Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus

1987 ◽  
Vol 15 (6) ◽  
pp. 1048-1049 ◽  
Author(s):  
ROBERT W. MacKINTOSH ◽  
CHARLES A. FEWSON
Keyword(s):  
Alcohol Dehydrogenase ◽  
Benzyl Alcohol ◽  
Acinetobacter Calcoaceticus ◽  
Benzaldehyde Dehydrogenase

scholarly journals Molecular characterization of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II of Acinetobacter calcoaceticus

1998 ◽  
Vol 330 (3) ◽  
pp. 1375-1381 ◽  
Author(s):  
J. David GILLOOLY ◽  
G. S. Alan ROBERTSON ◽  
A. Charles FEWSON
Keyword(s):  
Amino Acids ◽  
Alcohol Dehydrogenase ◽  
Benzyl Alcohol ◽  
Catalytic Mechanism ◽  
Acinetobacter Calcoaceticus ◽  
Higher Plant ◽  
Ph Optimum ◽  
Alcohol Dehydrogenases ◽  
Kinetic Coefficients ◽  
Benzaldehyde Dehydrogenase

The nucleotide sequences of xylB and xylC from Acinetobacter calcoaceticus, the genes encoding benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II, were determined. The complete nucleotide sequence indicates that these two genes form part of an operon and this was supported by heterologous expression and physiological studies. Benzaldehyde dehydrogenase II is a 51654 Da protein with 484 amino acids per subunit and it is typical of other prokaryotic and eukaryotic aldehyde dehydrogenases. Benzyl alcohol dehydrogenase has a subunit Mr of 38923 consisting of 370 amino acids, it stereospecifically transfers the proR hydride of NADH, and it is a member of the family of zinc-dependent long-chain alcohol dehydrogenases. The enzyme appears to be more similar to animal and higher-plant alcohol dehydrogenases than it is to most other microbial alcohol dehydrogenases. Residue His-51 of zinc-dependent alcohol dehydrogenases is thought to be necessary as a general base for catalysis in this category of alcohol dehydrogenases. However, this residue was found to be replaced in benzyl alcohol dehydrogenase from A. calcoaceticus by an isoleucine, and the introduction of a histidine residue in this position did not alter the kinetic coefficients, pH optimum or substrate specificity of the enzyme. Other workers have shown that His-51 is also absent from the TOL-plasmid-encoded benzyl alcohol dehydrogenase of Pseudomonas putida and so these two closely related enzymes presumably have a catalytic mechanism that differs from that of the archetypal zinc-dependent alcohol dehydrogenases.

scholarly journals Comparison of benzyl alcohol dehydrogenases and benzaldehyde dehydrogenases from the benzyl alcohol and mandelate pathways in Acinetobacter calcoaceticus and from the TOL-plasmid-encoded toluene pathway in Pseudomonas putida. N-terminal amino acid sequences, amino acid compositions and immunological cross-reactions

1991 ◽  
Vol 273 (1) ◽  
pp. 99-107 ◽  
Author(s):  
R M Chalmers ◽  
J N Keen ◽  
C A Fewson
Keyword(s):  
Amino Acid ◽  
Alcohol Dehydrogenase ◽  
Benzyl Alcohol ◽  
Aldehyde Dehydrogenase ◽  
Acinetobacter Calcoaceticus ◽  
Tol Plasmid ◽  
Alcohol Dehydrogenases ◽  
Aldehyde Dehydrogenases ◽  
Cross Reactions ◽  
Benzaldehyde Dehydrogenase

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.

scholarly journals Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus. Purification and preliminary characterization

1988 ◽  
Vol 250 (3) ◽  
pp. 743-751 ◽  
Author(s):  
R W MacKintosh ◽  
C A Fewson
Keyword(s):  
Alcohol Dehydrogenase ◽  
Benzyl Alcohol ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Acinetobacter Calcoaceticus ◽  
Reduction Reaction ◽  
Oxidation Reactions ◽  
Benzaldehyde Dehydrogenase ◽  
Blue Sepharose

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.

scholarly journals Regulation of the enzymes converting l-mandelate into benzoate in bacterium N.C.I.B. 8250

1972 ◽  
Vol 130 (4) ◽  
pp. 937-946 ◽  
Author(s):  
A. Livingstone ◽  
C. A. Fewson
Keyword(s):  
Alcohol Dehydrogenase ◽  
Single Cell ◽  
Benzyl Alcohol ◽  
Dehydrogenase Activity ◽  
The Other ◽  
Cell Free Extract ◽  
Mutant Strains ◽  
Benzaldehyde Dehydrogenase ◽  
Kinetics Of

l-Mandelate is oxidized to benzoate by the enzymes l-mandelate dehydrogenase, phenylglyoxylate carboxy-lyase and benzaldehyde dehydrogenase I. Conditions have been established for measuring these three enzymes as well as benzyl alcohol dehydrogenase, benzaldehyde dehydrogenase II and catechol 1,2-oxygenase in a single cell-free extract prepared from bacterium N.C.I.B. 8250. The kinetics of induction of all these enzymes have been measured under a variety of conditions. l-Mandelate dehydrogenase, phenylglyoxylate carboxy-lyase and benzaldehyde dehydrogenase I appear to be co-ordinately regulated because (a) their differential rates of synthesis are proportional to one another under various conditions of induction and repression, (b) they are specifically and gratuitously induced by thiophenoxyacetate and a number of other compounds, and (c) mutant strains have been isolated that lack all three enzymes. Phenylglyoxylate is the primary inducer of the regulon as mutant strains lacking phenylglyoxylate carboxy-lyase form the other two enzymes in the presence of l-mandelate or phenylglyoxylate, whereas in mutant strains devoid of l-mandelate dehydrogenase activity only phenylglyoxylate induces phenylglyoxylate carboxy-lyase and benzaldehyde dehydrogenase I.

