Kinetic characterization of acetone monooxygenase from Gordonia sp. strain TY-5.

Acetone monooxygenase (ACMO) is a unique member of the Baeyer-Villiger monooxygenase (BVMO) family based on its ability to act on small ketones, such as acetone. Herein, we performed a kinetic analysis of ACMO from the propane-utilizing bacterium Gordonia sp. strain TY-5 to assess its preference for smaller ketone substrates. Steady state kinetic analysis of ACMO with a range of linear (C3-C7) and cyclic ketones (C4-C6) reveals that the enzyme elicits the highest catalytic efficiency towards butanone and cyclobutanone. Stopped-flow and inhibition studies further revealed that ACMO has a relatively weak binding affinity for the coenzyme with a dissociation constant of 120 μM. We show through mutagenesis that sequence variation in the residue that coordinates to the 2'-phosphate of NADP(H) partially accounts for the weaker binding affinity observed. As for shown for related BVMOs, NADP stabilizes the C4a-peroxyflavin intermediate in ACMO; however, the rate of its formation is considerably slower in ACMO. The observed rate constant for NADPH-dependent flavin reduction was 60 s at 25 °C, and experiments performed with 4(R)-[4-H]NADPH confirm that the C4-pro-R-hydride from the nicotinamide ring is transferred to the FAD. The latter experimental result suggests that the nicotinamide ring rotates within the active site to carry out its two functional roles: reduction of the FAD cofactor and stabilization of the C4a-peroxyflavin adduct.