Methylmalonyl‐CoA Decarboxylase from Propionigenium Modestum

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Abstract
Methylmalonyl‐CoA decarboxylase catalyses the only energy‐conserving step during succinate fermentation by Propionigenium modestum: the decarboxylation of (S)‐methylmalonyl‐CoA to propionyl‐CoA is coupled to the vectorial transport of Na+ across the cytoplasmic membrane, thereby creating a sodium ion motive force that is used for ATP synthesis. By taking advantage of the sequence similarity between the β‐subunits of other Na+‐transport decarboxylases, a portion of the P.modestumβ‐subunit gene was amplified by PCR with degenerated primers. The cloned PCR product then served as homologous probe for cloning suitable fragments from genomic DNA. Sequence analysis of a 3.7‐kb region identified four genes which probably form a transcriptional unit, mmdADCB. Remarkably, a mmdE gene which is present in the homologous mmdADECB cluster from Veillonella parvula and encodes the 6‐kDa δ‐subunit, is missing in P.modestum. By sequence comparisons, the following functions could be assigned to the P.modestum proteins: MmdA (56.1 kDa; α‐subunit), carboxyltransferase; MmdB (41.2 kDa; β‐subunit), carboxybiotin‐carrier‐protein decarboxylase; MmdC (13.1 kDa; γ‐subunit), biotin carrier protein. MmdD (14.2 kDa; δ‐subunit) presumably is essential for the assembly of the complex, as shown for the corresponding V.parvula protein. Methylmalonyl‐CoA decarboxylase was solubilized from membranes of P.modestum with n‐dodecylmaltoside and enriched 15‐fold by affinity chromatography on monomeric avidin resin. The purified protein was composed of four subunits, three of which were identified by N‐terminal sequence analysis as MmdA, MmdD, and MmdC. The purified enzyme exhibited a specific activity of up to 25 U/mg protein and an apparent Km value for (S)‐methylmalonyl‐CoA of ≈ 12 μM. Compared to the five‐subunit complex of V.parvula, the four‐subunit enzyme of P.modestum appeared to be more labile, presumably a consequence of the lack of the ε‐subunit.