Biochemical characterization of the methylmercaptopropionate:cob(I)alamin methyltransferase fromMethanosarcina acetivorans

Abstract

ABSTRACTMethanogenesis from methylated substrates is initiated by substrate specific methyltransferases that generate the central metabolic intermediate methyl-coenzyme M. This reaction involves a methyl-corrinoid protein intermediate and one or two cognate methyltransferases. Based on genetic data, theMethanosarcina acetivoransMtpC (corrinoid protein) and MtpA (methyltransferase) proteins were suggested to catalyze the methylmercaptopropionate(MMPA):Coenzyme M (CoM) methyl transfer reaction without a second methyltransferase. To test this, MtpA was purified after overexpression in its native host and characterized biochemically. MtpA catalyzes a robust methyl transfer reaction using free methylcob(III)alamin as the donor and mercaptopropionate (MPA) as the acceptor, withkcatof 0.315 s-1and apparentKmfor MPA of 12 μM. CoM did not serve as a methyl acceptor, thus a second, unidentified methyltransferase is required to catalyze the full MMPA:CoM methyl transfer reaction. The physiologically relevant methylation of cob(I)alamin with MMPA, which is thermodynamically unfavorable, could also be demonstrated, but only at high substrate concentrations. Methylation of cob(I)alamin with methanol, dimethylsulfide, dimethylamine and methyl-CoM was not observed, even at high substrate concentrations. Although the corrinoid protein MtpC was poorly expressed alone, a stable MtpA/MtpC complex was obtained when both proteins were co-expressed. Biochemical characterization of this complex was not feasible because the corrinoid cofactor of this complex was in the inactive Co(II) state and could not be reactivated by incubation with strong reductants. The MtsF protein, comprised of both corrinoid and methyltransferase domains, co-purifies with the MtpA/MtpC, suggesting that it may be involved in MMPA metabolism.IMPORTANCEMMPA is an environmentally significant molecule produced by degradation of the abundant marine metabolite dimethylsulfoniopropionate, which plays a significant role in the biogeochemical cycles of both carbon and sulfur, with ramifications for ecosystem productivity and climate homeostasis. Detailed knowledge of the mechanisms for MMPA production and consumption is key to understanding steady state levels of this compound in the biosphere. Unfortunately, the biochemistry required for MMPA catabolism under anoxic conditions is poorly characterized. The data reported here validate the suggestion that the MtpA protein catalyzes the first step in methanogenic catabolism of MMPA. However, the enzyme does not catalyze a proposed second step required to produce the key intermediate methyl-CoM. Therefore, additional enzymes required for methanogenic MMPA catabolism await discovery.