Purification and properties of L-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus.
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SUBMITTER: Hoey ME
PROVIDER: S-EPMC1148630 | biostudies-other | 1987 Dec
REPOSITORIES: biostudies-other
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Project description:Procedures were developed for the optimal solubilization of D-lactate dehydrogenase, D-mandelate dehydrogenase, L-lactate dehydrogenase and L-mandelate dehydrogenase from wall + membrane fractions of Acinetobacter calcoaceticus. D-Lactate dehydrogenase and D-mandelate dehydrogenase were co-eluted on gel filtration, as were L-lactate dehydrogenase and L-mandelate dehydrogenase. All four enzymes could be separated by ion-exchange chromatography. D-Lactate dehydrogenase and D-mandelate dehydrogenase were purified by cholate extraction, (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography and chromatofocusing. The properties of D-lactate dehydrogenase and D-mandelate dehydrogenase were similar in several respects: they had relative molecular masses of 62 800 and 59 700 respectively, pI values of 5.8 and 5.5, considerable sensitivity to p-chloromercuribenzoate, little or no inhibition by chelating agents, and similar responses to pH. Both enzymes appeared to contain non-covalently bound FAD as cofactor.
| S-EPMC1152761 | biostudies-other
Project description:Acinetobacter calcoaceticus possesses an L(+)-lactate dehydrogenase and a D(-)-lactate dehydrogenase. Results of experiments in which enzyme activities were measured after growth of bacteria in different media indicated that the two enzymes were co-ordinately induced by either enantiomer of lactate but not by pyruvate, and repressed by succinate or L-glutamate. The two lactate dehydrogenases have very similar properties to L(+)-mandelate dehydrogenase and D(-)-mandelate dehydrogenase. All four enzymes are NAD(P)-independent and were found to be integral components of the cytoplasmic membrane. The enzymes could be solubilized in active form by detergents; Triton X-100 or Lubrol PX were particularly effective D(-)-Lactate dehydrogenase and D(-)-mandelate dehydrogenase could be selectively solubilized by the ionic detergents cholate, deoxycholate and sodium dodecyl sulphate.
| S-EPMC1144902 | biostudies-other
Project description:Acinetobacter calcoaceticus wild-type strain N.C.I.B. 8250 can grow on only the L(+)-isomer of mandelate but mutant strains have been isolated that can grow on D(-)-mandelate. These mutants contain a novel D(-)-mandelate dehydrogenase in addition to the original L(+)-mandelate dehydrogenase. A second wild-type strain, EBF 65/65, shows the opposite pattern and can grow on D(-)-mandelate but not on L(+)-mandelate; mutants have been isolated that possess an L(+)-mandelate dehydrogenase in addition to the original D(-)-mandelate dehydrogenase and can thus grow on L(+)-mandelate. Both L(+)- and D(-)-mandelate dehydrogenases, whether originally present or evolved, are very similar in many respects: they are membrane-bound and NAD(P)+-independent; their activities have similar dependence on temperature and pH; they are inhibited by oxalate but not by several metal-chelating agents; they are stereospecific in their action and are inhibited by the opposite stereoisomers. D(-)-Mandelate dehydrogenase is much more susceptible than L(+)-mandelate dehydrogenase to inhibition by HgCl2 and p-chloromercuribenzoate and is much more heat-labile.
