Abstract
Vitamin B12 acts as a cofactor for various metabolic reactions important in living organisms. The Vitamin B12 biosynthesis is restricted to prokaryotes, which means, all eukaryotic organisms must acquire this molecule through diet. This study presents the investigation of Vitamin B12 metabolism and the characterization of precorrin-4 C(11)-methyltransferase (CobM), an enzyme involved in the biosynthesis of Vitamin B12 in Corynebacterium pseudotuberculosis. The analysis of the C. pseudotuberculosis genome identified two Vitamin B12-dependent pathways, which can be strongly affected by a disrupted vitamin metabolism. Molecular dynamics, circular dichroism, and NMR-STD experiments identified regions in CobM that undergo conformational changes after s-adenosyl-L-methionine binding to promote the interaction of precorrin-4, a Vitamin B12 precursor. The binding of s-adenosyl-L-methionine was examined along with the competitive binding of adenine, dATP, and suramin. Based on fluorescence spectroscopy experiments the dissociation constant for the four ligands and the target protein could be determined; SAM (1.4 ± 0.7 µM), adenine (17.8 ± 1.5 µM), dATP (15.8 ± 2.0 µM), and Suramin (6.3 ± 1.1 µM). The results provide rich information for future investigations of potential drug targets within the C. pseudotuberculosis’s Vitamin B12 metabolism and related pathways to reduce the pathogen’s virulence in its hosts.
Highlights
Vitamin B12 acts as a cofactor for various metabolic reactions important in living organisms
This study presents the investigation of Vitamin B12 metabolism and the characterization of precorrin-4 C(11)-methyltransferase (CobM), an enzyme involved in the biosynthesis of Vitamin B12 in Corynebacterium pseudotuberculosis
Search in the GenBank-NCBI database revealed that all genes, with the exception of one (CobE), involved in the aerobic Vitamin B12 synthesis pathway are conserved in C. pseudotuberculosis strain 1002 and strain CIP52.97
Summary
Vitamin B12 acts as a cofactor for various metabolic reactions important in living organisms. Vitamin B12 participates as a coenzyme in various metabolic processes, such as the complex rearrangements and reductions through methylation[2] It functions as a cofactor of methyltranferases and is necessary for methionine synthesis, methanogenesis, CO2 fixation, and even in the regulation of bacterial gene expression, where it functions as a ligand of mRNA elements (riboswitches) for genetic control[2,3]. The enzymes that participate in the aerobic pathway contain the prefix Cob, whereas those of the anaerobic pathway are termed Cbi. The addition of eight S-adenosyl-L-methionine (SAM) methyl groups to the tetrapyrrole structure during the first part of Vitamin B12 biosynthesis is performed by six transmethylases, which are highly related in structure and in function. Besides the strong relation regarding the protein structure, a sequence alignment of the six transmethylases of C
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