Characterization of a self-sufficient trans-anethole oxygenase from Pseudomonas putida JYR-1.
A novel flavoprotein monooxygenase, trans-anethole oxygenase (TAO), from Pseudomonas putida JYR-1, which is capable of catalyzing the oxidation of trans-anethole to p-anisaldehyde, was heterologously expressed in E. coli and purified. Enzymatic kinetics of diverse substrates and cofactors revealed that TAO is likely to be a novel self-sufficient flavoprotein monooxygenase. Enzyme assays of GST-TAO demonstrated that TAO catalyzed a trans-anethole oxidation reaction without auxiliary component enzyme-like electron-transfer flavin reductases. The single component TAO had the ability to reduce flavin cofactors and simultaneously oxidize trans-anthole to p-anisaldehyde. In the processes of reduction of flavin and oxidation of trans-anethole, TAO accepted various flavin and NAD(P)H cofactors. TAO also catalyzed oxidation of isoeugenol, O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure with different catalytic efficiency. TAO had the greatest catalytic efficiency (k cat/K m) with the original substrate, trans-anethole. Investigation about partially deleted mutants of TAO indicated that reductase active sites appeared to be located near the N terminal. Site directed mutagenesis studies also proved that the proposed flavin binding sites, Trp-38, Thr-43, Tyr-55, were critical for flavin reduction. However, disruption of any portion of TAO eliminated the oxygenase activity.
School of Environmental Science and Engineering, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea.
Dongfei
D
Han
Michael J
MJ
Sadowsky
Youhoon
Y
Chong
Hor-Gil
HG
Hur
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D057075:Enzyme Assays; D010084:Oxidation-Reduction; D016958:Pseudomonas putida / Q000201:enzymology*
D001426:Bacterial Proteins