Carbon assimilation by Pseudomonas oxalaticus (OXI). 4. Metabolism of oxalate in cell-free extracts of the organism grown on oxalate

Abstract

Reactions of oxalate, glyoxylate, formate and bicarbonate in cell-free extracts of Pseudomonas oxalaticus (OXI) grown on oxalate have been studied. The extracts catalyse a decarboxylation of oxalate to formate which is dependent on catalytic quantities of succinate, adenosine triphosphate, coenzyme A and thiamine pyrophosphate. Magnesium ions and reduced glutathione accelerate the reaction. Succinate, adenosine triphosphate and coenzyme A can be replaced by catalytic quantities of succinyl-coenzyme A. The extracts catalyse the formation of succinyl-coenzyme A from succinate but no similar direct activation of oxalate, formate or acetate has been detected. During the decarboxylation of oxalate under the above experimental conditions both oxalylhydroxamic acid and formylhydroxamic acid have been isolated on addition of hydroxylamine, indicating the formation of coenzyme A esters of oxalic acid and formic acid. It is suggested that oxalyl-coenzyme A is synthesized by trans-ferase action between succinyl-coenzyme A and oxalate. Oxalyl-coenzyme A is decarboxylated to formyl-coenzyme A, the coenzyme A grouping of which can be transferred back to oxalate either directly, or indirectly via succinate. Whole cells of P. oxalaticus oxidize both formate and oxalate. Oxidation of oxalate is preceded by a rapid evolution of carbon dioxide suggesting that the oxidation proceeds via formate. Cell-free extracts possess a diphosphopyridine nucleotide-linked formic de-hydrogenase but no pyridine nucleotide-linked oxalic dehydrogenase was found. Extracts catalyse a triphosphopyridine nucleotide- and coenzyme A-dependent oxidation of glyoxylate to oxalate. The initial reaction product is considered to be oxalyl-coenzyme A which is hydrolysed under the experimental conditions to oxalate. Indirect evidence indicates the reaction to be reversible, and it is suggested that this is the primary reduction reaction in the conversion of oxalate into cell constituents. The extracts incorporate [C14]bicarbonate into malate and citrate in the presence of pyruvate and reduced triphosphopyridine nucleotide. [C14] -Bicarbonate is incorporated mainly into malate and aspartate in the presence of phosphopyruvate; this fixation is stimulated by inosine diphosphate.