Abstract
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.
Highlights
Bacterial oxygenases are important biological catalysts incorporating one or two oxygen atoms into organic compounds, which is often very difficult to perform via chemical reactions [1]
We observed that trans-anethole oxygenase (TAO) had both monooxygenase and reductase activities, no monooxygenase or flavin reductase catalytic subunits were found in TAO
TAO is characterized as a novel self-sufficient flavoprotein monooxygenase
Summary
Bacterial oxygenases are important biological catalysts incorporating one or two oxygen atoms into organic compounds, which is often very difficult to perform via chemical reactions [1]. It has been postulated that the closer location between the oxygenase and reductase components in the self-sufficient enzymes results in a reduction in auto-oxidation caused by reactive oxygen species, such as hydrogen peroxide, between the two components [11,14]. This subsequently results in an increased diffusion of the reduced FAD in the interprotein transfer process. Enzymatic kinetics of GST-TAO was investigated using diverse substrates and cofactors Results of these studies indicated that TAO is likely a novel self-sufficient flavoprotein monooxygenase
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