TABLE 2.

Relative activities of the wild-type and recombinant ONP 2-monooxygenases under various conditions or with different substrates

Conditions or substrate	Relative activity (%)
	Wild type	Recombinant
Conditions for assay with ONPa
Cell extract + NADPH	16	11
Cell extract + NADPH + Mgb	100	100
Cell extract + NADPH + Mg + FAD	108	108
Cell extract + NADH + Mg	8	8
Cell extract + NADPH + Mg (Tris-Cl buffer)	73	63
Substrates used for assayc
ONPb	100	100
4-Methyl-2-nitrophenol	109	107
5-Fluoro-2-nitrophenol	17	20

^aMost assay mixtures contained ONP (0.1 mM) and cell extract (about 50 μg/ml) in phosphate buffer (20 mM, pH 7.5); the exception was the mixture containing Tris-Cl buffer (50 mM, pH 7.5). The concentrations of other components were as follows: NAD(P)H, 0.4 mM; Mg²⁺, 4 mM; and flavin adenine dinucleotide (FAD), 0.05 mM.

^bThe activity determined in the enzyme assay (with about 50 μg/ml cell extract, 0.4 mM NADPH, and 4 mM Mg²⁺ in 500 μl [final volume] of 20 mM phosphate buffer [pH 7.5]) using 0.1 mM ONP was defined as 100%. The specific activities of cell extracts containing wild-type and recombinant ONP 2-monooxygenases were 57.3 and 44.6 U/g, respectively.

^cAll assay mixtures contained 0.1 mM substrate, 4 mM Mg²⁺, 0.4 mM NADPH, and cell extract (about 50 μg/ml) in phosphate buffer (20 mM, pH 7.5).