TABLE 2.

Oxidation rates of 26DNT, 2A46DNT, and 23DNT by wild-type NDO, NDO NagAc saturation variant F350T, and NagAc saturation and DNA shuffling variant F350T/G407S^a

Substrate	Product	Wild-type NDO		F350T			F350T/G407S
		Nitrite release rate	Product formation rate	Nitrite release rate	Product formation rate	Relative to NDO	Nitrite release rate	Product formation rate	Relative to NDO	Relative to F350T
26DNTb	3-methyl-4-nitrocatechol	0	0	0.36 ± 0.07	0.3 ± 0.1c (100%)	∞	1.1 ± 0.3	0.8 ± 0.1c (100%)	∞	3d
2A46DNTe	2-amino-4,6-dinitrobenzyl alcohol	NA	0	NA	70 ± 20f (73%)g	∞	NA	170 ± 10f (81%)g	∞	2
	3-amino-4-methyl-5-nitrocatechol	0	0	0.032 ± 0.006	0.043 ± 0.008c (27%)g	∞	0.12 ± 0.04	0.12 ± 0.03c (19%)g	∞	3d
23DNTe	2,3-dintrobenzyl alcohol	NA	115h (100%)i	NA	36h (100%)i	0.3	NA	104h (100%)i	0.9	3
	4-methyl-3-nitrocatechol	0.014 ± 0.003	NDj	0.08 ± 0.02	ND	6	0.15 ± 0.01	ND	11	2

^aNitrite release rate was determined in nmol NO₂⁻/min/mg protein. Product percentages are shown in parentheses with the product formation rate. NA, not available.

^bTransformations of 26DNT were performed using E. coli TG1 with 0.5 mM substrate.

^cRate determined in nmol product/min/mg protein.

^dRelative performance is average of nitrite release rate and product formation rate calculated from identical time course experiments.

^eTransformations of 2A46DNT and 23DNT were performed using E. coli JVQ2 with 0.5 mM substrate.

^fApproximate relative activity determined by comparison of slope for product peak area at 300 nm/min/incubating suspension OD 600 nm.

^gPercentages of each product generated are relative to the total product area formed, and are based on peak area (not concentration) at 300 nm.

^hApproximate relative activity determined by comparison of slope for product peak area at 260 nm/min/incubating suspension OD 600 nm.

ⁱPercentages of each product generated are relative to the total product area formed, and are based on peak area (not concentration) at 260 nm.

^j4-methyl-3-nitrocatechol peak not detected (ND) through HPLC.