Purification and properties of L-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus.
ABSTRACT: L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a 'wall + membrane' fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus.
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.
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.
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.
Project description:L(+)-Mandelate dehydrogenase was purified to homogeneity from the yeast Rhodotorula graminis KGX 39 by a combination of (NH4)2SO4 fractionation, ion-exchange and hydrophobic-interaction chromatography and gel filtration. The amino-acid composition and the N-terminal sequence of the enzyme were determined. Comprehensive details of the sequence determinations have been deposited as Supplementary Publication SUP 50172 (4 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. (1993) 289, 9. The enzyme is a tetramer as judged by comparison of its subunit M(r) value of 59,100 and native M(r) of 239,900, estimated by SDS/PAGE and gel filtration respectively. There is one molecule of haem and approx. one molecule of non-covalently bound FMN per subunit. 2,6-Dichloroindophenol, cytochrome c and ferricyanide can all serve as electron acceptors. L(+)-Mandelate dehydrogenase is stereospecific for its substrate. D(-)-Mandelate and L(+)-hexahydromandelate are competitive inhibitors. The enzyme has maximum activity at pH 7.9 and it has a pI value of 4.4. HgCl2 and 4-chloromercuribenzoate are potent inhibitors, but there is no evidence that the enzyme is subject to feedback inhibition by potential metabolic effectors. The evidence suggests that L(+)-mandelate dehydrogenase from R. graminis is a flavocytochrome b which is very similar to, and probably (at least so far as the haem domain is concerned) homologous with, certain well-characterized yeast L(+)-lactate dehydrogenases, and that the chief difference between them is their mutually exclusive substrate specificities.
Project description:Flavocytochrome b2 from Saccharomyces cerevisiae is an l-lactate dehydrogenase which exhibits only barely detectable activity levels towards another 2-hydroxyacid, l-mandelate. Using protein engineering methods we have altered the active site of flavocytochrome b2 and successfully introduced substantial mandelate dehydrogenase activity into the enzyme. Changes to Ala-198 and Leu-230 have significant effects on the ability of the enzyme to utilize l-mandelate as a substrate. The double mutation of Ala-198-->Gly and Leu-230-->Ala results in an enzyme with a kcat value (25 degrees C) with L-mandelate of 8.5 s-1, which represents an increase of greater than 400-fold over the wild-type enzyme. Perhaps more significantly, the mutant enzyme has a catalytic efficiency (as judged by kcat/Km values) that is 6-fold higher with l-mandelate than it is with L-lactate. Closer examination of the X-ray structure of S. cerevisiae flavocytochrome b2 led us to conclude that one of the haem propionate groups might interfere with the binding of L-mandelate at the active site of the enzyme. To test this idea, the activity with l-mandelate of the independently expressed flavodehydrogenase domain (FDH), was examined and found to be higher than that seen with the wild-type enzyme. In addition, the double mutation of Ala-198-->Gly and Leu-230-->Ala introduced into FDH produced the greatest mandelate dehydrogenase activity increase, with a kcat value more than 700-fold greater than that seen with the wild-type holoenzyme. In addition, the enzyme efficiency (kcat/Km) of this mutant enzyme was more than 20-fold greater with L-mandelate than with l-lactate. We have therefore succeeded in constructing an enzyme which is now a better mandelate dehydrogenase than a lactate dehydrogenase.
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.
Project description:L-Lactate dehydrogenase (L-LDH) from Saccharomyces cerevisiae and L-mandelate dehydrogenase (L-MDH) from Rhodotorula graminis are both flavocytochromes b2. The kinetic properties of these enzymes have been compared using steady-state kinetic methods. The most striking difference between the two enzymes is found by comparing their substrate specificities. L-LDH and L-MDH have mutually exclusive primary substrates, i.e. the substrate for one enzyme is a potent competitive inhibitor for the other. Molecular-modelling studies on the known three-dimensional structure of S. cerevisiae L-LDH suggest that this enzyme is unable to catalyse the oxidation of L-mandelate because productive binding is impeded by steric interference, particularly between the side chain of Leu-230 and the phenyl ring of mandelate. Another major difference between L-LDH and L-MDH lies in the rate-determining step. For S. cerevisiae L-LDH, the major rate-determining step is proton abstraction at C-2 of lactate, as previously shown by the 2H kinetic-isotope effect. However, in R. graminis L-MDH the kinetic-isotope effect seen with DL-[2-2H]mandelate is only 1.1 +/- 0.1, clearly showing that proton abstraction at C-2 of mandelate is not rate-limiting. The fact that the rate-determining step is different indicates that the transition states in each of these enzymes must also be different.
Project description:1. l-Mandelate dehydrogenase and mandelate racemase were partially purified from extracts of Pseudomonas fluorescens A-312 grown in media containing d-mandelate. 2. The activity of mandelate racemase, but not that of l-mandelate dehydrogenase, is greatly stimulated by Mg(2+), Mn(2+), Co(2+) and, though less effectively, by Ni(2+). Other metal ions are inactive or inhibitory. 3. Racemase activity is inhibited by phosphate, fluoride, pyrophosphate and EDTA. The inhibitions by pyrophosphate and EDTA are competitive with respect to the metal ion activator; those by phosphate and fluoride are competitive with respect to the substrate. 4. The addition of Mg(2+) diminishes the Michaelis constant of racemase. 5. The pH optimum of the racemase is at 7.8. The pH-activity curve of the dehydrogenase complex of enzymes has two peaks, at 7.0 and 8.2. 6. The enzymic racemization of d-mandelate is initially faster than that of l-mandelate. 7. The rates of oxidation of related substrates, catalysed by l-mandelate dehydrogenase, are in the decreasing order: l-p-hydroxymandelate; l-3,4-dihydroxymandelate; l-4-hydroxy-3-methoxymandelate. The racemase is active towards d-p-hydroxymandelate but inactive towards d-3,4-dihydroxymandelate and d-4-hydroxy-3-methoxymandelate. Since 4-hydroxy-3-methoxymandelate, and presumably also 3,4-dihydroxymandelate, arising from the metabolism of catechol-amines, have the d-configuration, the enzymes studied cannot be utilized for estimation of the last two acids in urine.
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.
Project description:1. Bacterium N.C.I.B. 8250 was grown on dl-mandelate, benzyl alcohol, benzoyl-formate, benzaldehyde and benzoate and also on 2-hydroxy, 4-hydroxy, 3,4-dihydroxy and 4-hydroxy-3-methoxy analogues of these compounds. The enzymic complements of the cells were determined and the specificities of some of the enzymes examined. 2. Growth on mandelate or benzoylformate induces l-mandelate dehydrogenase, benzoylformate decarboxylase, benzyl alcohol dehydrogenase and a heat-stable as well as a heat-labile benzaldehyde dehydrogenase. Growth on benzyl alcohol or benzaldehyde induces benzyl alcohol dehydrogenase and the heat-labile benzaldehyde dehydrogenase. 3. The enzymes of the mandelate-to-benzoate pathway are non-specifically active on, and induced by, all the substituted analogues that support growth. 4. Benzoate oxidase is induced by growth on benzoate or on 2-hydroxybenzoate. 2-Hydroxybenzoate hydroxylase, 4-hydroxybenzoate hydroxylase and 4-hydroxy-3-methoxybenzoate O-demethylase are induced only by growth on homologous substrates. 5. The results of the investigation are discussed with regard to the possible regulation of the enzyme systems.