Autotrophic acetyl coenzyme A biosynthesis in Methanococcus maripaludis.
To detect autotrophic CO2 assimilation in cell extracts of Methanococcus maripaludis, lactate dehydrogenase and NADH were added to convert pyruvate formed from autotrophically synthesized acetyl coenzyme A to lactate. The lactate produced was determined spectrophotometrically. When CO2 fixation was pulled in the direction of lactate synthesis, CO2 reduction to methane was inhibited. Bromoethanesulfonate (BES), a potent inhibitor of methanogenesis, enhanced lactate synthesis, and methyl coenzyme M inhibited it in the absence of BES. Lactate synthesis was dependent on CO2 and H2, but H2 + CO2-independent synthesis was also observed. In cell extracts, the rate of lactate synthesis was about 1.2 nmol min-1 mg of protein-1. When BES was added, the rate of lactate synthesis increased to 2.3 nmol min-1 mg of protein-1. Because acetyl coenzyme A did not stimulate lactate synthesis, pyruvate synthase may have been the limiting activity in these assays. Radiolabel from 14CO2 was incorporated into lactate. The percentages of radiolabel in the C-1, C-2, and C-3 positions of lactate were 73, 33, and 11%, respectively. Both carbon monoxide and formaldehyde stimulated lactate synthesis. 14CH2O was specifically incorporated into the C-3 of lactate, and 14CO was incorporated into the C-1 and C-2 positions. Low concentrations of cyanide also inhibited autotrophic growth, CO dehydrogenase activity, and autotrophic lactate synthesis. These observations are in agreement with the acetogenic pathway of autotrophic CO2 assimilation.
D000105:Acetyl Coenzyme A / Q000096:biosynthesis*; D002245:Carbon Dioxide / Q000378:metabolism; D003486:Cyanides / Q000494:pharmacology; D019605:Euryarchaeota / Q000201:enzymology* / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007773:Lactates / Q000096:biosynthesis; D009243:NAD / Q000378:metabolism; D050743:Pyruvate Synthase; D011773:Pyruvates / Q000096:biosynthesis / Q000378:metabolism; D013053:Spectrophotometry
D003486:Cyanides; D007773:Lactates; D011773:Pyruvates; D009243:NAD; D002245:Carbon Dioxide; D000105:Acetyl Coenzyme A; D007770:L-Lactate Dehydrogenase; D007658:Ketone Oxidoreductases; D050743:Pyruvate Synthase