Table 1. Steady-state Kinetic Parameters for Wild-Type Tfu-FNO and Selected Tfu-FNO G29X and P89X Variants toward Selected NCBs^a.

NCB	variant	kcat (min–1)	Km (mM)	kcat/Km (s–1 M–1)	(kcat/Km)mutant/(kcat/Km)wt
AmNA+	wt	1.4 ± 0.07	12 ± 1.7	1.9 ± 0.29
	G29S	9.6 ± 0.56	18 ± 2.5	8.8 ± 1.3	4.6 ± 0.98
	G29Y	3.2 ± 0.23	23 ± 3.5	2.3 ± 0.39	1.2 ± 0.28
	P89H	2.5 ± 0.21	14 ± 3.0	3.0 ± 0.69	1.6 ± 0.44
EtOHNA+	wt	1.4 ± 0.06	20 ± 2.5	1.2 ± 0.15
	G29Y	6.2 ± 0.58	23 ± 4.7	4.5 ± 1.0	3.8 ± 0.96
	P89H	2.9 ± 0.22	20 ± 3.3	2.4 ± 0.44	2.0 ± 0.44
	P89Y	5.0 ± 0.63	33 ± 9.2	2.5 ± 0.77	2.1 ± 0.69
ProOHNA+	wt	0.25 ± 0.013	25 ± 3.0	0.17 ± 0.022
	P89Y	0.71 ± 0.096	35 ± 10	0.34 ± 0.11	2.0 ± 0.7
EtNA+	wt	0.44 ± 0.01	25 ± 1.4	0.29 ± 0.018
ProNA+	wt	0.09 ± 0.007	10 ± 2.8	0.15 ± 0.044
	G29L	0.36 ± 0.057	18 ± 7.9	0.33 ± 0.16	2.2 ± 1.3
	P89H	0.27 ± 0.026	5.7 ± 1.9	0.79 ± 0.27	5.3 ± 2.4
	P89L	0.44 ± 0.048	27 ± 6.9	0.27 ± 0.075	1.8 ± 0.73
	P89Y	0.59 ± 0.11	35 ± 14	0.28 ± 0.12	1.9 ± 0.97
BNA+	wt	0.27 ± 0.01	6.4 ± 0.60	0.70 ± 0.071
	G29W	25 ± 1.1	4.3 ± 0.68	97 ± 15	139 ± 26
	G29Y	8.7 ± 0.64	9.5 ± 2.0	15 ± 3.4	21 ± 5.3
BAP+	G29W	252 ± 14	7.4 ± 1.2	568 ± 97
BAA+	G29W	NA	NA	NA

^aThe measurements were performed at 25 °C by adding 0.1–5 μM enzyme to 50 mM Tris-HCl, 100 mM NaCl, and 3% (v/v) DMSO at pH 8.0 with a constant F₄₂₀H₂ concentration of 40 μM and varying concentrations of NCBs between 0.1 and 250 mM. Absorbance traces over time were measured and fitted to the Michaelis–Menten model. Values are means from three independent measurements ± standard deviations. NA: not active. The corresponding Michaelis–Menten plots are shown in Figures S6 and S7 of the supporting information.