scholarly journals Enzymes of the mandelate pathway in bacterium N.C.I.B. 8250

1968 ◽  
Vol 107 (4) ◽  
pp. 497-506 ◽  
Author(s):  
S. I. T. Kennedy ◽  
C A Fewson
Keyword(s):  
Alcohol Dehydrogenase ◽  
Benzyl Alcohol ◽  
Benzoylformate Decarboxylase ◽  
Heat Stable ◽  
Enzyme Systems ◽  
Heat Labile ◽  
Hydroxybenzoate Hydroxylase ◽  
Benzaldehyde Dehydrogenase

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.

scholarly journals ntn Genes Determining the Early Steps in the Divergent Catabolism of 4-Nitrotoluene and Toluene in Pseudomonas sp. Strain TW3

1998 ◽  
Vol 180 (8) ◽  
pp. 2043-2049 ◽  
Author(s):  
Keith D. James ◽  
Peter A. Williams
Keyword(s):  
Alcohol Dehydrogenase ◽  
Benzyl Alcohol ◽  
Open Reading Frame ◽  
Pseudomonas Sp ◽  
Tol Plasmid ◽  
Reading Frame ◽  
Toluene Monooxygenase ◽  
Dehydrogenase Gene ◽  
Amino Acid Levels ◽  
Benzaldehyde Dehydrogenase

ABSTRACT Pseudomonas sp. strain TW3 is able to oxidatively metabolize 4-nitrotoluene and toluene via a route analogous to the upper pathway of the TOL plasmids. We report the sequence and organization of five genes, ntnWCMAB*, which are very similar to and in the same order as the xyl operon of TOL plasmid pWW0 and present evidence that they encode enzymes which are expressed during growth on both 4-nitrotoluene and toluene and are responsible for their oxidation to 4-nitrobenzoate and benzoate, respectively. These genes encode an alcohol dehydrogenase homolog (ntnW), an NAD+-linked benzaldehyde dehydrogenase (ntnC), a two-gene toluene monooxygenase (ntnMA), and part of a benzyl alcohol dehydrogenase (ntnB*), which have 84 to 99% identity at the nucleotide and amino acid levels with the corresponding xylWCMABgenes. The xylB homolog on the TW3 genome (ntnB*) appears to be a pseudogene and is interrupted by a piece of DNA which destroys its functional open reading frame, implicating an additional and as-yet-unidentified benzyl alcohol dehydrogenase gene in this pathway. This conforms with the observation that the benzyl alcohol dehydrogenase expressed during growth on 4-nitrotoluene and toluene differs significantly from the XylB protein, requiring assay via dye-linked electron transfer rather than through a nicotinamide cofactor. The further catabolism of 4-nitrobenzoate and benzoate diverges in that the former enters the hydroxylaminobenzoate pathway as previously reported, while the latter is further metabolized via the β-ketoadipate pathway.

scholarly journals Substrate-specificity of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase encoded by TOL plasmid pWW0. Metabolic and mechanistic implications

1992 ◽  
Vol 283 (3) ◽  
pp. 789-794 ◽  
Author(s):  
J P Shaw ◽  
F Schwager ◽  
S Harayama
Keyword(s):  
Alcohol Dehydrogenase ◽  
Pseudomonas Putida ◽  
Benzyl Alcohol ◽  
Tol Plasmid ◽  
Benzyl Alcohols ◽  
Substrate Specificities ◽  
Electronic Nature ◽  
Catalysed Oxidation ◽  
Substituted Benzaldehydes ◽  
Benzaldehyde Dehydrogenase

The substrate-specificities of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, encoded by TOL plasmid pWW0 of Pseudomonas putida mt-2, were determined. The rates of benzyl alcohol dehydrogenase-catalysed oxidation of substituted benzyl alcohols and reduction of substituted benzaldehydes were independent of the electronic nature of the substituents at positions 3 and 4. Substitutions at position 2 of benzyl alcohol affected the reactivity of benzyl alcohol dehydrogenase: the velocity of the benzyl alcohol dehydrogenase-catalysed oxidation was lower for compounds possessing electron-withdrawing substitutions. In the reverse reaction of benzyl alcohol dehydrogenase, none of the substitutions tested influenced the apparent kcat. values. The rates of benzaldehyde dehydrogenase-catalysed oxidation of substituted benzaldehydes were influenced by the electronic nature of the substitutions: electron-withdrawing groups at positions 3 and 4 favoured the oxidation of benzaldehydes. Substitution at position 2 of benzaldehyde greatly diminished the benzaldehyde dehydrogenase-catalysed oxidation. Substitution at position 2 with electron-donating groups essentially abolished reactivity, and only substitutions that were strongly electron-withdrawing, such as nitro and fluoro groups, permitted enzyme-catalysed oxidation. The influence of the electronic nature and the position of substitutions on the aromatic ring of the substrate on the velocity of the catalysed reactions provided some indications concerning the transition state during the oxidation of the substrates, and on the rate-limiting steps of the enzymes. Pseudomonas putida mt-2 containing TOL plasmid pWW0 cannot grow on toluene derivatives substituted at position 2, nor can it grow on 2-substituted benzyl alcohols or aldehydes. One of the reasons for this may be the substrate-specificities of the benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase.

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