Publications

• Enzymic and genetic basis for bacterial growth on malonate.

  Dimroth P., Hilbi H.

  Various bacteria are able to grow aerobically or anaerobically on malonate as sole source of carbon and energy. Independent of the mechanism for energy conservation, the decarboxylation of malonate is the key reaction in the decomposition of this compound. To achieve malonate decarboxylation under ... >> More

  Various bacteria are able to grow aerobically or anaerobically on malonate as sole source of carbon and energy. Independent of the mechanism for energy conservation, the decarboxylation of malonate is the key reaction in the decomposition of this compound. To achieve malonate decarboxylation under physiological conditions, the substrate must be converted into an activated (thioester) derivative. We report here on the malonate decarboxylases of Malonomonas rubra and Klebsiella pneumoniae. These enzymes perform an interesting substrate activation mechanism by generating a malonyl thioester with the enzyme. Formation of the malonyl-S-enzyme involves an 'activation module' that comprises the acetylation of a specific thiol group of an acyl carrier protein (ACP) and the transfer of the ACP moiety to malonate, yielding malonyl-S-ACP and acetate. The malonyl-S-ACP is subsequently decarboxylated with regeneration of the acetyl-ACP. The malonate activation mechanism is related to the activation of citrate by citrate lyase. The relationship extends to the identical 2'-(5''-phosphoribosyl)-3'-dephospho-CoA thiol cofactor that is bound covalently to the corresponding ACP subunit. In Klebsiella pneumoniae, malonate is decarboxylated by a water-soluble enzyme complex. In the anaerobic bacterium Malonomonas rubra, malonate decarboxylation is catalysed by a set of water-soluble as well as membrane-bound enzymes that function together in converting the free energy of the decarboxylation reaction into delta muNa+. Therefore, this malonate decarboxylase includes a biotin carrier protein that accepts the CO2 moiety from malonyl-S-ACP and delivers it to a membrane-bound decarboxylase acting as a Na+ pump. Genes encoding the individual protein components that perform the decarboxylation of malonate in K. pneumoniae or M. rubra have been identified within the mdc and mad gene clusters respectively. The function of most of the derived proteins could be envisaged from sequence similarities with proteins of known functions. The genetic evidence firmly supports the idea that malonate decarboxylation is carried out by the two different decarboxylases, as deduced from the biochemical studies of the enzymes. << Less

  Mol. Microbiol. 25:3-10(1997) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Aspartate 203 of the oxaloacetate decarboxylase beta-subunit catalyses both the chemical and vectorial reaction of the Na+ pump.

  Di Berardino M., Dimroth P.

  We report here a new mode of coupling between the chemical and vectorial reaction explored for the oxaloacetate decarboxylase Na+ pump from Klebsiella pneumoniae. The membrane-bound beta-subunit is responsible for the decarboxylation of carboxybiotin and the coupled translocation of Na+ ions acros ... >> More

  We report here a new mode of coupling between the chemical and vectorial reaction explored for the oxaloacetate decarboxylase Na+ pump from Klebsiella pneumoniae. The membrane-bound beta-subunit is responsible for the decarboxylation of carboxybiotin and the coupled translocation of Na+ ions across the membrane. The biotin prosthetic group which is attached to the alpha-subunit becomes carboxylated by carboxyltransfer from oxaloacetate. The two conserved aspartic acid residues within putative membrane-spanning domains of the beta-subunit (Asp149 and Asp203) were exchanged by site-directed mutagenesis. Mutants D149Q and D149E retained oxaloacetate decarboxylase and Na+ transport activities. Mutants D203N and D203E, however, had lost these two activities, but retained the ability to form the carboxybiotin enzyme. Direct participation of Asp203 in the catalysis of the decarboxylation reaction is therefore indicated. In addition, all previous and present data on the enzyme support a model in which the same aspartic acid residue provides a binding site for the metal ion catalysing its movement across the membrane. The model predicts that asp203 in its dissociated form binds Na+ and promotes its translocation, while the protonated residue transfers the proton to the acid-labile carboxybiotin which initiates its decarboxylation. Strong support for the model comes from the observation that Na+ transport by oxaloacetate decarboxylation is accompanied by H+ transport in the opposite direction. The inhibition of oxaloacetate decarboxylation by high Na+ concentrations in a pH-dependent manner is also in agreement with the model. << Less

  EMBO J 15:1842-1849(1996) [PubMed] [EuropePMC]

• Sequence of a gene cluster from Malonomonas rubra encoding components of the malonate decarboxylase Na+ pump and evidence for their function.

  Berg M., Hilbi H., Dimroth P.

  Malonate decarboxylation in Malonomonas rubra involves the formation of malonyl-S-[acyl-carrier protein] from acetyl-S-[acyl-carrier protein] and malonate, carboxyltransfer to a biotin protein and its decarboxylation that is coupled to delta mu Na+ generation. The genes encoding components of the ... >> More

  Malonate decarboxylation in Malonomonas rubra involves the formation of malonyl-S-[acyl-carrier protein] from acetyl-S-[acyl-carrier protein] and malonate, carboxyltransfer to a biotin protein and its decarboxylation that is coupled to delta mu Na+ generation. The genes encoding components of the malonate decarboxylase enzyme system have been cloned and sequenced. These are located within a gene cluster of approximately 11 kb comprising 14 genes that have been termed madYZGBAECDHKFLMN in the given order. Upstream of madY an open reading frame pointing into the opposite direction of the mad genes was found with structural similarities to insertion-sequence elements. The upstream region also contains DNA regions which are typical for an Escherichia coli sigma 70 promoter. Within 950 bp downstream of madN no other open reading frame was found. This region contains a putative terminator sequence. The intergenic regions within the mad gene cluster are short (usually < 70 bp, maximum 302 bp) and ribosome binding sites were defined before all 14 genes. Thus, this DNA region could form a transcriptional unit and all 14 genes could be translated into proteins. The genes madABCDEF encode the structural proteins of the malonate decarboxylase as yet identified. By comparing protein and DNA sequences and by data bank searches for related proteins with known function the following assignments could be made: MadA represents the acyl-carrier-protein-transferase component. MadB is the integral membrane-bound carboxybiotin protein decarboxylase, MadC and MadD are the two subunits of the carboxyltransferase, MadE is the acyl carrier protein and MadF is the biotin protein. Sequence comparison further indicates that MadH could be involved in the acetylation of the phosphoribosyl-dephospho-CoA prosthetic group and MadG could be involved in its biosynthesis. MadL and MadM are membrane proteins that could function as malonate carrier. The function of the madY,Z,K and N gene products is as yet unknown. << Less

  Eur. J. Biochem. 245:103-115(1997) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.