Purification and characterization of a microbial dehydrogenase: a vanillin:NAD(P)+ oxidoreductase.
Pseudomonas fluorescens (strain BTP9) was found to have at least two NAD(P)-dependent vanillin dehydrogenases: one is induced by vanillin, and the other is constitutive. The constitutive enzyme was purified by ammonium sulfate fractionation, gel-filtration, and Q-Sepharose chromatography. The subunit Mr value was 55,000, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native Mr value estimated by gel-filtration chromatography gave a value of 210,000. The enzyme made use of NAD+ less effectively than NADP+. Benzaldehyde, 4-hydroxybenzaldehyde, hexanal, and acetaldehyde were not oxidized at detectable rates in the presence of NAD+ or NADP+. The ultraviolet absorption spectrum indicated that there is no cofactor or prosthetic group bound. The vanillin oxidation reaction was essentially irreversible. The pH optimum was 9.5 and the pI of the enzyme was 4.9. Enzyme activity was not affected when assayed in the presence of salts, except FeCl2. The enzyme was inhibited by the thiol-blocking reagents 4-chloromercuribenzoate and N-ethylmaleimide. NAD+ and NADP+ protected the enzyme against such a type of inhibition along with vanillin to a lesser extent. The enzyme exhibited esterase activity with 4-nitrophenyl acetate as substrate and was activated by low concentrations of NAD+ or NADP+. We compared the properties of the enzyme with those of some well-characterized microbial benzaldehyde dehydrogenases.
Baré
Ghislain
G
Walloon Centre of Industrial Biology, University of Liege, Sart-Tilman, Belgium. g.bare@ulg.ac.be
Swiatkowski
Thierry
T
Moukil
Ahmed
A
Gerday
Charles
C
Thonart
Philippe
P
D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism*; D001547:Benzaldehydes / Q000494:pharmacology; D002474:Cell-Free System; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004790:Enzyme Induction; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000201:enzymology*
D001547:Benzaldehydes; D007527:Isoenzymes; D009249:NADP; C100058:vanillin; D000445:Aldehyde Oxidoreductases; C105452:vanillin dehydrogenase