One-Carbon Metabolism in Methanogens: Evidence for Synthesis of a Two-Carbon Cellular Intermediate and Unification of Catabolism and Anabolism in Methanosarcina barkeri

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Abstract
One-carbon metabolic transformations associated with cell C synthesis and methanogenesis were analyzed by long- and short-term 14CH3OH or 14CO2 incorporation studies during growth and by cell suspensions. 14CH3OH and 14CO2 were equivalently incorporated into the major cellular components (i.e., lipids, proteins and nucleic acids) during growth on H2-CO2-methanol. 14CH3OH was selectively incorporated into the C-3 of alanine with decreased amounts fixed in the C-1 and C-2 positions, whereas 14CO2 was selectively incorporated into the C1 moiety with decreasing amounts assimilated into the C-2 and C-3 atoms. Notably, 14CH4 and [3-14C]alanine synthesized from 14CH3OH during growth shared a common specific activity distinct from that of CO2 or methanol. Cell suspensions synthesized acetate and alanine from 14CO2. The addition of iodopropane inhibited acetate synthesis but did not decrease the amount of 14CH3OH or 14CO2 fixed into 1-carbon carriers (i.e., methyl coenzyme M or carboxydihydromethanopterin). Carboxydihydromethanopterin was only labeled from 14CH3OH in the absence of hydrogen. Cell extracts catalyzed the synthesis of acetate from 14CO (.apprx. 1 nmol/min per mg of protein) and an isotopic exchange between CO2 or CO and the C-1 of pyruvate. Acetate synthesis from 14CO was stimulated by methyl B12 but not by methyl tetrahydrofolate or methyl coenzyme M. Methyl coenzyme M and coenzyme M were inhibitory to acetate synthesis. Cell extracts contained high levels of phosphotransacetylase (> 6 .mu.mol/min per mg of protein) and acetate kinase (> 0.14 .mu.mol/min per mg of protein). It was not possible to distinguish between acetate and acetyl CoA as the immediate product of 2-carbon synthesis with the methods employed.