Publications

• Ferrous- or cobalt ion-dependent D-(-)-tartrate dehydratase of pseudomonads: purification and properties.

  Rode H., Giffhorn F.

  D-(-)-Tartrate dehydratases [D-(-)-tartrate hydro-lyase, EC 4.2.1...] were isolated from two Pseudomonas strains. The molecular weights of the native enzymes were determined to be 72,000 and 7 8,000, respectively, and each enzyme was composed of two subunits of identical size. The dehydratases had ... >> More

  D-(-)-Tartrate dehydratases [D-(-)-tartrate hydro-lyase, EC 4.2.1...] were isolated from two Pseudomonas strains. The molecular weights of the native enzymes were determined to be 72,000 and 7 8,000, respectively, and each enzyme was composed of two subunits of identical size. The dehydratases had no requirements for thiol compounds, were insensitive to oxygen, and required Fe2+ (0.1 mM) or Co2+ (0.5 mM) ions for optimal activity. << Less

  J Bacteriol 151:1602-1604(1982) [PubMed] [EuropePMC]

• D-(--)-tartrate dehydratase of Rhodopseudomonas sphaeroides: purification, characterization, and application to enzymatic determination of D-(--)-tartrate.

  Rode H., Giffhorn F.

  An isolate of Rhodopseudomonas sphaeroides was capable of growing phototrophically and chemotrophically (mu = 0.15 h(-1) for either condition) with d-(-)-tartrate as the carbon source. A d-(-)-tartrate dehydratase, (d-(-)-tartrate hydrolyase, EC 4.1.2.70) was induced in the presence of d-(-)-tartr ... >> More

  An isolate of Rhodopseudomonas sphaeroides was capable of growing phototrophically and chemotrophically (mu = 0.15 h(-1) for either condition) with d-(-)-tartrate as the carbon source. A d-(-)-tartrate dehydratase, (d-(-)-tartrate hydrolyase, EC 4.1.2.70) was induced in the presence of d-(-)-tartrate. The enzyme was purified 30-fold from cell extracts of R. sphaeroides to a specific activity of 7.5 U/mg of protein and was subsequently crystallized in the presence of 1 M KCl. The enzyme was homogeneous upon analytical electrophoresis in 5% polyacrylamide gels and by criteria of ultracentrifugation. The native enzyme had a molecular weight of 158,000 +/-1,000 as determined by gel filtration and ultracentrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single polypeptide chain with an estimated molecular weight of 39,500 +/-500, indicating that d-(-)-tartrate dehydratase was a tetramer. The isoelectric point of the native enzyme was at pH 5.5. The enzyme catalyzed irreversibly the conversion of d-(-)-tartrate to oxaloacetate and water, and the turnover number was calculated to be 1,185. The reaction followed Michaelis-Menten kinetics, and a K(m) value of 1.8 x 10(-4) M was determined. d-(-)-Tartrate dehydratase required Mg(2+) for activity. The pH optimum was within a range from 6.2 to 7.2, and the activation energy of the reaction (Delta H(0)) was 63.2 kJ/mol. The enzyme was specific for d-(-)-tartrate; it did not react with l-(+)-tartrate, meso-tartrate, and other hydroxycarboxylic acids. d-(-)-Tartrate dehydratase was strongly inhibited by meso-tartrate (50% at 0.6 mM). l-(+)-Tartrate and a variety of hydroxycarboxylic acids caused 50% inhibition at concentrations of >30 mM. << Less

  J Bacteriol 150:1061-1068(1982) [PubMed] [EuropePMC]

• Evolution of enzymatic activities in the enolase superfamily: D-tartrate dehydratase from Bradyrhizobium japonicum.

  Yew W.S., Fedorov A.A., Fedorov E.V., Wood B.M., Almo S.C., Gerlt J.A.

