. Author manuscript; available in PMC: 2025 Jan 7.

Published before final editing as: ACS Catal. 2025 Jan 2:1046–1060. doi: 10.1021/acscatal.4c05944

Table 2. Steady-state kinetics.

	Substrate	k_cat (s^-1)	K_M^substrate (μM)	k_cat/K_M (s^-1 mM^-1)	k_cat (s^-1)	K_M^substrate (μM)	k_cat/K_M (s^-1 mM^-1)
VAD ^a		Molecular oxygen ^b			2,6-Dichlorophenolindophenol ^c
WT	Vanillyl alcohol	Inactive	-	-	0.44±0.02	74.2±12.4	6
WT	Eugenol	Inactive	-	-	0.81±0.06	51.2±13.1	16
WT	4-Methoxy methylphenol	Inactive	-	-	0.06±0.003	330±40	0.2
WT	4-Hydroxy benzylalcohol	Inactive	-	-	0.25±0.01	208±9	1.2
P151L^d	Vanillyl alcohol	0.83±0.05	132±30	6	0.76±0.02	72.4±6.3	10.5
P151V	Vanillyl alcohol	0.09±0.01	174±20	0.52	0.90±0.04	42.2±6.6	21
P151I	Vanillyl alcohol	0.24±0.01	159±25	1.5	0.73±0.04	37.5±9.2	19
P151G	Vanillyl alcohol	0.86±0.02	60±9	14	1.49±0.07	316.5±39.7	5
P151N	Vanillyl alcohol	Inactive	-	-	0.14±0.07	151±24	1
P151T	Vanillyl alcohol	Inactive	-	-	0.48±0.03	17.6±5.5	27
nsVAO ^e		Molecular oxygen ^b			Bovine heart cytochrome c ^f
WT	Vanillyl alcohol	0.29±0.01	207±28	1.4	0.06±0.002	91.4±12.9	0.7
T181D	Vanillyl alcohol	0.28±0.01	203±22	1.4	0.07±0.004	337±67	0.2
EUGO ^g		Molecular oxygen ^b			2,6-Dichlorophenolindophenol ^c
WT	Vanillyl alcohol	9.68±0.43	204.4±28.9	47.4	0.48±0.017	17.6±3	27.2
P152L	Vanillyl alcohol	0.02±0.001	54±5.4	0.37	0.61±0.04	868±146	0.7

Measured at 25 °C in 50 mM potassium phosphate pH 7.5 and 150 mM NaCl. Data are shown as average values ± standard deviation. n = 3 independent experiments. The kinetics plots are shown in Figure S4.

Product formation was monitored spectroscopically.

2,6-Dichlorophenolindophenol (100 μM) reduction was monitored by following the absorbance at 600 nm. No activity by VAD was detected using bovine heart cytochrome c.

Data for more substrates are reported in Table S3. No activity was detected using p-cresol.

Measured at 35 °C in 50 mM Tris-HCl pH 9.0 and 300 mM NaCl. Data are shown as average values ± standard deviation. n = 3 independent experiments. The kinetics data are shown in Figure S5. nsVAO has low activities on eugenol and 4-hydroxybenzyl alcohol.

Cytochrome c (50 μM) reduction was monitored by following the absorbance at 550 nm.

Measured at 25 °C in 50 mM potassium phosphate pH 7.5 and 150 mM NaCl. Data are shown as average values ± standard deviation. n = 3 independent experiments. The k_cat and K_M values for wild-type EUGO are very similar to the previously reported values [9]. The kinetics plots are shown in Figure S6.