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.

• Malonate decarboxylase of Malonomonas rubra, a novel type of biotin-containing acetyl enzyme.

  Hilbi H., Dehning I., Schink B., Dimroth P.

  Cell suspensions or crude extracts of Malonomonas rubra grown anaerobically on malonate catalyze the decarboxylation of this substrate at a rate of 1.7-2.5 mumol.min-1.mg protein-1 which is consistent with the malonate degradation rate during growth. After fractionation of the cell extract by ultr ... >> More

  Cell suspensions or crude extracts of Malonomonas rubra grown anaerobically on malonate catalyze the decarboxylation of this substrate at a rate of 1.7-2.5 mumol.min-1.mg protein-1 which is consistent with the malonate degradation rate during growth. After fractionation of the cell extract by ultracentrifugation, neither the soluble nor the particulate fraction alone catalyzed the decarboxylation of malonate, but on recombination of the two fractions 87% of the activity of the unfractionated extract was restored. The decarboxylation pathway did not involve the intermediate formation of malonyl-CoA, but decarboxylation proceeded directly with free malonate. The catalytic activity of the enzyme was completely abolished on incubation with hydroxylamine or NaSCN. Approximately 50-65% of the original decarboxylase activity was restored by incubation of the extract with ATP in the presence of acetate, and the extent of reactivation increased after incubation with dithioerythritol. Reactivation of the enzyme was also obtained by chemical acetylation with acetic anhydride. These results indicate modification of the decarboxylase by deacetylation leading to inactivation and by acetylation of the inactivated enzyme specimens leading to reactivation. It is suggested that the catalytic mechanism involves exchange of the enzyme-bound acetyl residues by malonyl residues and subsequent decarboxylation releasing CO2 and regenerating the acetyl-enzyme. The decarboxylase was inhibited by avidin but not by an avidin-biotin complex indicating that biotin is involved in catalysis. A single biotin-containing 120-kDa polypeptide was present in the extract and is a likely component of malonate decarboxylase. << Less

  Eur J Biochem 207:117-123(1992) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The acyl carrier protein of malonate decarboxylase of Malonomonas rubra contains 2'-(5''-phosphoribosyl)-3'-dephosphocoenzyme A as a prosthetic group.

  Berg M., Hilbi H., Dimroth P.

  Malonate decarboxylase of Malonomonas rubra is composed of soluble and membrane-bound components and contains an acetyl residue that is essential for catalytic activity. Upon incubation with hydroxylamine, the acetyl residue is removed, forming an inactive thiol enzyme, which is reactivated by ace ... >> More

  Malonate decarboxylase of Malonomonas rubra is composed of soluble and membrane-bound components and contains an acetyl residue that is essential for catalytic activity. Upon incubation with hydroxylamine, the acetyl residue is removed, forming an inactive thiol enzyme, which is reactivated by acetylation with ATP, acetate, and a specific ligase. After incubation of the thiol enzyme with iodoacetate in the presence of excess dithioerythritol, the prosthetic group thiol residue was carboxymethylated and reactivation by acetylation was impaired. Radioactive labeling with [1-14C] iodoacetate revealed the site of carboxymethyation on a distinct cytoplasmic protein with the apparent molecular mass of 14 000 Da. The same protein was specifically labeled by enzymic acetylation of the thiol enzyme with [1-14C]acetate and ATP. Malonate decarboxlyation by [14C]acetyl malonate decarboxlyation resulted in the release of the radioactive acetyl residue from the enzyme,indicating that this acetyl residue is exchanged for a malonyl residue during catalysis. The acyl carrier protein has been purified as its [14C]carboxymethylated derivative to apparent homogeneity. The prosthetic group of the acyl carrier protein was isolated after alkaline hydrolysis, and its chemical structure was identified by high-performance liquid chromatography (HPLC) with the corresponding compound from citrate lyase from Klebsiella pneumoniae as reference and by mass spectrometry. Malonate decarboxylase was found to carry the same prosthetic group as citrate lyase, i.e. 2'-(5"-phosphoribosyl)-3'-dephospho-CoA. << Less

  Biochemistry 35:4689-4696(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.