Amrita B. Hazra

Cofactor biosynthesis – still yielding fascinating new biological chemistry

This mini review covers recent advances in the mechanistic enzymology of cofactor biosynthesis.

A “Radical Dance” in Thiamin Biosynthesis: Mechanistic Analysis of the Bacterial Hydroxymethylpyrimidine Phosphate Synthase

Thiamin pyrophosphate is an important cofactor in all forms of life, where it plays a central role in the stabilization of the acyl-carbanion biosynthon1, 2. Its biosynthesis involves separate synthesis of the thiazole and the pyrimidine hererocycles, which are then linked to form the cofactor. Thiamin-thiazole biosynthesis is relatively well-understood3-7. In prokaryotes, 1-deoxy-D-xylulose-5-phosphate, cysteine and glycine or tyrosine are utilized by five proteins to construct the thiazole moiety, whereas in Saccharomyces cerevisiae, just one gene product converts NAD and glycine to thiazole, obtaining sulfur from a source yet unknown. In comparison, the mechanistic understanding of thiamin-pyrimidine (HMP) biosynthesis, in both prokaryotes and eukaryotes, is still at an early stage. In yeast, a single gene product THI5p is implicated in HMP biosynthesis from PLP and histidine, however this reaction has not yet been successfully reconstituted in vitro. In bacteria and plants HMP-P synthase (ThiC) catalyzes the conversion of aminoimidazole ribonucleotide (AIR 1), an intermediate in the purine nucleotide biosynthesis pathway, to hydroxylmethyl pyrimidine phosphate (HMP-P 2)8. In vivo and in vitro studies on the reaction catalyzed by ThiC, using labeled AIR, have revealed the involvement of a rearrangement reaction of remarkable complexity (Figure 1A)9. The ThiC catalyzed reaction has recently been reconstituted in a defined biochemical system. Spectroscopic, structural and biochemical studies established this enzyme as a unique member of the [4Fe-4S] cluster dependent radical-SAM superfamily10-11.

Cobamide structure depends on both lower ligand availability and CobT substrate specificity.

Cobamides are members of the vitamin B12 family of cofactors that function in a variety of metabolic processes and are synthesized only by prokaryotes. Cobamides produced by different organisms vary in the structure of the lower axial ligand. Here we explore the molecular factors that control specificity in the incorporation of lower ligand bases into cobamides. We find that the cobT gene product, which activates lower ligand bases for attachment, limits the range of lower ligand bases that can be incorporated by bacteria. Furthermore, we demonstrate that the substrate specificity of CobT can be predictably altered by changing two active site residues. These results demonstrate that sequence variations in cobT homologs contribute to cobamide structural diversity. This analysis could open new routes to engineering specific cobamide production and understanding cobamide-dependent processes.

Mechanisms of direct inhibition of the respiratory sulfate-reduction pathway by (per)chlorate and nitrate.

We investigated perchlorate (ClO4−) and chlorate (ClO3−) (collectively (per)chlorate) in comparison with nitrate as potential inhibitors of sulfide (H2S) production by mesophilic sulfate-reducing microorganisms (SRMs). We demonstrate the specificity and potency of (per)chlorate as direct SRM inhibitors in both pure cultures and undefined sulfidogenic communities. We demonstrate that (per)chlorate and nitrate are antagonistic inhibitors and resistance is cross-inducible implying that these compounds share at least one common mechanism of resistance. Using tagged-transposon pools we identified genes responsible for sensitivity and resistance in Desulfovibrio alaskensis G20. We found that mutants in Dde_2702 (Rex), a repressor of the central sulfate-reduction pathway were resistant to both (per)chlorate and nitrate. In general, Rex derepresses its regulon in response to increasing intracellular NADH:NAD+ ratios. In cells in which respiratory sulfate reduction is inhibited, NADH:NAD+ ratios should increase leading to derepression of the sulfate-reduction pathway. In support of this, in (per)chlorate or nitrate-stressed wild-type G20 we observed higher NADH:NAD+ ratios, increased transcripts and increased peptide counts for genes in the core Rex regulon. We conclude that one mode of (per)chlorate and nitrate toxicity is as direct inhibitors of the central sulfate-reduction pathway. Our results demonstrate that (per)chlorate are more potent inhibitors than nitrate in both pure cultures and communities, implying that they represent an attractive alternative for controlling sulfidogenesis in industrial ecosystems. Of these, perchlorate offers better application logistics because of its inhibitory potency, solubility, relative chemical stability, low affinity for mineral cations and high mobility in environmental systems.

