4-Hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB - A novel flavoprotein catalyzing Baeyer-Villiger oxidation of aromatic compounds

A novel flavoprotein that catalyses the NADPH-dependent oxidation of 4-hydr oxyacetophenone to 4-hydroxyphenyl acetate, was purified to homogeneity fro m Pseudomonas fluorescens ACB. Characterization of the purified enzyme show ed that 4-hydroxyacetophenone monooxygenase (HAPMO) is a homodimer of appro ximate to 140 kDa with each subunit containing a noncovalently bound FAD mo lecule. HAPMO displays a tight coupling between NADPH oxidation and substra te oxygenation. Besides 4-hydroxyacetophenone a wide range of other acetoph enones are readily converted via a Baeyer-Villiger rearrangement reaction i nto the corresponding phenyl acetates. The P. fluorescens HAPMO gene (hapE) was characterized. It encoded a 640 amino-acid protein with a deduced mass of 71 884 Da. Except for an N-terminal extension of approximate to 135 res idues, the sequence of HAPMO shares significant similarity with two known t ypes of Baeyer-Villiger monooxygenases: cyclohexanone monooxygenase (27-33% sequence identity) and steroid monooxygenase (33% sequence identity). The HAPMO sequence contains several sequence motifs indicative for the presence of two Rossman fold domains involved in FAD and NADPH binding. The functio nal role of a recently identified flavoprotein sequence motif (ATG) was exp lored by site-directed mutagenesis. Replacement of the strictly conserved g lycine (G490) resulted in a dramatic effect on catalysis. From a kinetic an alysis of the G490A mutant it is concluded that the observed sequence motif serves a structural function which is of importance for NADPH binding.