Purification and characterization of benzaldehyde dehydrogenase I from Acinetobacter calcoaceticus.
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.
Chalmers
R M
RM
Department of Biochemistry, University of Glasgow, U.K.
Fewson
C A
CA
D000150:Acinetobacter / Q000201:enzymology*; D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D050727:Benzaldehyde Dehydrogenase (NADP+); D001547:Benzaldehydes / Q000378:metabolism; D002412:Cations / Q000494:pharmacology; D002845:Chromatography; D004789:Enzyme Activation; D004950:Esterases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D001547:Benzaldehydes; D002412:Cations; D046911:Macromolecular Substances; D013439:Sulfhydryl Reagents; D009243:NAD; D000445:Aldehyde Oxidoreductases; D050727:Benzaldehyde Dehydrogenase (NADP+); D004950:Esterases; C032175:benzaldehyde