title: Purification and characterization of an NAD+-dependent XylB-like aryl alcohol dehydrogenase identified in Acinetobacter baylyi ADP1.
abstract: The gene xylB(ADP1) from Acinetobacter baylyi ADP1 (gene annotation number ACIAD1578), coding for a putative aryl alcohol dehydrogenase, was heterologously expressed in Escherichia coli BL21(DE3). The respective aryl alcohol dehydrogenase was purified by fast protein liquid chromatography to apparent electrophoretic homogeneity. The predicted molecular weight of 39,500 per subunit was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. According to the native M(w) as determined by gel filtration, the enzyme forms dimers and therefore seems to be XylB related. The enzyme showed the highest activity at 40°C. For both the reduction and the oxidation reactions, the pH for optimum activity was 6.5. The enzyme was NADH dependent and able to reduce medium- to long-chain n-alkylaldehydes, methyl-branched aldehydes, and aromatic aldehydes, with benzaldehyde yielding the highest activity. The oxidation reaction with the corresponding alcohols showed only 2.2% of the reduction activity, with coniferyl alcohol yielding the highest activity. Maximum activities for the reduction and the oxidation reaction were 104.5 and 2.3 U mg(-1) of protein, respectively. The enzyme activity was affected by low concentrations of Ag(+) and Hg(2+) and high concentrations of Cu(2+), Zn(2+), and Fe(2+). The gene xylB(ADP1) seems to be expressed constitutively and an involvement in coniferyl alcohol degradation is suggested. However, the enzyme is most probably not involved in the degradation of benzyl alcohol, anisalcohol, salicyl alcohol, vanillyl alcohol, cinnamyl alcohol, or aliphatic and isoprenoid alcohols.
authors.affiliation: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Uthoff
authors.forename: Alexander
authors.initials: A
authors.lastname: Steinbüchel
mesh_terms: D000150:Acinetobacter / Q000201:enzymology*; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D002853:Chromatography, Liquid; D003067:Coenzymes / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000378:metabolism; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002413:Cations, Divalent; D003067:Coenzymes; D008670:Metals; D011994:Recombinant Proteins; D009243:NAD; D000429:Alcohol Oxidoreductases; C055882:aryl-alcohol dehydrogenase