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Chemically similar yet functionally distinct: Selectivity of methylmalonyl-CoA mutase among naturally diverse cobamide cofactors


Vitamin B12 is predominantly associated with human health, and many people are surprised to learn that B12 is produced by bacteria. Like humans, bacteria have metabolic enzymes that require B12 as a cofactor. B12-dependent enzymes catalyze chemical reactions required for DNA, amino acid, and secondary metabolite synthesis, as well as for use of various carbon and energy sources. Curiously, in addition to producing B12, bacteria and archaea produce and use chemical analogs of B12 called cobamides, which share the key structural features of B12 but have small chemical differences in a part of the molecule called the lower ligand. Differences in lower ligand structure, while small, are sufficient to make cobamides functionally distinct. Microorganisms are known to prefer certain cobamides over others for growth, which is ecologically important and could also be harnessed as a way to promote or inhibit the growth of specific organisms as an alternative to probiotics or antibiotics. However, the biochemical mechanisms that drive differential cobamide use in bacteria are not understood sufficiently to pursue such an application. Moreover, the response of humans to the diverse cobamides produced by microorganisms has not been fully explored.

I have used a combination of in vitro biochemistry and bacterial growth assays to dissect the functional differences between cobamides. The central hypothesis of this work is that a major determinant of the specific cobamide requirements of an organism is the ability of its cobamide-dependent enzymes to use diverse cobamides as cofactors. To better understand the mechanisms by which enzymes are affected by lower ligand structure, I selected a model enzyme, methylmalonyl-CoA mutase (MCM), which is one of the two cobamide-dependent enzymes in humans and is also relatively widespread in bacteria. Using bacterial MCM orthologs from Sinorhizobium meliloti, Escherichia coli, and Veillonella parvula, I found that the major effect of changes in lower ligand structure is alteration of the binding affinity of cobamides for MCM, with smaller effects on enzyme activity. I observed different cobamide-binding selectivity in MCM from different bacteria, which correlated with the cobamides produced by each respective organism, consistent with potential coevolution of cobamide production and use in cobamide-producing bacteria. Importantly, the cobamide-dependent growth of S. meliloti was largely consistent with the cobamide selectivity of the MCM enzyme in this organism, supporting the hypothesis that enzyme selectivity is an important mechanism by which cobamides differentially affect bacterial growth.

In addition to examining bacterial MCM orthologs, I analyzed the ability of human MCM to use cobamides with diverse lower ligands. It is assumed that humans require exclusively B12, but prior to this work the ability of human MCM to use diverse cobamides had not been tested. I found that, in fact, human MCM is able to bind to, and is catalytically active with, several cobamides other than B12, suggesting that other cobamides could be relevant for human physiology. Having discovered this, I tested whether any cobamides besides cobalamin improved the activity of six MCM variants containing disease-associated mutations. Although I did not observe any significant rescue of activity in the mutated MCM enzymes, I did find that, like the WT enzyme, multiple cobamides supported activity of these mutants, reinforcing the potential for application of diverse cobamides in studies of human cobalamin metabolism.

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