| The diversity in the nucleoside base in cobamides (Cbas) is facilitated in part by the lack of substrate specificity of a family of enzymes called nicotinate mononucleotide (NaMN):base phosphoribosyltransferase (Pfam 02277, EC 2.4.2.21). These enzymes synthesize an unusual alpha-linked riboside monophosphate (alpha-ribotides) using benzimidazole, purine and phenolic aromatic bases with NaMN as the phosphoribosyl donor that are incorporated into cobamide biosynthesis. Before the work in this thesis, the Salmonella enterica homolog CobT was characterized to incorporate a number of benzimidazole and purine bases into alpha-ribotides. However, CobT cannot use phenolic base substrates. Phenolic Cbas were isolated in the acetogenic bacterium Sporomusa ovata and in this organism we isolated from a genomic library a heterodimeric homolog of CobT we named ArsAB. ArsAB not only incorporates phenolics, but also benzimidazole and purine bases into alpha-ribotides. Mutational studies and structures of variant CobT homologs (S. enterica and S. ovata) showed the contribution of two acidic residues in maintaining the enzyme in an active conformation and other key residues involved in substrate binding. This work led to the engineering of a S. enterica CobT variant that can synthesize alpha- p-cresolyl-ribotide. The research presented in this thesis expands our understanding in the mechanism of the CobT family of enzymes during adenosylcobamide biosynthesis. |