| S-EPMC1154104 | biostudies-other
Project description:Benzaldehyde dehydrogenase I was purified from Acinetobacter calcoaceticus by DEAE-Sephacel, phenyl-Sepharose and f.p.l.c. gel-filtration chromatography. The enzyme was homogeneous and completely free from the isofunctional enzyme benzaldehyde dehydrogenase II, as judged by denaturing and non-denaturing polyacrylamide-gel electrophoresis. The subunit Mr value was 56,000 (determined by SDS/polyacrylamide-gel electrophoresis). Estimations of the native Mr value by gel-filtration chromatography gave values of 141,000 with a f.p.l.c. Superose 6 column, but 219,000 with Sephacryl S300. Chemical cross-linking of the enzyme subunits indicated that the enzyme is tetrameric. Benzaldehyde dehydrogenase I was activated more than 100-fold by K+, Rb+ and NH4+, and the apparent Km for K+ was 11.2 mM. The pH optimum in the presence of K+ was 9.5 and the pI of the enzyme was 5.55. The apparent Km values for benzaldehyde and NAD+ were 0.69 microM and 96 microM respectively, and the maximum velocity was approx. 110 mumol/min per mg of protein. Various substituted benzaldehydes were oxidized at significant rates, and NADP+ was also used as cofactor, although much less effectively than NAD+. Benzaldehyde dehydrogenase I had an NAD+-activated esterase activity with 4-nitrophenol acetate as substrate, and the dehydrogenase activity was inhibited by a range of thiol-blocking reagents. The absorption spectrum indicated that there was no bound cofactor or prosthetic group. Some of the properties of the enzyme are compared with those of other aldehyde dehydrogenases, specifically the very similar isofunctional enzyme benzaldehyde dehydrogenase II from the same organism.
| S-EPMC1133517 | biostudies-other
Project description:A quick, reliable, purification procedure was developed for purifying both benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from a single batch of Acinetobacter calcoaceticus N.C.I.B. 8250. The procedure involved disruption of the bacteria in the French pressure cell and preparation of a high-speed supernatant, followed by chromatography on DEAE-Sephacel, affinity chromatography on Blue Sepharose CL-6B and Matrex Gel Red A, and finally gel filtration through a Superose 12 fast-protein-liquid-chromatography column. The enzymes co-purified as far as the Blue Sepharose CL-6B step were separated on the Matrex Gel Red A column. The final preparations of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II gave single bands on electrophoresis under non-denaturing conditions or on SDS/polyacrylamide-gel electrophoresis. The enzymes are tetramers, as judged by comparison of their subunit (benzyl alcohol dehydrogenase, 39,700; benzaldehyde dehydrogenase II, 55,000) and native (benzyl alcohol dehydrogenase, 155,000; benzaldehyde dehydrogenase II, 222,500) Mr values, estimated by SDS/polyacrylamide-gel electrophoresis and gel filtration respectively. The optimum pH values for the oxidation reactions were 9.2 for benzyl alcohol dehydrogenase and 9.5 for benzaldehyde dehydrogenase II. The pH optimum for the reduction reaction for benzyl alcohol dehydrogenase was 8.9. The equilibrium constant for oxidation of benzyl alcohol to benzaldehyde by benzyl alcohol dehydrogenase was determined to be 3.08 x 10(-11) M; the ready reversibility of the reaction catalysed by benzyl alcohol dehydrogenase necessitated the development of an assay procedure in which hydrazine was used to trap the benzaldehyde formed by the NAD+-dependent oxidation of benzyl alcohol. The oxidation reaction catalysed by benzaldehyde dehydrogenase II was essentially irreversible. The maximum velocities for the oxidation reactions catalysed by benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II were 231 and 76 mumol/min per mg of protein respectively; the maximum velocity of the reduction reaction of benzyl alcohol dehydrogenase was 366 mumol/min per mg of protein. The pI values were 5.0 for benzyl alcohol dehydrogenase and 4.6 for benzaldehyde dehydrogenase II. Neither enzyme activity was affected when assayed in the presence of a range of salts. Absorption spectra of the two enzymes showed no evidence that they contain any cofactors such as cytochrome, flavin, or pyrroloquinoline quinone. The kinetic coefficients of the purified enzymes with benzyl alcohol, benzaldehyde, NAD+ and NADH are also presented.
