TABLE 3.

Oxidation rates of 4A2NT and 23DNT by wild-type NDO, NDO NagAc saturation variant L225R, and DNA shuffling variant G50S/L225R/A269T^a

Substrate	Product	Wild-Type NDO		L225R				G50S/L225R/A269T
		Nitrite release rate	Product formation rate	Nitrite release rate	Product formation rate	Relative to NDO	Relative to G50S/L225R/A269T	Nitrite release rate	Product formation rate	Relative to NDO
4A2NTb	4-amino-2-nitrocresol	NA	90 ± 60c (100%)d	NA	1100 ± 140c (92%)d	12	1	NA	1000 ± 40c (93%)d	11
	4-amino-2-nitrobenzyl alcohol	NA	0	NA	0.7 ± 0.1e (8%)d	∞	1	NA	0.53 ± 0.08e (7%)d	∞
23DNTb	2,3-dintrobenzyl alcohol	NA	115f (100%)g	NA	1709f (98%)g	15	3	NA	498f (100%)g	4
	4-methyl-3-nitrocatechol	0.014 ± 0.003	NDh	0.3 ± 0.1	−i (2%)g	24	2	0.19 ± 0.05	ND	14

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

^bTransformations of 4A2NT and 23DNT were performed using E. coli JVQ2 with 0.5 mM substrate.

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

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

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

^fApproximate relative activity determined by comparison of slope for product peak area at 260 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 260 nm.

^h4-methyl-3-nitrocatechol peak not detected (ND).

ⁱ4-methyl-3-nitrocatechol peak detected only after 60 minutes of incubation with 23DNT, relative activity could not be quantified through HPLC analyses.