Table 2.

Initial rate kinetics of active AA5_2 enzymes*

Enzyme	Substrate	KM (mM)	kcat (s−1)	kcat/KM (M−1.s−1)	Condition
ExeGalOx	Galactose	25 ± 4.0	130 ± 3.7	(5.2 ± 0.8) × 103	pH 6.0, 49 °C
	Melibiose	25 ± 0.6	37 ± 0.2	(1.5 ± 0.03) × 103
MreGalOx	Galactose	64 ± 4.8	32 ± 0.7	(5.0 ± 0.4) × 102	pH 6.0, 55 °C
	Melibiose	n.d.§	n.d.§	(1.8 ± 0.8) × 102
FoxGalOxB	Galactose	24 ± 1.3	81 ± 1.0	(3.4 ± 0.2) × 103	pH 8.0, 39 °C
	Melibiose	n.d.§	n.d.§	(5.9 ± 1.7) × 103
PfeGalOx	Glycerol	69 ± 1.6	140 ± 1.1	(2.0 ± 0.1) × 103	pH 8.5, 30 °C
	Galactose	28 ± 6.7	140 ± 4.5	(5.0 ± 1.2) × 103
UmaRafOx	Glycerol	2900 ± 720	130 ± 24	45 ± 14	pH 8.5, 30 °C
	Raffinose	450 ± 94	130 ± 15	290 ± 69
PhuRafOx	Glycerol	2900 ± 390	56 ± 3.8	19 ± 2.9	pH 8.5, 30 °C
	Raffinose	410 ± 33	8.8 ± 0.5	21 ± 2.1
AflAlcOx	Benzyl Alcohol	19 ± 1.9	410 ± 11	(2.1 ± 0.2) × 104	pH 8.5, 39 °C
	HMF	5.0 ± 1.3	190 ± 20	(3.8 ± 1.1) × 104
	Galactose	980 ± 61	400 ± 10	(4.1 ± 0.3) × 102
PruAA5_2A (PruAlcOx)	Glycerol	34 ± 1.7	170 ± 3.0	(4.9 ± 0.3) × 103	pH 7.5, 35 °C
	Galactose	110 ± 18	220 ± 8.6	(2.0 ± 0.3) × 103
	Benzyl Alcohol	9.2 ± 1.8	110 ± 5.0	(1.2 ± 0.2) × 104
FoxAlcOx	Benzyl Alcohol	230 ± 70	24 ± 4.5	100 ± 37	pH 8.5, 45 °C
	Galactose	2000 ± 650	8.3 ± 1.7	4.1 ± 1.6
PorAlcOx	Benzyl Alcohol	1.3 ± 0.10	8.6 ± 0.15	(6.6 ± 0.5) × 103	pH 7.5, 30 °C
	Butanol	4.6 ± 0.30	7.9 ± 0.09	(1.7 ± 0.1) × 103
	Glycerol	57 ± 8.1	8.5 ± 0.32	(1.5 ± 0.2) × 102
	1,4-Butanediol	4.3 ± 0.56	9.5 ± 0.51	(2.2 ± 0.3) × 103
CglAlcOx	Benzyl Alcohol	3.6 ± 0.8	350 ± 20	(9.7 ± 2.2) × 104	pH 8.0, 30 °C
	HMF	0.09 ± 0.01	240 ± 16	(2.7 ± 0.3) × 106
	1,4-Butanediol	3.9 ± 0.4	330 ± 13	(8.5 ± 0.9) × 104
AsyAlcOx	Benzyl Alcohol	79 ± 22	17 ± 1.8	(2.2 ± 0.6) × 102	pH 8.0, 30 °C
	Glycerol	700 ± 80	38 ± 2.0	54 ± 6.8

^*Error bars represent the standard deviation from the mean values

§ Individual K_M and k_cat values not determinable; k_cat/K_M values obtained from slope of linear v₀ versus [S] plots (see panels B in Figs. S7–S18)