Files
Abstract
Cobamides are used as cofactors by diverse enzymes across all domains of life to catalyze numerous reactions including carbon skeleton rearrangements, methylations and eliminations. Cobamides are modified tetrapyrroles, and belong to a family of coenzymes known as the “pigments of life” which includes chlorophylls, bilins, heme and coenzyme F430. As such, cobamides are one of the most complex molecules produced in nature, and are composed of a cobalt-containing cyclic tetrapyrrole with an upper (Co) and lower (Co) ligand. Perhaps one of the best-known cobamides is coenzyme B12, or adenosylcobalamin, which critical to numerous metabolic reactions including methionine synthesis, methanogenesis, and catabolism of ethanolamine and 1,2-propanediol. De novo biosynthesis of coenzyme B12 requires approximately 30 enzymes, and is only performed by some prokaryotes. Two different pathways have been defined for synthesis of the central corrin ring, in which the Co+2 may be inserted “early” or “late” in the pathway. These pathways are further differentiated by a requirement for molecular oxygen in the “late” insertion pathway, while the “early” insertion pathway occurs under anaerobic conditions. Once the corrin ring is formed, the axial ligands are attached. The final steps of biosynthesis include the activation and attachment of the lower ligand through a process known as nucleotide loop assembly (NLA). Many prokaryotes are incapable of de novo corrin ring biosynthesis, but are capable of salvaging ring precursors into a complete coenzyme using this pathway.
In this work, we investigate the biosynthesis and use of coenzyme B12 in a number of prokaryotes. We identified a metabolosome-free system for ethanolamine utilization in Acinetobacter baumannii. We investigate the interactions between the NLA enzymes CobU and the integral membrane protein CobS, and propose that the cis bond formed between Glu80 and Cys81 of CobU is involved in facilitating this interaction. We further characterize CbiS, a fusion protein identified in Methanopyrus kandleri that catalyzes both amidohydrolase and phosphatase reactions necessary for precursor salvaging in archaea. This work expands our understanding of enzymes that make and require cobamides in multiple prokaryotic species.