Demonstration of carbon-carbon bond cleavage of acetyl coenzyme A by using isotopic exchange catalyzed by the CO dehydrogenase complex from acetate-grown Methanosarcina thermophila

Abstract

The purified nickel-containing CO dehydrogenase complex isolated from methanogenic Methanosarcina thermophila grown on acetate is able to catalyze the exchange of [1-14C] acetyl-coenzyme A (CoA) (carbonyl group) with 12CO as well as the exchange of [3'-32P]CoA with acetyl-CoA. Kinetic parameters for the carbonyl exchange have been determined: Km (acetyl-CoA) = 200 ,uM, Vmax = 15 min-'. CoA is a potent inhibitor of this exchange (K1 = 25 ,uM) and is formed under the assay conditions because of a slow but detectable acetyl-CoA hydrolase activity of the enzyme. Kinetic parameters for both exchanges are compared with those previously determined for the acetyl-CoA synthase/CO dehydrogenase from the acetogenic Clostridium thermoaceticum. Collectively, these results provide evidence for the postulated role of CO dehydrogenase as the key enzyme for acetyl-CoA degradation in acetotrophic bacteria.Acetogens and methanogens possess novel pathways for the synthesis of acetyl-coenzyme A (acetyl-CoA) from single carbon units which allow these organisms to grow on CO2 as the sole carbon source (5). Other strains of methanogenic bacteria such as Methanosarcina species are able to grow on analogous Cl feedstocks including methanol and methylamine by using variations of this same pathway for carbon assimilation. The key enzyme in these cases is carbon monoxide dehydrogenase, a complex nickel enzyme so named for its ability to catalyze the reduction of CO2 to CO. However, the enzyme has been implicated in a number of cases as the catalyst for the carbon-carbon bond formation from suitable methyl donors and enzymatically generated CO from CO2 to give acetyl units.In the acetogenic Clostridium thermoaceticum, one molecule of CO2 is reduced to formate and formate is reduced to a methyl group by using tetrahydrofolate as a carrier; enzymatic transfer to a corrinoid iron/sulfur protein yields the methylated organocobalt species (19). Nickel-containing CO dehydrogenase (more appropriately called acetyl-CoA synthase) then acts as the site of carbon-carbon bond coupling by accepting the methyl group and catalyzing its carbonylation in the presence of CoA to give acetyl-CoA as product. The enzyme from C. thermoaceticum is the most well characterized acetogenic enzyme containing 2 Ni, 11 Fe, and 1 Zn atoms per active site; there is some discussion about the subunit composition of the functional complex as isolated: c433 [

Methane from acetate

“…After cleavage, the methyl group is transferred to the Co"+ atom of the Co/Fe-S component. The M. thermophila enzyme complex catalyzes an exchange of CoA with acetylCoA at rates fivefold greater than that of C. thermoaceticum acetyl-CoA synthase (46). The difference in rates may reflect the acetyl-CoA cleavage function for the M. thermophila enzyme, as opposed to the C. thermoaceticum enzyme which functions in the biosynthesis of acetyl-CoA.…”

“…In M. thermophila, a COOH enzyme complex cleaves the C-C and C-S bonds of acetyl-CoA, as demonstrated by an exchange of CO with the carbonyl group of acetyl-CoA (46). The complex also oxidizes CO to CO2 and, therefore, is referred to as a CODH enzyme complex; however, the primary function is cleavage of acetyl-CoA during growth on *CH #COO- acetate.…”

Evidence for Intersubunit Communication during Acetyl-CoA Cleavage by the Multienzyme CO Dehydrogenase/Acetyl-CoA Synthase Complex from Methanosarcina thermophila

“…5 and 6 for review). The acetyl-CoA/CoA exchange reaction of the Methanosarcina barkeri (1,7) and Methanosarcina thermophila (8) CODH/ACS complex also has been studied in great detail. The CO/acetyl-CoA exchange activity of the CODH/ACS complex also has been previously studied (8).…”

Identification of a cysteine involved in the interaction between carbon monoxide dehydrogenase and corrinoid/Fe‐S protein from Clostridium thermoaceticum

“…It has been recently demonstrated that the CODH:corrinoid complex from Methanosarcina thermophila synthesizes acetyl-CoA from methyl iodide, CO and CoA [16,17]. Further, Raybuck et al [18] have shown that a similar complex from M. thermophila also catalyzes the acetyl-CoA-CO exchange as does CODH from Clostridium thermoaceticum. Our present findings clearly provide evidence for the existence of a functionally active complex of CODH and corrinoid in Clostridium thermoaceticum as in certain methanogens.…”