Purification, characterization and gene cloning of isoeugenol-degrading enzyme from Pseudomonas putida IE27.
An isoeugenol-degrading enzyme was purified to homogeneity from Pseudomonas putida IE27, an isoeugenol-assimilating bacterium. The purified enzyme was a 55 kDa monomer and catalyzed the initial step of isoeugenol degradation, the oxidative cleavage of the side chain double-bond of isoeugenol, to form vanillin. Another reaction product of isoeugenol degradation besides vanillin was identified to be acetaldehyde. The values of Km and k (cat) for isoeugenol were 175 muM and 5.18 s(-1), respectively. The purified enzyme catalyzed the incorporation of an oxygen atom from either molecular oxygen or water into vanillin, suggesting that the isoeugenol-degrading enzyme is a kind of monooxygenase. The gene encoding the isoeugenol-degrading enzyme and its flanking regions were isolated from P. putida IE27. The amino acid sequence of the enzyme was similar to those of lignostilbene-alpha,beta-dioxygenases, carotenoid monooxygenases and 9-cis-epoxycarotenoid dioxygenases.
Yamada
Mamoru
M
Department of Biomolecular Science, Gifu University, Yanagido 1-1, Gifu, 501-1193, Japan.
Okada
Yukiyoshi
Y
Yoshida
Toyokazu
T
Nagasawa
Toru
T
D000079:Acetaldehyde / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001709:Biotechnology / Q000379:methods; D003001:Cloning, Molecular; D005054:Eugenol / Q000031:analogs & derivatives* / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D005054:Eugenol; C036643:isoeugenol; D006899:Mixed Function Oxygenases; D000079:Acetaldehyde