Table 1 Kinetic properties of AKRtyl and its mutants to cofactor NADPH and substrate tylosin.^a

Ligands	Proteins	K_m or K_0.5 (μM)^d	k_cat (s⁻¹)^d	k_cat/K_m (s⁻¹ μM⁻¹)^d	K_i (μM)^d	Hill coeff n^e
NADPH^b	WT	2.98 ± 0.16	4.38 ± 0.09	1470.0 × 10^–3	148.6 ± 8.9 (Inhibition)	-
	W331A	11.03 ± 1.09	0.18 ± 0.01	16.3 × 10^–3	1541.0 ± 699.1 (Loss inhibition)	-
	W331A^f	8.33 ± 0.60	0.16 ± 0.01	19.3 × 10^–3	-	1.17 ± 0.08 (Slight pos. coop.)
Tylosin^c	WT	214.51 ± 17.17	1.44 ± 0.05	6.71 × 10^–3	-	1.76 ± 0.19 (Pos. coop.)
	WT^g	278.00 ± 12.32	4.65 ± 0.09	16.72 × 10^–3	-	1.76 ± 0.11 (Pos. coop.)
	E193A	501.70 ± 176.30	1.10 ± 0.15	2.20 × 10^–3	-	0.99 ± 0.18 (Loss of coop.)
	E193W	1147.98 ± 334.21	0.56 ± 0.07	0.49 × 10^–3	-	1.05 ± 0.12 (Loss of coop.)
	R195A	333.70 ± 32.77	1.28 ± 0.05	3.84 × 10^–3	-	1.17 ± 0.09 (Reduced coop.)
	R195W	690.83 ± 112.26	1.37 ± 0.10	1.98 × 10^–3	-	1.08 ± 0.09 (Loss of coop.)
	Q254W	329.70 ± 46.26	1.20 ± 0.65	3.63 × 10^–3		1.12 ± 0.11 (Reduced coop.)
	R257A	382.19 ± 57.17	1.39 ± 0.09	3.64 × 10^–3	-	1.38 ± 0.20 (Reduced coop.)
	R257W	372.01 ± 99.22	1.31 ± 0.13	3.52 × 10^–3	-	0.95 ± 0.13 (Loss of coop.)

All measurements were carried out in triplicate and are shown as mean ± SD (standard deviation, n = 3 independent experiments).
^aThis table lists kinetic data of the important mutants and the comprehensive mutants can be found in the Supplementary Table 2.
^bThe saturation concentration of tylosin in NADPH kinetic assays: 5 mM.
^cThe saturation concentration of NADPH in most tylosin kinetic assays: 200 μM.
^dThe Michaelis-Menten constant K_m and K_0.5 in the Hill equation, the catalytic rate constant k_cat, the catalytic efficiency k_cat/K_m and the inhibition constant K_i.
^eThe Hill coefficient n of substrate binding as a measure of cooperativity (n > 1, positive cooperativity; n = 1, no cooperativity; n < 1, negative cooperativity).
^fKinetic data of the W331A mutant on NADPH are fitted with the Hill equation.
^gThe saturation concentration of NADPH in this tylosin kinetic assays: 25 μM.

Search