Publications

• The methylcitric acid pathway in Ralstonia eutropha: new genes identified involved in propionate metabolism.

  Bramer C.O., Steinbuchel A.

  From Ralstonia eutropha HF39 null-allele mutants were created by Tn5 mutagenesis and by homologous recombination which were impaired in growth on propionic acid and levulinic acid. From the molecular, physiological and enzymic analysis of these mutants it was concluded that in this bacterium propi ... >> More

  From Ralstonia eutropha HF39 null-allele mutants were created by Tn5 mutagenesis and by homologous recombination which were impaired in growth on propionic acid and levulinic acid. From the molecular, physiological and enzymic analysis of these mutants it was concluded that in this bacterium propionic acid is metabolized via the methylcitric acid pathway. The genes encoding enzymes of this pathway are organized in a cluster in the order prpR, prpB, prpC, acnM, ORF5 and prpD, with prpR transcribed divergently from the other genes. (i) prpC encodes a 2-methylcitric acid synthase (42720 Da) as shown by the measurement of the respective enzyme activity, complementation of a prpC mutant of Salmonella enterica serovar Typhimurium and high sequence similarity. (ii) For the translational product of acnM the function of a 2-methyl-cis-aconitic acid hydratase (94726 Da) is proposed. This protein and also the ORF5 translational product are essential for growth on propionic acid, as revealed by the propionic-acid-negative phenotype of Tn5-insertion mutants, and are required for the conversion of 2-methylcitric acid into 2-methylisocitric acid as shown by the accumulation of the latter, which could be purified as its calcium salt from the supernatants of these mutants. In contrast, inactivation of prpD did not block the ability of the cell to use propionic acid as carbon and energy source, as shown by the propionic acid phenotype of a null-allele mutant. It is therefore unlikely that prpD from R. eutropha encodes a 2-methyl-cis-aconitic acid dehydratase as proposed recently for the homologous prpD gene from S. enterica. (iii) The translational product of prpB encodes 2-methylisocitric acid lyase (32314 Da) as revealed by measurement of the respective enzyme activity and by demonstrating accumulation of methylisocitric acid in the supernatant of a prpB null-allele mutant. (iv) The expression of prpC and probably also of the other enzymes is regulated and is induced during cultivation on propionic acid or levulinic acid. The putative translational product of prpR (70895 Da) exhibited high similarities to PrpR of Escherichia coli and S. enterica, and might represent a transcriptional activator of the sigma-54 family involved in the regulation of the other prp genes. Since the prp locus of R. eutropha was very different from those of E. coli and S. enterica, an extensive comparison of prp loci available from databases and literature was done, revealing two different classes of prp loci. << Less

  Microbiology 147:2203-2214(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The acnD genes of Shewenella oneidensis and Vibrio cholerae encode a new Fe/S-dependent 2-methylcitrate dehydratase enzyme that requires prpF function in vivo.

  Grimek T.L., Escalante-Semerena J.C.

  The propionate utilization operons of several bacteria differ from each other in the occurrence of two genes, acnD and prpF, in place of or in addition to the prpD gene encoding an Fe/S-independent 2-methylcitrate dehydratase enzyme. We cloned the acnD and prpF genes from two organisms, Shewanella ... >> More

  The propionate utilization operons of several bacteria differ from each other in the occurrence of two genes, acnD and prpF, in place of or in addition to the prpD gene encoding an Fe/S-independent 2-methylcitrate dehydratase enzyme. We cloned the acnD and prpF genes from two organisms, Shewanella oneidensis and Vibrio cholerae, and found that, together, the AcnD and PrpF proteins restored the ability of a prpD mutant strain of Salmonella enterica to grow on propionate as a source of carbon and energy. However, neither acnD nor prpF alone was able to substitute for prpD. The AcnD and PrpF proteins were isolated and biochemically analyzed. The AcnD protein required reconstitution of an Fe/S cluster for activity. All detectable AcnD activity was lost after incubation with iron-chelating agents, and no AcnD activity was observed after attempted reconstitution without iron. Nuclear magnetic resonance spectroscopy and in vitro activity assay data showed that AcnD dehydrated 2-methylcitrate and citrate to 2-methyl-cis-aconitate and cis-aconitate, respectively; AcnD also hydrated cis-aconitate. However, 2-methylisocitrate and isocitrate were not substrates for AcnD, indicating that AcnD only catalyzes the first half of the aconitase-like dehydration reactions. No aconitase-like activity was found for PrpF. It is hypothesized that, in vivo, PrpF is an accessory protein required to prevent oxidative damage of the Fe/S center of active AcnD enzyme or that it may be involved in synthesis or repair of the Fe/S cluster present in AcnD. << Less

  J. Bacteriol. 186:454-462(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The three-dimensional crystal structure of the PrpF protein of Shewanella oneidensis complexed with trans-aconitate: insights into its biological function.

  Garvey G.S., Rocco C.J., Escalante-Semerena J.C., Rayment I.

  In bacteria, the dehydration of 2-methylcitrate to yield 2-methylaconitate in the 2-methylcitric acid cycle is catalyzed by a cofactor-less (PrpD) enzyme or by an aconitase-like (AcnD) enzyme. Bacteria that use AcnD also require the function of the PrpF protein, whose function was previously unkno ... >> More

  In bacteria, the dehydration of 2-methylcitrate to yield 2-methylaconitate in the 2-methylcitric acid cycle is catalyzed by a cofactor-less (PrpD) enzyme or by an aconitase-like (AcnD) enzyme. Bacteria that use AcnD also require the function of the PrpF protein, whose function was previously unknown. To gain insights into the function of PrpF, the three-dimensional crystal structure of the PrpF protein from the bacterium Shewanella oneidensis was solved at 2.0 A resolution. The protein fold of PrpF is strikingly similar to those of the non-PLP-dependent diaminopimelate epimerase from Haemophilus influenzae, a putative proline racemase from Brucella melitensis, and to a recently deposited structure of a hypothetical protein from Pseudomonas aeruginosa. Results from in vitro studies show that PrpF isomerizes trans-aconitate to cis-aconitate. It is proposed that PrpF catalysis of the cis-trans isomerization proceeds through a base-catalyzed proton abstraction coupled with a rotation about C2-C3 bond of 2-methylaconitate, and that residue Lys73 is critical for PrpF function. The newly identified function of PrpF as a non-PLP-dependent isomerase, together with the fact that PrpD-containing bacteria do not require PrpF, suggest that the isomer of 2-methylaconitate that serves as a substrate of aconitase must have the same stereochemistry as that synthesized by PrpD. From this, it follows that the 2-methylaconitate isomer generated by AcnD is not a substrate of aconitase, and that PrpF is required to generate the correct isomer. As a consequence, the isomerase activity of PrpF may now be viewed as an integral part of the 2-methylcitric acid cycle. << Less

  Protein Sci. 16:1274-1284(2007) [PubMed] [EuropePMC]