  We focus on the assignment of function to and elucidation of structure-function relationships for a member of the mechanistically diverse enolase superfamily encoded by the Bradyrhizobium japonicum genome (bll6730; GI:27381841). As suggested by sequence alignments, the active site contains the sam ... >> More

  We focus on the assignment of function to and elucidation of structure-function relationships for a member of the mechanistically diverse enolase superfamily encoded by the Bradyrhizobium japonicum genome (bll6730; GI:27381841). As suggested by sequence alignments, the active site contains the same functional groups found in the active site of mandelate racemase (MR) that catalyzes a 1,1-proton transfer reaction: two acid/base catalysts, Lys 184 at the end of the second beta-strand, and a His 322-Asp 292 dyad at the ends of the seventh and sixth beta-strands, respectively, as well as ligands for an essential Mg2+, Asp 213, Glu 239, and Glu 265 at the ends of the third, fourth, and fifth beta-strands, respectively. We screened a library of 46 acid sugars and discovered that only d-tartrate is dehydrated, yielding oxaloacetate as product. The kinetic constants (kcat = 7.3 s(-1); kcat/KM = 8.5 x 10(4) M(-1) s(-1)) are consistent with assignment of the d-tartrate dehydratase (TarD) function. The kinetic phenotypes of mutants as well as the structures of liganded complexes are consistent with a mechanism in which Lys 184 initiates the reaction by abstraction of the alpha-proton to generate a Mg2+-stabilized enediolate intermediate, and the vinylogous beta-elimination of the 3-OH group is general acid-catalyzed by the His 322, accomplishing the anti-elimination of water. The replacement of the leaving group by solvent-derived hydrogen is stereorandom, suggesting that the enol tautomer of oxaloacetate is the product; this expectation was confirmed by its observation by 1H NMR spectroscopy. Thus, the TarD-catalyzed reaction is a "simple" extension of the two-step reaction catalyzed by MR: base-catalyzed proton abstraction to generate a Mg2+-stabilized enediolate intermediate followed by acid-catalyzed decomposition of that intermediate to yield the product. << Less

  Biochemistry 45:14598-14608(2006) [PubMed] [EuropePMC]

• Biochemical similarities and differences between the catalytic [4Fe-4S] cluster containing fumarases FumA and FumB from Escherichia coli.

  van Vugt-Lussenburg B.M., van der Weel L., Hagen W.R., Hagedoorn P.L.

  <h4>Background</h4>The highly homologous [4Fe-4S] containing fumarases FumA and FumB, sharing 90% amino acid sequence identity, from Escherichia coli are differentially regulated, which suggests a difference in their physiological function. The ratio of FumB over FumA expression levels increases b ... >> More

  <h4>Background</h4>The highly homologous [4Fe-4S] containing fumarases FumA and FumB, sharing 90% amino acid sequence identity, from Escherichia coli are differentially regulated, which suggests a difference in their physiological function. The ratio of FumB over FumA expression levels increases by one to two orders of magnitude upon change from aerobic to anaerobic growth conditions.<h4>Methodology/principal findings</h4>To understand this difference in terms of structure-function relations, catalytic and thermodynamic properties were determined for the two enzymes obtained from homologous overexpression systems. FumA and FumB are essentially identical in their Michaelis-Menten kinetics of the reversible fumarate to L-malate conversion; however, FumB has a significantly greater catalytic efficiency for the conversion of D-tartrate to oxaloacetate consistent with the requirement of the fumB gene for growth on D-tartrate. Reduction potentials of the [4Fe-4S](2+) Lewis acid active centre were determined in mediated bulk titrations in the presence of added substrate and were found to be approximately -290 mV for both FumA and FumB.<h4>Conclusions/significance</h4>This study contradicts previously published claims that FumA and FumB exhibit different catalytic preferences for the natural substrates L-malate and fumarate. FumA and FumB differ significantly only in the catalytic efficiency for the conversion of D-tartrate, a supposedly non-natural substrate. The reduction potential of the substrate-bound [4Fe-4S] active centre is, contrary to previously reported values, close to the cellular redox potential. << Less

  PLoS ONE 8:E55549-E55549(2013) [PubMed] [EuropePMC]