Anaerobic biosynthesis of the lower ligand of vitamin B12

Significance Vitamin B12 is required by humans and a variety of other organisms for diverse metabolic processes, but is produced only by a subset of microorganisms. The anaerobic biosynthesis of the “lower ligand” of B12, 5,6-dimethylbenzimidazole (DMB), is the only unknown component of the B12 biosynthetic pathway. We report the identification of the bzaABCDE genes that are necessary and sufficient for the anaerobic biosynthesis of DMB. We have characterized the role of each of the bza genes and identified three intermediates in the pathway. This finding not only completes the B12 biosynthesis pathway but also enables the sequence-based prediction of cobamides synthesized by anaerobic microorganisms. Vitamin B12 (cobalamin) is required by humans and other organisms for diverse metabolic processes, although only a subset of prokaryotes is capable of synthesizing B12 and other cobamide cofactors. The complete aerobic and anaerobic pathways for the de novo biosynthesis of B12 are known, with the exception of the steps leading to the anaerobic biosynthesis of the lower ligand, 5,6-dimethylbenzimidazole (DMB). Here, we report the identification and characterization of the complete pathway for anaerobic DMB biosynthesis. This pathway, identified in the obligate anaerobic bacterium Eubacterium limosum, is composed of five previously uncharacterized genes, bzaABCDE, that together direct DMB production when expressed in anaerobically cultured Escherichia coli. Expression of different combinations of the bza genes revealed that 5-hydroxybenzimidazole, 5-methoxybenzimidazole, and 5-methoxy-6-methylbenzimidazole, all of which are lower ligands of cobamides produced by other organisms, are intermediates in the pathway. The bza gene content of several bacterial and archaeal genomes is consistent with experimentally determined structures of the benzimidazoles produced by these organisms, indicating that these genes can be used to predict cobamide structure. The identification of the bza genes thus represents the last remaining unknown component of the biosynthetic pathway for not only B12 itself, but also for three other cobamide lower ligands whose biosynthesis was previously unknown. Given the importance of cobamides in environmental, industrial, and human-associated microbial metabolism, the ability to predict cobamide structure may lead to an improved ability to understand and manipulate microbial metabolism.

Analysis of Substrate Specificity in CobT Homologs Reveals Widespread Preference for DMB, the Lower Axial Ligand of Vitamin B12

Cobamides such as vitamin B12 (cobalamin) are produced exclusively by prokaryotes and used by many other organisms as cofactors for diverse metabolic processes. Cobamides are cobalt-containing tetrapyrroles with upper and lower axial ligands. The structure of the lower ligand varies in cobamides produced by different bacteria. We investigated the biochemical basis of this structural variability by exploring the reactivity of homologs of CobT, the enzyme responsible for activating lower ligand bases for incorporation into cobamides. Our results show that CobT enzymes can activate a range of lower ligand substrates, and the majority of the enzymes tested preferentially attach 5,6-dimethylbenzimidazole (DMB), the lower ligand of cobalamin. This suggests that many bacteria that synthesize cobamides other than cobalamin in pure culture may produce cobalamin in mixed communities by attaching DMB when it is available in the environment.

Thiamin pyrimidine biosynthesis in Candida albicans : a remarkable reaction between histidine and pyridoxal phosphate.

In Saccharomyces cerevisiae , thiamin pyrimidine is formed from histidine and pyridoxal phosphate (PLP). The origin of all of the pyrimidine atoms has been previously determined using labeling studies and suggests that the pyrimidine is formed using remarkable chemistry that is without chemical or biochemical precedent. Here we report the overexpression of the closely related Candida albicans pyrimidine synthase (THI5p) and the reconstitution and preliminary characterization of the enzymatic activity. A structure of the C. albicans THI5p shows PLP bound at the active site via an imine with Lys62 and His66 in close proximity to the PLP. Our data suggest that His66 of the THI5 protein is the histidine source for pyrimidine formation and that the pyrimidine synthase is a single-turnover enzyme.

A Missing Enzyme in Thiamin Thiazole Biosynthesis: Identification of TenI as a Thiazole Tautomerase.

In many bacteria tenI is found clustered with genes involved in thiamin thiazole biosynthesis. However, while TenI shows high sequence similarity with thiamin phosphate synthase, the purified protein has no thiamin phosphate synthase activity, and the role of this enzyme in thiamin biosynthesis remains unknown. In this contribution, we identify the function of TenI as a thiazole tautomerase, describe the structure of the enzyme complexed with its reaction product, identify the substrates phosphate and histidine 122 as the acid/base residues involved in catalysis, and propose a mechanism for the reaction. The identification of the function of TenI completes the identification of all of the enzymes needed for thiamin biosynthesis by the major bacterial pathway.

Biosynthesis of the thiamin thiazole in Bacillus subtilis: Identification of the product of the thiazole synthase-catalyzed reaction

In this paper, we describe an optimized reconstitution of the thiamin thiazole synthase (ThiG) catalyzed reaction and demonstrate that the enzymatic product is an unanticipated dearomatized thiazole tautomer.

Anaerobic 5-Hydroxybenzimidazole Formation from Aminoimidazole Ribotide: An Unanticipated Intersection of Thiamin and Vitamin B12 Biosynthesis

Comparative genomics of the bacterial thiamin pyrimidine synthase (thiC) revealed a paralogue of thiC (bzaF) clustered with anaerobic vitamin B12 biosynthetic genes. Here we demonstrate that BzaF is a radical S-adenosylmethionine enzyme that catalyzes the remarkable conversion of aminoimidazole ribotide (AIR) to 5-hydroxybenzimidazole (5-HBI). We identify the origin of key product atoms and propose a reaction mechanism. These studies represent the first step in solving a long-standing problem in anaerobic vitamin B12 assembly and reveal an unanticipated intersection of thiamin and vitamin B12 biosynthesis.