The Synthesis of One‐Carbon Units from CO₂ in Clostridium kluyveri

Abstract

Clostridium kluyveri was grown (a) on standard media, in which alternately [14C]bicarbonate, [U-14C], [1-14C], and 2-14C]acetate plus ethanol were the labeled substrates, and (b) on standard media supplemented with either formate, glycine or serine, in which bicarbonate or formate were 14C-labeled. Adenine, guanine and methionine were isolated from the cells and degraded.(a) Carbon atoms 2 and 8 of the purines were found to be synthesized from CO, (about 70 O/,) and from the methyl group (about 2O0lO) and the carboxyl group (about loo/,) of acetate. The X-methyl group of methionine was shown to be derived almost equally from CO, (about 5O0iO) and from the two-carbon substrates, the methyl group of acetate accounting for about 3501, and the carboxyl group for about 15 ,lo.(b) The participation of CO, in the synthesis of the X-methyl group of methionine remained unaffected (about SO0/,) in the presence of serine, while it was decreased to below 5 0 / , in the presence of formate and increased to over 80 in the presence of glycine. The participation of formate was in the order of 55 01,.These data demonstrate that (1) two major competitive pathways for the synthesis of onecarbon units exist in Clostridium kluyveri : C,-Units are formed predominantly by a reduction of CO, and to a minor degree by a cleavage of serine, the usual pathway ((3,-serine = C,-acetate) and (2) the relative rates of the two pathways are controlled by the glycine requirement of the growing organism. [l] is a strict anaerobe growing on synthetic media with ethanol, acetate, and bicarbonate as the sole energy-and carbonsubstrates [2-41. Clostridium IcluyveriIts carbon assimilation is characterized by a high net CO, fixation. About 30°/, of the cell carbon is derived from CO,, about 70°/, from acetate [5]. It has been found that the reductive, ferredoxin-dependent carboxylation of acetyl CoA to pyruvate [6] leads to the synthesis of both amino acids [7-91 and carbohydrates [9-111. I n this paper it will be shown that in Clostridium kluyveri CO, and acetate are also incorporated into the X-methyl group of methionine and into the positions 2 and 8 of purines, CO, being the major, acetate the minor source of one carbon units. The CO, assimilation is unexpected, as normally the hydroxymethyl group of serine and the aminomethyl group of glycine, which in Clostridium kluyveri are derived from the methyl-and carboxyl group of acetate [7], are the only precursors of reduced one carbon units.Studies on the "CO," and the "acetate" pathway and their regulation will be presented.

13C‐NMR study of autotrophic CO2 fixation pathways in the sulfur‐reducing Archaebacterium Thermoproteus neutrophilus and in the phototrophic Eubacterium Chloroflexus aurantiacus

“…Acetate feeding experiments afforded 3-hydroxypropionate in which the carboxyl C1 was mainly derived from CO,, C2 mainly from the methyl of acetate. and C3 mainly from the carboxyl of acetate [37]. This labelling would be consistent with acetyl-CoA carboxylation to malonyl-CoA and its reductive conversion to 3-hydroxypropionate.…”

“…These C1 units could originate from C3 of serine or from C2 of glycine. It is also conceivable that formate would be incorporated via formyl tetrahydrofolate synthetase [37]. Since glycine was labelled it follows that C1 of succinate was incorporated into glycine.…”

CO2‐reduction to formate by NADPH. The initial step in the total synthesis of acetate from CO2 in Clostridium thermoaceticum

“…In the anabolism of C. kluyveri [2,3] and of C'. pasteurianum [4] CO* conversion to formate is a key step in the formation of the S-methyl group of methionine and the CZ and Cs positions of the purines.…”

FAD Is Covalently Attached to Peptidyl‐tRNA during Cell‐free Synthesis of 6‐Hydroxy‐d‐nicotine Oxidase

“…. ' At the end of growth, the cells were harvested and adenine isolated as reported earlier [18]. Its concentration was determined spectrophotometrically using a value of ~2 6 0 of 15 800 cm2 x mol-' and its radioactivity by liquid scintillation counting.…”