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Journal of Bacteriology, December 2000, p. 6565-6569, Vol. 182, No. 23
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Conversion of 4-Hydroxyacetophenone into 4-Phenyl Acetate by a Flavin Adenine Dinucleotide-Containing Baeyer-Villiger-Type Monooxygenase

Adam Tanner and David J. Hopper*

Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion SY23 3DD, United Kingdom

Received 23 June 2000/Accepted 24 August 2000

An arylketone monooxygenase was purified from Pseudomonas putida JD1 by ion exchange and affinity chromatography. It had the characteristics of a Baeyer-Villiger-type monooxygenase and converted its substrate, 4-hydroxyacetophenone, into 4-hydroxyphenyl acetate with the consumption of one molecule of oxygen and oxidation of one molecule of NADPH per molecule of substrate. The enzyme was a monomer with an Mr of about 70,000 and contained one molecule of flavin adenine dinucleotide (FAD). The enzyme was specific for NADPH as the electron donor, and spectral studies showed rapid reduction of the FAD by NADPH but not by NADH. Other arylketones were substrates, including acetophenone and 4-hydroxypropiophenone, which were converted into phenyl acetate and 4-hydroxyphenyl propionate, respectively. The enzyme displayed Michaelis-Menten kinetics with apparent Km values of 47 µM for 4-hydroxyacetophenone, 384 µM for acetophenone, and 23 µM for 4-hydroxypropiophenone. The apparent Km value for NADPH with 4-hydroxyacetophenone as substrate was 17.5 µM. The N-terminal sequence did not show any similarity to other proteins, but an internal sequence was very similar to part of the proposed NADPH binding site in the Baeyer-Villiger monooxygenase cyclohexanone monooxygenase from an Acinetobacter sp.

* Corresponding author. Mailing address: Institute of Biological Sciences, University of Wales, Aberystwyth, Cledwyn Bldg., Ceredigion SY23 3DD, United Kingdom. Phone: 01970 622292. Fax: 01970 622307. E-mail: dvh{at}aber.ac.uk.

Journal of Bacteriology, December 2000, p. 6565-6569, Vol. 182, No. 23
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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