A study on the functional and genomic context of CobT in eukaryotes and the molecular factors governing its regioselectivity

Abstract

Vitamin B12, a cofactor belonging to the cobamide family is required by a myriad of organisms for catalyzing diverse sets of reactions but is believed to be synthesized only by a subset of prokaryotes. It is one of nature’s largest small molecules consisting of a tetrapyrrolic corrin ring,an upper ligand and a lower ligand 5,6 dimethyl benzimidazole (DMB), that is connected to the corrin ring via a nucleotide loop. NaMN: DMB phosphoribosyltransferase CobT is an enzyme that is involved in the activation of the lower ligand prior to its attachment to the corrin ring. The gene encoding CobT enzyme has been found present in some eukaryotes though they are thought to be incapable of synthesizing vitamin B12. In this study, we seek to determine the function of cobT in eukaryotes. As a first step, gene neighbourhood studies were carried out to look out for B12 related genes in the vicinity of CobT. Since eukaryotic B12 related genes were not present in clusters, subsequent studies involved using comparative genomic methods to search for these genes in the whole genome of organisms. We showed that a group of eukaryotes have genes associated with nucleotide loop assembly and therefore might be capable of partial cobamide biosynthesis. Previous studies revealed that the gene neighbourhood encoding different CobT homologs correlates with the ratio of the two regioisomers it forms. The second part of the study thus involved finding the molecular basis for the regioselective nature of CobT. To this end, specificity determining positions (SDPs) were identified and mutations were made on the SDPs of CobT. The mutant CobT was reconstituted in vitro and observed for changes in regioselectivity. In this way, key amino acids contributing to the regioselectivity of CobT were determined.