Conversion of 4-hydroxyacetophenone into 4-phenyl acetate by a flavin adenine dinucleotide-containing Baeyer-Villiger-type monooxygenase

Abstract

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 M(r) 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 K(m) values of 47 microM for 4-hydroxyacetophenone, 384 microM for acetophenone, and 23 microM for 4-hydroxypropiophenone. The apparent K(m) value for NADPH with 4-hydroxyacetophenone as substrate was 17.5 microM. 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.

FIG. 1

The reaction catalyzed by 4-hydroxyacetophenone monooxygenase to convert 4-hydroxyacetophenone into 4-phenyl acetate.

FIG. 2

SDS-PAGE of samples from the stages in purification of 4-hydroxyacetophenone monooxygenase. Lane a, crude extract (20 μg); lane b, protein after Q-Sepharose FF chromatography (10 μg); lane c, pure enzyme (7 μg); lane d, high-molecular-weight markers; lane e, pure enzyme (3.5 μg); lane f, low-molecular-weight markers.

FIG. 3

UV/visible absorbance spectra of enzyme (A) and extracted flavin (B). The spectra in A are of concentrated enzyme (2.3 mg/ml) (–––) to show the features between 300 and 550 nm and of diluted enzyme (0.56 mg/ml) (——) to show the UV peak.

FIG. 4

A comparison of an internal amino acid sequence from 4-hydroxyacetophenone with sequences from a putative cyclohexanone monooxygenase from P. fluorescens DSM 50106 (11) and the proposed NADP⁺-binding site in cyclohexanone monooxygenase from Acinetobacter sp. strain NCIMB 9871 (2).