Characterization of a Pseudomonas putidaAllylic Alcohol Dehydrogenase Induced by Growth on 2-Methyl-3-Buten-2-ol

ABSTRACT We have been working to develop an enzymatic assay for the alcohol 2-methyl-3-buten-2-ol (232-MB), which is produced and emitted by certain pines. To this end we have isolated the soil bacteriumPseudomonas putida MB-1, which uses 232-MB as a sole carbon source. Strain MB-1 contains inducible 3-methyl-2-buten-1-ol (321-MB) and 3-methyl-2-buten-1-al dehydrogenases, suggesting that 232-MB is metabolized by isomerization to 321-MB followed by oxidation. 321-MB dehydrogenase was purified to near-homogeneity and found to be a tetramer (151 kDa) with a subunit mass of 37,700 Da. It catalyzes NAD+-dependent, reversible oxidation of 321-MB to 3-methyl-2-buten-1-al. The optimum pH for the oxidation reaction was 10.0, while that for the reduction reaction was 5.4. 321-MB dehydrogenase oxidized a wide variety of aliphatic and aromatic alcohols but exhibited the highest catalytic specificity with allylic or benzylic substrates, including 321-MB, 3-chloro-2-buten-1-ol, and 3-aminobenzyl alcohol. The N-terminal sequence of the enzyme contained a region of 64% identity with the TOL plasmid-encoded benzyl alcohol dehydrogenase of P. putida. The latter enzyme and the chromosomally encoded benzyl alcohol dehydrogenase ofAcinetobacter calcoaceticus were also found to catalyze 321-MB oxidation. These findings suggest that 321-MB dehydrogenase and other bacterial benzyl alcohol dehydrogenases are broad-specificity allylic and benzylic alcohol dehydrogenases that, in conjunction with a 232-MB isomerase, might be useful in an enzyme-linked assay for 232-MB.

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

Biochemical Journal ◽

10.1042/bj3301375 ◽

1998 ◽

Vol 330 (3) ◽

pp. 1375-1381 ◽

Cited By ~ 11

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.

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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

Biochemical Journal ◽

10.1042/bj2730099 ◽

1991 ◽

Vol 273 (1) ◽

pp. 99-107 ◽

Cited By ~ 10

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.

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

Biochemical Journal ◽

10.1042/bj2500743 ◽

1988 ◽

Vol 250 (3) ◽

pp. 743-751 ◽

Cited By ~ 29

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.

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Regulation of Synthesis of Benzyl Alcohol Dehydrogenase in Acinetobacter calcoaceticus NCIB8250

Journal of General Microbiology ◽

10.1099/00221287-103-1-127 ◽

1977 ◽

Vol 103 (1) ◽

pp. 127-140 ◽

Cited By ~ 13

Author(s):

J. D. Beggs ◽

C. A. Fewson

Keyword(s):

Alcohol Dehydrogenase ◽

Benzyl Alcohol ◽

Acinetobacter Calcoaceticus ◽

Regulation Of Synthesis

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Characterization of four Rhodococcus alcohol dehydrogenase genes responsible for the oxidation of aromatic alcohols

Applied Microbiology and Biotechnology ◽

10.1007/s00253-005-0204-6 ◽

2005 ◽

Vol 71 (6) ◽

pp. 824-832 ◽

Cited By ~ 13

Author(s):

Xue Peng ◽

Hironori Taki ◽

Syoko Komukai ◽

Mitsuo Sekine ◽

Kaneo Kanoh ◽

...

Keyword(s):

Alcohol Dehydrogenase ◽

Aromatic Alcohols

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Conversion of Pyruvate Under Natural and Artificial Anaerobiosis in Maize

Functional Plant Biology ◽

10.1071/pp9760755 ◽

1976 ◽

Vol 3 (6) ◽

pp. 755 ◽

Cited By ~ 2

Author(s):

S Leblova ◽

J Zima ◽

E Perglerova

Keyword(s):

Molecular Weight ◽

Alcohol Dehydrogenase ◽

Chelating Agents ◽

Competitive Inhibitor ◽

Cyclohexanone Oxime ◽

Alcohol Dehydrogenases ◽

Sh Groups ◽

Competitive Inhibitors ◽

Optimum Ph ◽

Probable Presence

Lactate is formed during the initial hours of seed swelling during natural anaerobiosis in maize. Ethanol is formed later, at a concentration greater by one order. With 7-day-old seedlings, first lactate and then ethanol are also formed after transfer of the plants to an atmosphere of nitrogen. Lactate and alcohol dehydrogenases are active in the germinating seed. The molecular weight of maize alcohol dehydrogenase (EC 1.1.1.1) is 62 000 � 5000. Inhibition by chelating agents and 'sulphydryl poisons' indicates the probable presence of metal and -SH groups. The enzyme oxidizes ethanol at an optimum pH of 8.7 with a Km of 1.8 x 10-2 M and reduces acetaldehyde at an optimum pH of 6.7 with a Km of 1.0 x 10-3M. It is inhibited by succinate, malate, lactate and acetate, non-competitively with respect to the substrate. Acetoxime is a competitive inhibitor and butyrylamide, acetamide and cyclohexanone oxime are non-competitive inhibitors.

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The aromatic alcohol dehydrogenases in Pseudomonas putida N.C.I.B. 9869 grown on 3,5-xylenol and p-cresol

Biochemical Journal ◽

10.1042/bj1750659 ◽

1978 ◽

Vol 175 (2) ◽

pp. 659-667 ◽

Cited By ~ 6

Author(s):

M J Keat ◽

D J Hopper

Keyword(s):

Alcohol Dehydrogenase ◽

Pseudomonas Putida ◽

The Other ◽

Alcohol Dehydrogenases ◽

Whole Cells ◽

Aromatic Alcohol ◽

Aromatic Alcohols ◽

Hydroxybenzyl Alcohol

Whole cells of Pseudomonas putida N.C.I.B 9869, when grown on either 3,5-xylenol or p-cresol, oxidized both m- and p-hydroxybenzyl alcohols. Two distinct NAD+-dependent m-hydroxybenzyl alcohol dehydrogenases were purified from cells grown on 3,5-xylenol. Each is active with a range of aromatic alcohols, including both m- and p-hydroxybenzyl alcohol, but differ in their relative rates with the various substrates. An NAD+-dependent alcohol dehydrogenase was also partially purified from p-cresol grown cells. This too was active with m- and p-hydroxybenzyl alcohol and other aromatic alcohols, but was not identical with either of the other two dehydrogenases. All three enzymes were unstable, but were stabilized by dithiothreitol and all were inhibited with p-chloromercuribenzoate. All were specific for NAD+ and each was shown to catalyse conversion of alcohol into aldehyde.

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