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Eur J Biochem. 1997 Dec 1;250(2):590-9.

Methylmalonyl-CoA decarboxylase from Propionigenium modestum--cloning and sequencing of the structural genes and purification of the enzyme complex.

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Mikrobiologisches Institut der Eidgenössischen Technischen Hochschule Zürich, Switzerland.


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 beta-subunits of other Na+-transport decarboxylases, a portion of the P. modestum beta-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 epsilon-subunit, is missing in P. modestum. By sequence comparisons, the following functions could be assigned to the P. modestum proteins: MmdA (56.1 kDa; alpha-subunit), carboxyltransferase; MmdB (41.2 kDa; beta-subunit), carboxybiotin-carrier-protein decarboxylase; MmdC (13.1 kDa; gamma-subunit), biotin carrier protein. MmdD (14.2 kDa; delta-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 approximately 12 microM. 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 epsilon-subunit.

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