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

作者：Bott M; Pfister K; Burda P; Kalbermatter O; Woehlke G; Dimroth P

来源：European Journal of Biochemistry, 1997, 250(2): 590-599.

DOI：10.1111/j.1432-1033.1997.0590a.x

• methylmalonyl-CoA decarboxylase
• Propionigenium modestum
• mmdADCB genes
• biotin
• sodium-ion pump

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 K-m value for (S)-methylmalonyl-CoA of approximate to 12 mu M. 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.

• 出版日期1997-12-1