Guardian of cobamide diversity: Probing the role of CobT in lower ligand activation in the biosynthesis of vitamin B and other cobamide cofactors.

Enzymes catalyze a wide variety of reactions with exquisite precision under crowded conditions within cellular environments. When encountered with a choice of small molecules in their vicinity, even though most enzymes continue to be specific about the substrate they pick, some others are able to accept a range of substrates and subsequently produce a variety of products. The biosynthesis of Vitamin B, an essential nutrient required by humans involves a multi-substrate α-phosphoribosyltransferase enzyme CobT that activates the lower ligand of B. Vitamin B is a member of the cobamide family of cofactors which share a common tetrapyrrolic corrin scaffold with a centrally coordinated cobalt ion, and an upper and a lower ligand. The structural difference between B and other cobamides mainly arises from variations in the lower ligand, which is attached to the activated corrin ring by CobT and other downstream enzymes. In this chapter, we describe the steps involved in identifying and reconstituting the activity of new CobT homologs by deriving lessons from those previously characterized. We then highlight biochemical techniques to study the unique properties of these homologs. Finally, we describe a pairwise substrate competition assay to rank CobT substrate preference, a general method that can be applied for the study of other multi-substrate enzymes. Overall, the analysis with CobT provides insights into the range of cobamides that can be synthesized by an organism or a community, complementing efforts to predict cobamide diversity from complex metagenomic data.