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

Abstract

One-carbon metabolic transformations associated with cell carbon synthesis and methanogenesis were analyzed by long- and short-term 14 CH 3 OH or 14 CO 2 incorporation studies during growth and by cell suspensions. 14 CH 3 OH and 14 CO 2 were equivalently incorporated into the major cellular components (i.e., lipids, proteins, and nucleic acids) during growth on H 2 -CO 2 -methanol. 14 CH 3 OH was selectively incorporated into the C-3 of alanine with decreased amounts fixed in the C-1 and C-2 positions, whereas 14 CO 2 was selectively incorporated into the C 1 moiety with decreasing amounts assimilated into the C-2 and C-3 atoms. Notably, 14 CH 4 and [3- 14 C]alanine synthesized from 14 CH 3 OH during growth shared a common specific activity distinct from that of CO 2 or methanol. Cell suspensions synthesized acetate and alanine from 14 CO 2 . The addition of iodopropane inhibited acetate synthesis but did not decrease the amount of 14 CH 3 OH or 14 CO 2 fixed into one-carbon carriers (i.e., methyl coenzyme M or carboxydihydromethanopterin). Carboxydihydromethanopterin was only labeled from 14 CH 3 OH in the absence of hydrogen. Cell extracts catalyzed the synthesis of acetate from 14 CO (∼1 nmol/min per mg of protein) and an isotopic exchange between CO 2 or CO and the C-1 of pyruvate. Acetate synthesis from 14 CO was stimulated by methyl B 12 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.