Study of the Mechanism of Coenzyme Specificity of Phenylacetone Monooxygenase from Thermobifida fusca by Site-Directed Mutagenesis

Phenylacetone monooxygenase from Thermobifida fusca (EC 1.14.13.92, PAMO) belongs to the Baeyer–Villiger family of monooxygenases and catalyzes the oxidation of various aromatic ketones to the corresponding esters using NADPH as a cofactor. In this study we analyzed the structure of the cofactor binding site, selected the most important amino acid residues for recognition of the phosphate group of the cofactor, and simulated enzyme structures with amino acid substitutions that could potentially lead to a change in the coenzyme specificity of the enzyme from NADPH to NADH. Based on the modeling, the most promising amino acid substitutions, T218D, T218E, K336A, and K336R, were proposed. Using site-directed mutagenesis we obtained genetic constructs containing genes encoding PAMOs with the corresponding amino acid substitutions, and the enzymes were expressed and purified. The resulting mutant PAMOs are able to bind NADH, but lack the ability to catalyze the oxidation of benzylacetone in the presence of NADH and show a deterioration of the Michaelis constants for NADPH. The catalytic constants of the mutant enzymes studied decrease slightly, and remain within the allowed experimental error.