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

AMB Express

Table 1 Steady-state kinetic parameters of ACMO and GSTACMO

Enzyme	Variable substrate	k_cat (s⁻¹)	K_m (µM)	k_cat/K_m (M⁻¹ s⁻¹) × 10³
ACMO	NADPH	2.0 ± 0.1	6.7 ± 0.8	300 ± 36
ACMO	Acetone	1.4 ± 0.2	170 ± 11	8.5 ± 0.59
ACMO	Butanone	2.1 ± 0.1	0.34 ± 0.03	6000 ± 550
ACMO	2-Pentanone	1.9 ± 0.1	0.37 ± 0.06	4800 ± 760
ACMO	2-Heptanone	3.9 ± 0.1	1.5 ± 0.1	2600 ± 130
ACMO	3-Methylbutanone	2.2 ± 0.1	4.4 ± 0.5	510 ± 55
ACMO	2,4-Dimethyl-3-pentanone	1.5 ± 0.1	1500 ± 220	1.0 ± 0.15
ACMO	Cyclobutanone	2.0 ± 0.1	1.5 ± 0.2	1400 ± 190
ACMO	Cyclopentanone	4.3 ± 0.1	120 ± 11	35 ± 3.2
ACMO	Cyclohexanone	3.6 ± 0.2	2400 ± 400	1.5 ± 0.3
ACMO	Bicyclo[3.2.0]hept-2-en-6-one	1.5 ± 0.1	6.7 ± 1.3	220 ± 45
ACMO	Phenylacetone	1.0 ± 0.1	8.9 ± 1.5	112 ± 20
GSTACMO	Cyclohexanone	2.8 ± 0.1	2300 ± 380	1.2 ± 0.2
H325K	NADPH	1.6 ± 0.1	0.48 ± 0.05	3250 ± 320
H325K	Butanone	1.6 ± 0.1	0.48 ± 0.05	3250 ± 320

The experiments were performed in 50 mM HEPES–NaOH pH 7.5 at 25 °C as described in “Materials and methods”. Butanone was present at a fixed saturating concentration of 200 μM in steady-state experiments where NADPH served as the variable substrate. For experiments where the ketone served as the variable substrate, NADPH was present at 100 µM