Autotrophic synthesis of activated acetic acid from CO₂ in Methanobacterium thermoautotrophicum

Abstract

The synthesis of acetyl-CoA from C02, H2, and various C1 compounds was studied in vitro with extracts and with protein fractions of Methanobacterium thermoautotrophicum. Acetyl-CoA synthesis from C 0 2 and H2 by extracts required C 0 2 reduction to CH4 to proceed. Both processes were highly stimulated by formaldehyde which served as the carbon precursor of both CH4 and the CH3 group of acetate. Carbon monoxide in combination with formaldehyde dramatically stimulated the acetyl-CoA synthesis up to 1 %-fold. In t h s system, which did not require C 0 2 reduction to the formaldehyde and CO level, acetyl-CoA synthesis was no longer dependent on CH4 formation. The soluble (100000 x g supernatant) cell protein was resolved into a protein fraction [45-60% (NH4),S04-fraction] which catalyzed acetyl-CoA synthesis at a specific rate of 15 nmol . min-' . (equivalent of mg cell protein)-' (60 "C). This oxygen-sensitive enzyme reaction required dithioerythritol for activity and was strictly dependent on (a) coenzyme A, (b) CO, and (c) N5,N"-methylene tetrahydromethanopterin, N5-methyl tetrahydromethanopterin or formaldehyde plus tetrahydromethanopterin. The incorporation of formaldehyde is explained by the spontaneous formation of methylene tetrahydromethanopterin. The product of the reaction, acetyl-CoA, was quantitatively derived from CO (carboxyl of acetate) and a C1 derivative of tetrahydromethanopterin (methyl of acetate). The C1 derivative of tetrahydromethanopterin could not be replaced by a C1 derivative of tetrahydrofolate or by methyl-coenzyme M; ATP was not required. The active protein fraction contained CO dehydrogenase and at least one corrinoid protein. These results provide strong biochemical arguments for the proposed mechanism of autotrophic acetyl-CoA synthesis in Methanobacterium.Methanogenic bacteria are strict anaerobic archaebacteria which in general are able to obtain energy simply from the reduction of C 0 2 with H2 (C02 + 4 H2 --t CH4 + 2 H20).Most species grow autotrophically, i.e. in addition they use CO, as sole carbon compound for biosyntheses [I]. In Methanobacterium thermoautotrophicum, a thermophilic microorganism [2], C 0 2 assimilation does not proceed via the pentose phosphate cycle (Calvin cycle) [3 -81. Rather, a novel non-cyclic pathway for a de novo synthesis of acetyl-coenzyme A from 2 C 0 2 + 4 H2 was established termed the 'acetylCoA pathway' [8 -1 I]. This process was found to have many features in common with acetate synthesis from CO, in the acetogenic bacterium Clostridium thermoaceticum (for reviews see [12-141. C. thermoaceticum ferments hexoses to acetates: two acetate molecules are generated from two pyruvate molecules and one from (20,. The validity of this model was questioned when carbon monoxide was found to replace pyruvate as C 0 2 donor, and acetylCoA was shown to be the reaction product [16]. Studies by the laboratory of Thauer [17 -201 had indicated that the nickel enzyme carbon monoxide dehydrogenase is involved in this process. It is now postulated that CO dehy...

Synthesis of acetyl coenzyme A by carbon monoxide dehydrogenase complex from acetate-grown Methanosarcina thermophila

“…It is proposed that CODH catalyzes the synthesis of acetyl-CoA during growth of methanogens on one-carbon substrates (4,10,13). CODH activity is present in methanolgrown M. thermophila (27), but it is not known whether the CODH complex described here is required for acetyl-CoA synthesis for cell carbon.…”

“…Cell suspensions of acetate-grown Methanosarcina barkeri synthesize acetate from CH3I, CO2, and a reductant (14) and catalyze exchange of the carbonyl group of acetyl-CoA with CO2 (7), suggesting that the CODH from this acetotrophic methanogen catalyzes both the synthesis and the cleavage of acetyl-CoA. Methanogens grown on one-carbon substrates also contain CODH, which is postulated to catalyze the synthesis of acetyl-CoA for cell carbon (4,10,13,23). However, neither the synthesis nor the cleavage of acetyl-CoA has been demonstrated with any of the CODHs purified from methanogens (4,9,12,27).…”

Pseudoauxotrophy of Methanococcus voltae for acetate, leucine, and isoleucine

“…CH20 reacts chemically with tetrahydromethanopterin to form methylenetetrahydromethanopterin (7). Under a H2 atmosphere, methylenetetrahydromethanopterin is converted to methyltetrahydromethanopterin, which is a methyl donor for the C-2 of acetyl-CoA (14,19). CO reacts with CO dehydrogenase, which is the donor for the C-1 of acetyl-CoA (19,22).…”

“…The biosynthesis of acetyl-CoA and CO2 reduction to methane share a common C1 pool (21). The methyl group of the acetate is derived from methyltetrahydromethanopterin, an intermediate in methanogenesis (7,14,19,30). Thus, the syntheses of acetate and methanogenesis are linked processes.…”

Folic acid and pteroylpolyglutamate contents of archaebacteria

“…Methanogens contain extremely low levels of folic acid activity and tetrahydrofolate-dependent enzymes (9,21). Traces of folate activity have been attributed to a hydrolytic product of methanopterin (a folate analog) which functions as a one-carbon carrier (8,12,20,21,33).…”