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

摘要

One-carbon metabolic transformations associated with cell carbon 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 one-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 (∼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 μmol/min per mg of protein) and acetate kinase (>0.14 μmol/min per mg of protein). It was not possible to distinguish between acetate and acetyl coenzyme A as the immediate product of two-carbon synthesis with the methods employed.