Publications

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

  Bott M., Pfister K., Burda P., Kalbermatter O., Woehlke G., Dimroth P.

  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 ... >> More

  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. << Less

  Eur. J. Biochem. 250:590-599(1997) [PubMed] [EuropePMC]

• Conversion of the chemical energy of methylmalonyl-CoA decarboxylation into a Na+ gradient.

  Hilpert W., Dimroth P.

  Nature 296:584-585(1982) [PubMed] [EuropePMC]

• The carboxyltransferase activity of the sodium-ion-translocating methylmalonyl-CoA decarboxylase of Veillonella alcalescens.

  Hoffmann A., Hilpert W., Dimroth P.

  Methylmalonyl-CoA decarboxylase of Veillonella alcalescens catalyzed the isotopic exchange between methylmalonyl-CoA and [1-14C]propionyl-CoA or between malonyl-CoA and [1-14C]acetyl-CoA. The exchange was independent of sodium ions and was abolished by avidin. The enzyme also catalyzed the carboxy ... >> More

  Methylmalonyl-CoA decarboxylase of Veillonella alcalescens catalyzed the isotopic exchange between methylmalonyl-CoA and [1-14C]propionyl-CoA or between malonyl-CoA and [1-14C]acetyl-CoA. The exchange was independent of sodium ions and was abolished by avidin. The enzyme also catalyzed the carboxyl transfer reaction from methylmalonyl-CoA to acetyl-CoA yielding propionyl-CoA and malonyl-CoA, and vice versa. The beta subunit was dissociated from methylmalonyl-CoA decarboxylase by prolonged washing of the enzyme while bound via its biotin prosthetic group to monomeric avidin-Sepharose. The beta-chain-depleted enzyme was inactive as a methylmalonyl-CoA decarboxylase but retained carboxyltransferase activity. The beta subunits were specifically protected by Na+ ions from tryptic hydrolysis. Based on these and other observations the following functions may be assigned to the different polypeptide chains of methylmalonyl-CoA decarboxylase: carboxyltransferase (alpha), carboxybiotin-carrier-protein decarboxylase (beta), biotin carrier protein (gamma). The function of the delta chain is unknown. << Less

  Eur. J. Biochem. 179:645-650(1989) [PubMed] [EuropePMC]

• Expression of the sodium ion pump methylmalonyl-coenzyme A-decarboxylase from Veillonella parvula and of mutated enzyme specimens in Escherichia coli.

  Huder J.B., Dimroth P.

  The structural genes of the sodium ion pump methylmalonyl-coenzyme A (CoA)-decarboxylase from Veillonella parvula have recently been cloned on three overlapping plasmids (pJH1, pJH20, and pJH40) and sequenced. To synthesize the complete decarboxylase in Escherichia coli, the genes were fused in th ... >> More

  The structural genes of the sodium ion pump methylmalonyl-coenzyme A (CoA)-decarboxylase from Veillonella parvula have recently been cloned on three overlapping plasmids (pJH1, pJH20, and pJH40) and sequenced. To synthesize the complete decarboxylase in Escherichia coli, the genes were fused in the correct order (mmdADECB) on a single plasmid (pJH70). A DNA region upstream of mmdA apparently served as promoter in E. coli because expression of the mmd genes was not dependent on the correct orientation of the lac promoter present on the pBluescript KS(+)-derived expression plasmid. To allow controlled induction of the mmd genes, the upstream region was deleted and the mmd genes were cloned behind a T7 promoter. The derived plasmid, pT7mmd, was transformed into E. coli BL21(DE3) expressing T7 RNA polymerase under the control of the lac promoter. The synthesized proteins showed the typical properties of methylmalonyl-CoA-decarboxylase, i.e., the same migration behavior during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, stimulation of the decarboxylation activity by sodium ions, and inhibition with avidin. In methylmalonyl-CoA-decarboxylase expressed in E. coli from pT7mmd, the gamma subunit was only partially biotinylated and the alpha subunit was present in substoichiometric amounts, resulting in a low catalytic activity. This activity could be considerably increased by coexpression of biotin ligase and by incubation with separately expressed alpha subunit. After these treatments methylmalonyl-CoA-decarboxylase with a specific activity of about 5 U/mg of protein was isolated by adsorption and elution from monomeric avidin-Sepharose. To analyze the function of the delta and epsilon subunits, the corresponding genes were deleted from plasmid pT7mmd. E. coli cells transformed with pJHdelta2, which lacks mmdE and the 3' -terminal part of mmdD, showed no methylmalonyl-CoA-decarboxylase activity. In addition, a contrast, catalytically active methylmalonyl-CoA-decarboxylase was expressed in E. coli from plasmid pJHdelta1, which contained a deletion of the mmdE gene only. The mutant enzyme could be isolated, reconstituted into proteolipsomes, and shown to function in the transport of Na+ ions coupled to methylmalonyl-CoA decarboxylation. The small epsilon subunit therefore has no catalytic function within the methylmalonyl-CoA-decarboxylase complex but appears to increase the stability of this complex. << Less

  J. Bacteriol. 177:3623-3630(1995) [PubMed] [EuropePMC]

• Methylmalonyl coenzyme A decarboxylase. Its role in succinate decarboxylation by Micrococcus lactilyticus.

  Galivan J.H., Allen S.H.

  J Biol Chem 243:1253-1261(1968) [PubMed] [EuropePMC]