Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 295, Issue 31, Pages 10522-10534Publisher
ELSEVIER
DOI: 10.1074/jbc.RA120.014197
Keywords
vitamin B-12; cobamide; CobT; phosphoribosyltransferase; BzaC; methyltransferase; 5-hydroxybenzimidazole; 5-methoxybenzimidazole; 5; 6-dimethylbenzimidazole; Moorella thermoacetica; enzyme; microbiology; S-adenosylmethionine (SAM); substrate specificity; adenosylcobalamin (AdoCbl); benzimidazoles
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Funding
- Department of Science and Technology (DST), Ministry of Science and Technology, India -Science and Engineering Research Board Early Career Research Award [ECR/2016/000466]
- Department of Biotechnology -Ramalingaswami Re-entry Fellowship
- Council for Scientific and Industrial Research, India
- National Science Foundation
- DST-Kishor Vaigyanik Protsahan Yojana fellowship
- DST-INSPIRE fellowships
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Vitamin B(12)and other cobamides are essential cofactors required by many organisms and are synthesized by a subset of prokaryotes via distinct aerobic and anaerobic routes. The anaerobic biosynthesis of 5,6-dimethylbenzimidazole (DMB), the lower ligand of vitamin B-12, involves five reactions catalyzed by thebzaoperon gene products, namely the hydroxybenzimidazole synthase BzaAB/BzaF, phosphoribosyltransferase CobT, and three methyltransferases, BzaC, BzaD, and BzaE, that conduct three distinct methylation steps. Of these, the methyltransferases that contribute to benzimidazole lower ligand diversity in cobamides remain to be characterized, and the precise role of thebzaoperon protein CobT is unclear. In this study, we used thebzaoperon from the anaerobic bacteriumMoorella thermoacetica(comprisingbzaA-bzaB-cobT-bzaC) to examine the role of CobT and investigate the activity of the first methyltransferase, BzaC. We studied the phosphoribosylation catalyzed byMtCobT and found that it regiospecifically activates 5-hydroxybenzimidazole (5-OHBza) to form the 5-OHBza-ribotide (5-OHBza-RP) isomer as the sole product. Next, we characterized the domains ofMtBzaC and reconstituted its methyltransferase activity with the predicted substrate 5-OHBza and with two alternative substrates, theMtCobT product 5-OHBza-RP and its riboside derivative 5-OHBza-R. Unexpectedly, we found that 5-OHBza-R is the most favoredMtBzaC substrate. Our results collectively explain the long-standing observation that the attachment of the lower ligand in anaerobic cobamide biosynthesis is regiospecific. In conclusion, we validateMtBzaC as a SAM:hydroxybenzimidazole-riboside methyltransferase (HBIR-OMT). Finally, we propose a new pathway for the synthesis and activation of the benzimidazolyl lower ligand in anaerobic cobamide biosynthesis.
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