A study into the cobalamin-dependent methionine synthase reactivation mechanism in corynebacterium glutamicum

Abstract

Methionine is one of the seven essential amino-acids for humans and is also used in feed supplements, predominantly for poultry and felines. Thus there is commercial interest in producing this nutrient. The final step in the biological synthesis of methionine is the addition of a methyl group to the sulphur of homocysteine; two enzymes can perform this step. They are referred to as the cobalamin-dependent and cobalamin-independent enzymes. Cobalamin-dependent Methionine Synthase (cdMS) catalyzes the transfer of a methyl group from methyl-tetrahydrofolate (MTHF) via the (corrin-bound) cobalt of cobalamin to homocysteine to form methionine and tetrahydrofolate. During the catalytic cycle of the B12-dependent methionine synthase the cobalt ion shunts between Co1+ and Co3+ states, but occasionally the cobalt ion becomes derailed due to oxidative stress to the Co2+ state. The bacterium of interest in this study, Corynebacterium glutamicum, possesses a genome which encodes genes for both a cobalamin independent and cobalamin dependent methionine synthase. However, it does not possess a methionine synthase reductase (MSR) remotely similar to those of Escherichia coli or Homo sapiens, both of which utilise a flavin to reduce the Co2+ centrally-ligated cobalt of cobalamin to Co1+ during reactivation. The investigations into this unknown methionine synthase reductase lead to the identification of three ferredoxins, FdxA, B and C, of which FdxC was identified as a potential MSR by complementation studies. This ferredoxin is unlike any encoded for by E. coli and further lead to the identification of a ferredoxin reductase in C. glutamicum which was capable of reducing the highly electronegative 2[4Fe4S] clusters contained within itself and FdxA, which are highly similar. Further assay-based studies by HPLC-MS, HPLC-MS/MS and EPR revealed that despite their similarities, only FdxC could act in the role of MSR with any great efficacy.