SUBSTRATE SPECIFICITY OF NUCLEOTIDE LOOP ASSEMBLY PATHWAY ENZYMES IN VITAMIN B12 BIOSYNTHESIS

Abstract

Vitamin B12 belongs to a class of cofactors referred to as cobamides. Structural variations amongst cobamides are introduced via the lower ligand. Although cobamides are cofactors required by organisms from all domains of life, they can only be produced by a small subset of microbes. As all microbes that utilise cobamides cannot produce them, cobamides become important shared nutrients in microbial communities. Different microbes produce and preferentially use cobamides with different lower ligands which allows cobamides to affect the structure of microbial communities. In this study, we try to understand whether this preference for certain lower ligands over others can be observed in the proteins involved in the biosynthesis of cobamides. In order to do so, we look at the proteins that are involved specifically in the portion of the biosynthetic pathway that deals with the attachment of the lower ligand, that is, the Nucleotide Loop Assembly Pathway. We have performed in vivo studies with protein orthologs from four different organisms heterologously expressed in Escherichia coli under conditions where the salvaging of cobamide precursors and synthesis of a complete cobamide using the heterologously expressed protein was crucial to the survival of the microbe. Differences observed in either the final biomass, based on OD600 readings, or the growth rate, based on fitting growth curve data with Gompertz model, when cells expressing different protein orthologs were grown in the presence of the same lower ligand indicate that the orthologs may have different preferences for the given lower ligands.