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

An arylketone monooxygenase was purified from Pseudomonas putida JD1 by ion exchange and affinity chromatography. It had the characteristics of a Baey er-Villiger-type monooxygenase and converted its substrate, 4-hydroxyacetop henone, into 4-hydroxyphenyl acetate with the consumption of one molecule o f 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 mole cule of flavin adenine dinucleotide (FAD). The enzyme was specific for NADP H 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 muM for 4-hydroxya cetophenone, 384 muM for acetophenone, and 23 muM for 4-hydroxypropiophenon e. The apparent K-m value for NADPH with 4-hydroxyacetophenone as substrate was 17.5 muM. The N-terminal sequence did not show any similarity to other proteins, but an internal sequence was very similar to part of the propose d NADPH binding site in the Baeyer-Villiger monooxygenase cyclohexanone mon ooxygenase from an Acinetobacter sp.