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Piecing together Vitamin B-12

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recent paper from Dr Amrita Hazra’s group at IISER Pune offers new insights into the puzzle of how vitamin B-12, one of the largest ‘small molecules' made in nature, is pieced together. They do this by reconstituting the activity of two novel enzymes involved in B-12 synthesis and find that the enzymes methylate a part of the B-12 molecule in a site-specific manner. This paper has been selected as an Editor's Pick (July 31, 2020) by the journal.


Vitamin B-12 is an essential nutrient we get from animal products such as dairy and meat. As is the case for all vitamins, the human body cannot synthesize vitamin B-12. This vitamin, which is massive in size as compared to average small molecules, can be produced only by a subset of bacteria and archaea.


The structure of B-12 is similar to chlorophyll or heme and is composed of three main components: the tetrapyrrolic corrin ring with a cobalt ion at the center; a nucleotide loop; and two ligands, an upper and a lower ligand which are coordinated to cobalt. The lower ligand which also forms the base of the nucleotide loop can be a benzimidazole, purine, or phenol, and this chemical diversity of the lower ligand leads to a set of diverse family of biomolecules called the cobamides.


Note the three main components of the vitamin B-12 molecule (left). Anaerobic bacterium Moorella thermoacetica's bzaABC-cobT genes determine the production of components that go on to piece together vitamin B-12, in a substrate and region specific manner. The mechanism for the specificity is described in this paper by the Hazra group (Image Courtesy: Dr Amrita Hazra, IISER Pune)


“Like in an assembly line, different components of the cobamide molecule are synthesized independently and then assembled. The synthesis can take place either through a pathway that requires oxygen—the aerobic pathway, or through the one that occurs in the absence of oxygen—the anaerobic pathway,” explains Yamini Mathur, PhD student in Dr Amrita Hazra’s group.


“In our lab, we are studying the mechanism of enzymes on the recently discovered pathway for the anaerobic biosynthesis of cobamide lower ligands and their attachment to the corrin ring, says Dr Amrita Hazra. “We believe this will help us unravel new paradigms of enzyme catalysis and provide exciting insights in terms of biological chemistry and an important window into addressing disease,” she says.


The anaerobic biosynthesis pathway of the lower ligand involves the bza operon. Operon is a cluster of genes that is controlled together in a regulated manner through common structural elements (referred to as promoter and operator). The bza operon consists of six genes namely bzaABCDE and cobT. Together, these genes code for enzymes that produce four different benzimidazole derivatives which are derived by consecutive methylations, and each of them are found as lower ligands in anaerobic microorganisms.


A benzimidazole derivative, 5-methoxybenzimidazole (5-OMeBza), is produced as the lower ligand by Moorella thermoaceticaa bacterium that can live only in environments that lack oxygen, and which possesses bzaABC-cobT genes (Read more here and here).


Previous findings had predicted that the enzyme BzaC methylates the first intermediate 5-hydroxybenzimidazole (5-OHBza) to yield 5-OMeBza which then undergoes activation by the phosphoribosyltransferase-CobT for attachment to the corrin ring.


In this study, Yamini Mathur and three IISER Pune undergraduate students Sheryl Sreyas, Prathamesh Datar and Manjima Sathian reconstituted the activity of enzymes CobT and BzaC from the Moorella thermoacetica bza operon and probed their substrate specificity and regiospecificity.


The CobT enzyme is instrumental in determining the diversity of cobamides produced in microorganisms (more here and here). CobT’s role is to attach one of the two nitrogens of the lower ligand benzimidazole moiety to a ribose ring. In the case of asymmetric lower ligands such as 5-OMeBza and 5-OHBza, the CobT reaction can form two regioisomers of the activated lower ligands which in turn, can lead to formation of two different cobamides.


Extensive labelling studies conducted in 1980s established that the anaerobic lower ligand biosynthesis pathway is highly regisospecific and produces only one of the cobamides. However, the molecular basis for such unique regiospecificity remained unknown.


This present study reports that CobT from M. thermoacetica is the first of its known homologs to regiospecifically activate 5-OHBza to form 5-OHBza-ribotide. The paper also shows that BzaC from M. thermoacetica is a functional methyltransferase that preferentially methylates 5-OHBza-riboside, which is spontaneously formed from the CobT product 5-OHBza-ribotide. This results in the regiospecific activation of the lower ligand 5-OHBza before it is methylated, which is then likely attached to the corrinoid ring.


Thus, a missing piece of the cobamide biosynthesis puzzle—how the regiospecific biosynthesis of cobamide lower ligand is achieved—is explained by this study.


‘Through this study, we propose a revised pathway for the anaerobic biosynthesis of cobamides such as vitamin B-12. Further, this study has set up the stage for exploring the substrates and reactions involved in the anaerobic biosynthesis and assembly of diverse cobamide cofactors in microorganisms,” says Dr Hazra, indicating that this broadens the field and will help in understanding the mechanistic details of how the bza group of proteins affects the structure and function of molecules such as vitamin B-12.


This research received funding from DBT and DST-SERB.


Article Citation: CobT and BzaC catalyze the regiospecific activation and methylation of the 5-hydroxybenzimidazole lower ligand in anaerobic cobamide biosynthesis.  Yamini Mathur, Sheryl Sreyas, Prathamesh M Datar, Manjima B Sathian and Amrita B Hazra (2020) Journal of Biological Chemistry 295:10522-10534.


See also the Author Profile of the first author Yamini Mathur in the same issue. 


With inputs from Dr Amrita Hazra