Table ED1.

Summary of kinetic analyses of XyGUL glycoside hydrolases.^*

Enzyme	Substrate b	kcat (s−1)†	Km (mM)‡	kcat/Km (s−1 mM−1)‡,§	Assay||
BoGH2A	Gal-β-PNP	4.0 ± 0.092	0.087 ± 0.0074	46.0	PNP
BoGH3A	Glc-β-PNP	Trace activity			PNP
	cellobiose	Trace activity			Glc-kit
	cellotetraose	65.0 ± 3.81	2.49 ± 0.42	21.3	Glc-kit
	cellohexaose	0.62 ± 0.15	0.698 ± 0.52	0.90	Glc-kit
	GLLG	n.d.¶	n.d.	0.15	Glc-kit
	GXXG	n.d.	n.d.	3.41	Glc-kit
BoGH3B	Glc-β-PNP	1.74 ± 0.21	0.155 ± 0.039	11.2	PNP
	Gal-β-PNP	Trace activity			PNP
	cellobiose	1.57 ± 0.11	0.68 ± 0.16	2.3	Glc-kit
	cellotetraose	3.99 ± 0.31	0.23 ± 0.060	17.3	Glc-kit
	cellohexaose	4.91 ± 0.71	0.47 ± 0.21	10.4	Glc-kit
	GLLG	n.d.	n.d.	0.18	Glc-kit
	GXXG	n.d.	n.d.	3.34	Glc-kit
BoGH5A	XXXG-β-CNP	10.5 ± 0.14	0.036 ± 0.0027	291.7	CNP
	XLLG-β-CNP	11.1 ± 1.13	0.145 ± 0.024	76.5	CNP
	GGGG-β-CNP	0.12 ± 0.025	3.59 ± 1.27	0.034	CNP
	Tamarind XyG	435.3 ± 25.6	0.82 ± 0.17 mg ml−1	534.0 s−1 mg−1 ml	DNSA
	Lettuce XyG	n.d.	n.d.	543.1 s−1 mg−1 ml	DNSA
	Tomato XyG	n.d.	n.d.	501.5 s−1 mg−1 ml	DNSA
BoGH9A	Tomato XG	Active (MALDI-MS data)			DNSA
BoGH31A	Xyl-α-PNP	1.60 ± 0.031	7.7 ± 0.273	0.21	PNP
	Glc-α-PNP	0.071 ± 0.015	31.8 ± 5.1	0.0022	PNP
	XXXG	32.6 ± 2.1	0.223 ± 0.046	146.2	HPLC
	XLLG	31.0 ± 1.9	0.378 ± 0.075	82.0	HPLC
	Isoprimeverose	1.78 ± 0.21	38.1 ± 7.1	0.047	Glc-kit
BoGH43A	L-Araf-α-PNP	0.057 ± 0.001	0.71 ± 0.03	0.081	PNP
	Xyl-β-PNP	0.26 ± 0.005	6.58 ± 0.21	0.039	PNP
	Tomato XyGOs	n.d.	n.d.	0.013 s−1 mg−1 ml	GDh-kit
	Tomato XyG	Active			GDh-kit
BoGH43B	L-Araf-α-PNP	5.0 × 10−4 ± 8.1 × 10−5	6.6 ± 2.3	7.6 × 10−5	Cont. assay
	Tomato XyGOs	n.d.	n.d.	0.0024 s−1 mg−1 ml	GDh-kit
	Tomato XyG	Active			GDh-kit

^*pH-rate profiles, full kinetic data plots, and carbohydrate product analyses are available in Supplementary Figures S1–S20 for all enzymes.

^†Data is only given for those substrates on which a given enzyme was active. For example, BoGH5A and BoGH9A were not active on arabinoxylan, barley mixed-linkage glucan, curdlan, galactan, gluco- and galactomannan, lichenan, carboxymethyl cellulose, or hydroxyethyl cellulose. The full panel of chromogenic and natural substrates against which enzymes were tested is described in the Methods (Supplementary Information).

^‡These units apply to all values in the column, unless otherwise stated. Standard errors of means are indicated.

^§For substrates for which individual k_cat and K_m values are not reported, k_cat/K_m values were obtained from linear curve fitting to initial rate data in the [S] ≪ K_m(apparent) regime, where the standard Michaelis-Menten equation reduces to v_o = k_cat[E][S]/K_m

^¶n.d.: not determined.