| S-EPMC1148920 | biostudies-other
Project description:Quinoprotein glucose dehydrogenase (EC 1.1.99.17) from Acinetobacter calcoaceticus L.M.D. 79.41 was purified to homogeneity. It is a basic protein with an isoelectric point of 9.5 and an Mr of 94,000. Denaturation yields two molecules of PQQ/molecule and a protein with an Mr of 48000, indicating that the enzyme consists of two subunits, which are probably identical because even numbers of aromatic amino acids were found. The oxidized enzyme form has an absorption maximum at 350 nm, and the reduced form, obtained after the addition of glucose, at 338 nm. Since double-reciprocal plots of initial reaction rates with various concentrations of glucose or electron acceptor show parallel lines, and substrate inhibition is observed for glucose as well as for electron acceptor at high concentrations, a ping-pong kinetic behaviour with the two reactants exists. From the plots, Km values for glucose and Wurster's Blue of 22 mM and 0.78 mM respectively, and a Vmax. of 7.730 mumol of glucose oxidized/min per mg of protein were derived. The enzyme shows a broad substrate specificity for aldose sugars. Cationic electron acceptors are active in the assay, anionic acceptors are not. A pH optimum of 9.0 was found with Wurster's Blue and 6.0 with 2,6-dichlorophenol-indophenol. Two types of quinoprotein glucose dehydrogenases seem to exist: type I enzymes are acidic proteins from which PQQ can be removed by dialysis against EDTA-containing buffers (examples are found in Escherichia coli, Klebsiella aerogenes and Pseudomonas sp.); type II enzymes are basic proteins from which PQQ is not removed by dialysis against EDTA-containing buffers (examples are found in A. calcoaceticus and Gluconobacter oxydans).
| S-EPMC1147254 | biostudies-other
Project description:The x-ray structure of the monotopic membrane protein (S)-mandelate dehydrogenase (MDH) from Pseudomonas putida reveals an inherent flexibility of its membrane binding segment that might be important for its biological activity. The surface of MDH exhibits a concentration of the positive charges on one side and the negative charges on the other side. The putative membrane binding surface of MDH has a concentric circular ridge, formed by positively charged residues, which projects away from the protein surface by ∼4 Å; this is an unique structural feature and not observed in other monotopic membrane proteins to our knowledge. There are three α-helixes in the membrane binding region. Based on the structure of MDH, it is possible to propose that the interaction of MDH with the membrane is stabilized by coplanar electrostatic interactions, between the positively charged concentric circular ridge and the negatively charged head-groups of the phospholipid bilayer, along with three α-helixes that provide additional stability by inserting into the membrane. The structure reveals the possible orientation of these helixes along with possible roles for the individual residues which form those helixes. These α-helixes may play a role in the enzyme's mobility. A detergent molecule, N-Dodecyl-β-maltoside, is inserted between the membrane binding region and rest of the molecule and may provide structural stability to intra-protein regions by forming hydrogen bonds and close contacts. From the average B-factor of the MDH structure, it is likely that MDH is highly mobile, which might be essential for its interaction in membrane and non-membrane environments, as its substrate (S)-mandelate, is from the cytoplasm, while its electron acceptor is a component of the membrane electron transport chain.
| S-EPMC6204087 | biostudies-literature
Project description:BACKGROUND: The interconversion of two important energy metabolites, 3-hydroxybutyrate and acetoacetate (the major ketone bodies), is catalyzed by D-3-hydroxybutyrate dehydrogenase (BDH1: EC 1.1.1.30), a NAD+-dependent enzyme. The eukaryotic enzyme is bound to the mitochondrial inner membrane and harbors a unique lecithin-dependent activity. Here, we report an advanced purification method of the mammalian BDH applied to the liver enzyme from jerboa (Jaculus orientalis), a hibernating rodent adapted to extreme diet and environmental conditions. RESULTS: Purifying BDH from jerboa liver overcomes its low specific activity in mitochondria for further biochemical characterization of the enzyme. This new procedure is based on the use of polyclonal antibodies raised against BDH from bacterial Pseudomonas aeruginosa. This study improves the procedure for purification of both soluble microbial and mammalian membrane-bound BDH. Even though the Jaculus orientalis genome has not yet been sequenced, for the first time a D-3-hydroxybutyrate dehydrogenase cDNA from jerboa was cloned and sequenced. CONCLUSION: This study applies immunoaffinity chromatography to purify BDH, the membrane-bound and lipid-dependent enzyme, as a 31 kDa single polypeptide chain. In addition, bacterial BDH isolation was achieved in a two-step purification procedure, improving the knowledge of an enzyme involved in the lipid metabolism of a unique hibernating mammal. Sequence alignment revealed conserved putative amino acids for possible NAD+ interaction.
