbon atoms of GTP to give the final pterin. Mechanistic studies involved characterization of the products of the reaction, identification of the position of hydrogen atom abstraction by 5’-deoxyadenosyl radical and trapping of intermediates by using 2’,3’-dideoxyGTP, 2’-deoxyGTP and 2’-chloroGTP as substrate analogs.

Thiamin pyrimidine synthase, ThiC, catalyzes a complex rearrangement reaction involving the conversion of aminoimidazole ribotide (AIR) to thiamin pyrimidine (HMP-P). A hydrogen atom transfer from S-adenosylmethionine (AdoMet) to HMP-P was demonstrated. Also, the stereochemistry of this transfer was elucidated.

Bioinformatics studies on ThiC revealed that a paralog of ThiC was clustered with vitamin B12 biosynthetic genes in several anaerobic microorganisms. The gene responsible for the anaerobic vitamin B12 – benzimidazole biosynthesis was previously unknown. We demonstrate that the gene product of this ThiC paralog is a radical S- adenosylmethionine enzyme. Remarkably it catalyzes the conversion of aminoimidazole ribotide (AIR) to 5-hydroxybenzimidazole (5-HBI) and formate, and S-adenosylmethionine to 5’-deoxyadenosine. We determine the hydrogen atom abstracted by 5’-deoxyadenosyl radical. We also performed carbon, nitrogen and hydrogen labeling studies and characterized the labeling pattern on 5-HBI. Based on these studies we propose a reaction mechanism of this remarkable conversion of AIR to 5-HBI.