Angad P. Mehta

Radical S-Adenosylmethionine (SAM) Enzymes in Cofactor Biosynthesis: A Treasure Trove of Complex Organic Radical Rearrangement Reactions

In this minireview, we describe the radical S-adenosylmethionine enzymes involved in the biosynthesis of thiamin, menaquinone, molybdopterin, coenzyme F420, and heme. Our focus is on the remarkably complex organic rearrangements involved, many of which have no precedent in organic or biological chemistry.

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.

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.

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.

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 nisA structural gene, cyclase (nisC) and dehydratase (nisB), 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.

IIdentification of the first Riboflavin Catabolic Gene Cluster isolated from Microbacterium maritypicum G10

Riboflavin is a common cofactor, and its biosynthetic pathway is well characterized. However, its catabolic pathway, despite intriguing hints in a few distinct organisms, has never been established. This article describes the isolation of a Microbacterium maritypicum riboflavin catabolic strain, and the cloning of the riboflavin catabolic genes. RcaA, RcaB, RcaD, and RcaE were overexpressed and biochemically characterized as riboflavin kinase, riboflavin reductase, ribokinase, and riboflavin hydrolase, respectively. Based on these activities, a pathway for riboflavin catabolism is proposed.