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

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

Molybdopterin biosynthesis - Mechanistic studies on a novel MoaA catalyzed insertion of a purine carbon into the ribose of GTP

Abstract The first step in the biosynthesis of the molybdopterin cofactor involves an unprecedented insertion of the purine C8 carbon between the C2′ and C3′ carbons of the ribose moiety of GTP. Here we review mechanistic studies on this remarkable transformation. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications

Molybdopterin biosynthesis: Trapping an unusual purine ribose adduct in the MoaA-catalyzed reaction

MoaA/MoaC catalyze a remarkable rearrangement reaction in which guanosine-5′-triphosphate (GTP) is converted to cyclic pyranopterin monophosphate (cPMP). In this reaction, the C8 of GTP is inserted between the C2′ and the C3′ carbons of the GTP ribose. Previous experiments with GTP isotopomers demonstrated that the ribose C3′ hydrogen atom is abstracted by the adenosyl radical. This led to a novel mechanistic proposal involving an intermediate with a bond between the C8 of guanine and C3′ of the ribose. This paper describes the use of 2′,3′-dideoxyGTP to trap this intermediate. © 2013 American Chemical Society

Molybdopterin biosynthesis: Trapping of intermediates for the MoaA-catalyzed reaction using 2′-deoxyGTP and 2′-chloroGTP as substrate analogues.

MoaA is a radical S-adenosylmethionine (AdoMet) enzyme that catalyzes a complex rearrangement of guanosine-5\u27-triphosphate (GTP) in the first step of molybdopterin biosynthesis. In this paper, we provide additional characterization of the MoaA reaction product, describe the use of 2′-chloroGTP to trap the GTP C3′ radical, generated by hydrogen atom transfer to the 5′-deoxyadenosyl radical, and the use of 2′-deoxyGTP to block a late step in the reaction sequence. These probes, coupled with the previously reported trapping of an intermediate in which C3′ of the ribose is linked to C8 of the purine, allow us to propose a plausible mechanism for the MoaA-catalyzed reaction. © 2014 American Chemical Society

Molybdopterin Biosynthesis: Trapping an Unusual Purine Ribose Adduct in the MoaA-Catalyzed Reaction

MoaA/MoaC catalyze a remarkable rearrangement reaction in which guanosine-5′-triphosphate (GTP) is converted to cyclic pyranopterin monophosphate (cPMP). In this reaction, the C8 of GTP is inserted between the C2′ and the C3′ carbons of the GTP ribose. Previous experiments with GTP isotopomers demonstrated that the ribose C3′ hydrogen atom is abstracted by the adenosyl radical. This led to a novel mechanistic proposal involving an intermediate with a bond between the C8 of guanine and C3′ of the ribose. This paper describes the use of 2′,3′-dideoxyGTP to trap this intermediate

High-resolution crystal structure of the eukaryotic HMP-P synthase (THIC) from Arabidopsis thaliana

Vitamin B1 is an essential compound in all organisms acting as a cofactor in key metabolic reactions. It is formed by the condensation of two independently biosynthesized molecules referred to as the pyrimidine and thiazole moieties. In bacteria and plants, the biosynthesis of the pyrimidine moiety, 4- amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P), requires a single enzyme, THIC (HMP-P synthase). The enzyme uses an iron–sulfur cluster as well as a 50-deoxyadenosyl radical as cofactors to rearrange the 5-amino-imidazole ribonucleotide (AIR) substrate to the pyrimidine ring. So far, the only structure reported is the one from the bacteria Caulobacter crescentus. In an attempt to structurally characterize an eukaryotic HMP-P synthase, we have determined the high-resolution crystal structure of THIC from Arabidopsis thaliana at 1.6 Å. The structure is highly similar to its bacterial counterpart although several loop regions show significant differences with potential implications for the enzymatic properties. Furthermore, we have found a metal ion with octahedral coordination at the same location as a zinc ion in the bacterial enzyme. Our high-resolution atomic model shows a metal ion with multiple coordinated water molecules in the close vicinity of the substrate binding sites and is an important step toward the full characterization of the chemical rearrangement occurring during HMP-P biosynthesis

Recombinant Macrocyclic Lanthipeptides Incorporating Non-Canonical Amino Acids

Nisin is a complex lanthipeptide that has broad spectrum antibacterial activity. In efforts to broaden the structural diversity of this ribosomally synthesized lantibiotic, we now report the recombinant expression of Nisin variants that incorporate noncanonical amino acids (ncAAs) at discrete positions. This is achieved by expressing the <i>nisA</i> structural gene, cyclase (<i>nisC</i>) and dehydratase (<i>nisB</i>), together with an orthogonal nonsense suppressor tRNA/aminoacyl-tRNA synthetase pair in Escherichia coli. A number of ncAAs with novel chemical reactivity were genetically incorporated into NisA, including an α-chloroacetamide-containing ncAA that allowed for the expression of Nisin variants with novel macrocyclic topologies. This methodology should allow for the exploration of lanthipeptide variants with new or enhanced activities

Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase

Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5′-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies