When [¹⁴C]propionate was incubated with a cell-free extract of Rhodospirillum rubrum in the presence of glyoxylate, ATP, CoA, Mg²⁺, and Mn²⁺, radioactivity was incorporated into mesaconate (MSA) as well as into β-methylmalate (MMA) and citramalate (CMA). MSA was suggested to be an intermediate of the conversion of MMA to CMA based on the following observations. (i) When non-labeled MSA was added to the CMA-forming reaction system, radioactivity was trapped in MSA. (ii) When MSA was incubated with the cell-free extract, CMA was formed. (iii) The α-carboxyl group of CMA was shown to be derived from the β-carboxyl group of MMA, implying that CMA was formed from MMA via MSA through successive dehydration and hydration. From the results of Sephadex G-10 column chro-matography of the reaction products, β-methylmalyl-CoA and mesaconyl-CoA were presumed to be involved in the reaction. A new CMA-forming metabolic pathway is proposed as follows:
erythro-β-methylmalyl-CoA→mesaconyl-CoA→MSA→L-CMA.