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 MichaelisMenten kinetics with apparent K m values of 47 M for 4-hydroxyacetophenone, 384 M for acetophenone, and 23 M for 4-hydroxypropiophenone. The apparent K m value for NADPH with 4-hydroxyacetophenone as substrate was 17.5 M. 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.One of the strategies used by microorganisms for the aerobic metabolism of ketones in degradative processes is to perform the biological equivalent of the Baeyer-Villiger reaction. This involves the insertion of an oxygen between the keto carbon and an adjacent carbon to form an ester, which can then be readily hydrolyzed. This sequence has been described for the degradation of a number of cyclic ketones, for example cyclohexanone and camphor, which yield lactones on oxygenation (6,20), and for the splitting of aliphatic ketones such as 2-tridecanone (1) or the removal of the side chain of progesterone (13). Several of the enzymes involved in the oxygenating reaction have been purified, and the cyclohexanone monooxygenase from an Acinetobacter species has been studied in great detail and particularly well characterized (2, 7, 21). Generally they have the properties of flavoprotein monooxygenases with a requirement for reduced NAD or NADP, although the enzymes that lactonize the enantiomers of camphor require an additional protein to transfer electrons from the NADH to the monooxygenase component (20). Some of these enzymes have broad substrate specificities, including the ability to oxidize sulfur or selenium atoms in organic compounds, and they are capable of forming chiral products in high enantiomeric excess. Consequently, there has been considerable interest in this class of enzyme because of the potential use for the biological production of chiral synthons, and some of these enzymes are now produced commercially (14,15).In addition to the metabolism of cyclic and aliphatic ketones, the degradation of aryl ketones can also proceed by formation of an ester, apparently by the action of a Baeyer-Villiger type of monooxygenase. In the...

Cloning and Characterization of a Gene Cluster Involved in Cyclopentanol Metabolism in Comamonas sp. Strain NCIMB 9872 and Biotransformations Effected by Escherichia coli -Expressed Cyclopentanone 1,2-Monooxygenase

“…(11,12); a novel 4-hydroxyacetophenone monooxygenase from two Pseudomonas spp. (37,65); and CHMO activities from a black yeast (29) and the fungus Cunninghamella echinulata (3). To date, the complete pathway genes of only the cyclohexanol metabolism in Acinetobacter sp.…”

Anaerobic Degradation of p -Ethylphenol by “ Aromatoleum aromaticum ” Strain EbN1: Pathway, Regulation, and Involved Proteins

“…In Pseudomonas putida JD1, the aerobic p-ethylphenol degradation pathway is initiated by an oxygen-independent hydroxylation (9). However, subsequent reaction steps include monooxygenase activity (48), necessitating a different pathway under anoxic conditions.…”

Baeyer−Villiger Monooxygenases: More Than Just Green Chemistry

“…revealed high similarity in an internal part of the protein. 52 Purification, gene cloning, sequence analysis, and biochemical characterization of 4-hydroxyacetophenone monooxygenase (HAPMO) from P. fluorescens ACB was reported by the group of Janssen one year after purification of the aryl ketone MO. 49 Purified HAPMO was reported to be a FAD-containing homodimer of 140 kDa and significant similarities (∼33%) in the protein sequence were found with CHMO and SMO.…”

“…strain 101 indicated three metabolic pathways, two of them involving enzymatic BV reactions. 136 The first aromatic ketone degrading enzyme to be purified was the aryl ketone MO from P. putida JD1, 52 a strain which was previously shown to be responsible for the conversion of aromatic ketones. 133 Biochemical characterization showed that the enzyme contains one molecule of FAD and has a molecular weight of M r ≈ 70 kDa.…”