| S-EPMC2572057 | biostudies-literature
Project description:1. N-Terminal sequences were determined for benzyl alcohol dehydrogenase, benzaldehyde dehydrogenase I and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus N.C.I.B. 8250, benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase encoded by the TOL plasmid pWW53 in Pseudomonas putida MT53 and yeast K(+)-activated aldehyde dehydrogenase. Comprehensive details of the sequence determinations have been deposited as Supplementary Publication SUP 50161 (5 pages) at the British Library Document Supply Centre, Boston Spa. Wetherby. West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273. 5. The extent of sequence similarity suggests that the benzyl alcohol dehydrogenases are related to each other and also to established members of the family of long-chain Zn2(+)-dependent alcohol dehydrogenases. Benzaldehyde dehydrogenase II from Acinetobacter appears to be related to the Pseudomonas TOL-plasmid-encoded benzaldehyde dehydrogenase. The yeast K(+)-activated aldehyde dehydrogenase has similarity of sequence with the mammalian liver cytoplasmic class of aldehyde dehydrogenases but not with any of the Acinetobacter or Pseudomonas enzymes. 2. Antisera were raised in rabbits against the three Acinetobacter enzymes and both of the Pseudomonas enzymes, and the extents of the cross-reactions were determined by immunoprecipitation assays with native antigens and by immunoblotting with SDS-denatured antigens. Cross-reactions were detected between the alcohol dehydrogenases and also among the aldehyde dehydrogenases. This confirms the interpretation of the N-terminal sequence comparisons and also indicates that benzaldehyde dehydrogenase I from Acinetobacter may be related to the other two benzaldehyde dehydrogenases. 3. The amino acid compositions of the Acinetobacter and the Pseudomonas enzymes were determined and the numbers of amino acid residues per subunit were calculated to be: benzyl alcohol dehydrogenase and TOL-plasmid-encoded benzyl alcohol dehydrogenase, 381; benzaldehyde dehydrogenase I and benzaldehyde dehydrogenase II, 525; TOL-plasmid-encoded benzaldehyde dehydrogenase, 538.
| S-EPMC1149885 | biostudies-other
Project description:The Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex has emerged as a high priority among hospital-acquired pathogens in intensive care units (ICUs), posing a challenge to infection management practices. In this study, the clinical characteristics, antimicrobial susceptibility patterns, and patients outcome among genospecies were retrospectively compared. Samples were taken from the tracheal secretions of 143 patients in the ICU. Genospecies of the ACB complex were discriminated by analysis of the 16S-23S rRNA gene intergenic spacer (ITS) sequence. Univariate and multiple variable logistic regression analyses were performed to identify risk factors for infection and mortality. Three genospecies were isolated: A. baumannii (73, 51.0%), A. nosocomialis (29, 20.3%), and A. pittii (41, 28.7%). The results showed that the distribution of infection and colonization among the three genospecies were the same, while A. baumannii was more resistant to common antibiotics than A. nosocomialis and A. pittii. Advanced age, a long stay in the ICU, acute physiology and chronic health evaluation (APACHE) II score, the use of a mechanical ventilator, and previous antibiotic use were risk factors for patient infection. The APACHE II score was a risk factor for mortality in patients with ACB complex isolated from tracheal secretions. Poor outcome of patients with ACB complex isolated from tracheal secretion appears to be related to the APACHE II score rather than genospecies.
| S-EPMC5794090 | biostudies-literature