1

Biochemical and structural characterization of a thermostable family GH62 α-L-1

arabinofuranosidase from Streptomyces thermoviolaceus to elucidate the molecular basis for 2

activity towards arabinoxylan 3

4

5

Running title: Structure functional analysis of SthAbf62A 6

7

Weijun Wang1, Galina Mai-Gisondi2, Peter J. Stogios1, Amrit Kaur1, Xiaohui Xu1, Hong Cui1, Ossi 8

Turunen2, Alexei Savchenko1, Emma R. Master 1, 2# 9

10

11

1Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College 12

Street, Toronto, Ontario, M5S 3E5, Canada. 13

2Department of Biotechnology and Chemical Technology, Aalto University, Kemistintie 1 D1, Espoo 14

02150, Finland. 15

16

#Corresponding Author 17

Emma R. Master 18

Telephone: 416-946-7861 19

Fax: 416-978-8605 20

Email: emma.master@utoronto.ca 21

22

23

AEM Accepts, published online ahead of print on 20 June 2014Appl. Environ. Microbiol. doi:10.1128/AEM.00685-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 24

Xylan debranching enzymes facilitate the complete hydrolysis of xylan and can be used to alter 25

xylan chemistry. Herein, the GH62 family α-L-arabinofuranosidase from Streptomyces thermoviolaceus 26

(SthAbf62A) was shown to have a half-life of 60 min at 60 °C, and ability to cleave α-1,3 L-27

arabinofuranose (L-Araf ) from singly-substituted xylopyranosyl (Xylp) backbone residues in wheat 28

arabinoxylan; low activity towards arabinan as well as 4-nitrophenyl α-L-arabinofuranoside was also 29

detected. After selectively removing α-1,3 L-Araf substituents from di-substituted Xylp residues present 30

in wheat arabinoxylan, SthAbf62A could also cleave the remaining α-1,2 L-Araf substituents, 31

confirming the ability of SthAbf62A to remove α-L-Araf residues that are (1→2) and (1→3) linked to 32

mono-substituted β-D-Xylp sugars. Three-dimensional structures of SthAbf62A and its complex with 33

xylotetraose and L-arabinose confirmed a five-bladed β-propeller fold and revealed a molecular Velcro 34

in blade V between the β1 and β21 strands, a disulfide bond between Cys 27 and Cys 297, and a 35

calcium ion coordinated in the central channel of the fold. The enzyme-arabinose complex structure 36

further revealed a narrow and seemingly rigid L-arabinose binding pocket situated at the center of one 37

side of the β propeller, which stabilized the arabinofuranosyl substituent through several hydrogen-38

bonding and hydrophobic interactions. The predicted catalytic amino acids were oriented towards this 39

binding pocket and the catalytic essentiality of Asp53 and Glu213 was confirmed by site-specific 40

mutagenesis. Complex structures with xylotetraose revealed a shallow cleft for xylan backbone binding 41

which is open at both ends and comprises multiple binding subsites above and flanking the L-arabinose 42

binding pocket. 43

44

45

46

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INTRODUCTION 47

Xylan is a major plant cell wall polysaccharide that has been harnessed to produce different 48

chemicals, fuels and materials; the bioactive properties of xylans are also valued in food applications 49

(1-3). Xylan consists of a linear β-1,4-linked xylopyranosyl (β-D-Xylp) backbone, which depending on 50

the source, can be partially substituted at O-2 positions and/or O-3 positions with α-L-arabinofuranosyl 51

(α-L-Araf) substituents and acetyl groups, and at O-2 positions with α-D-glucuronic acid (GlcA) or 4-52

O-methyl GlcA (MeGlcA) (4-6). The O-5 position of some arabinose residues can be further esterified 53

with p-coumaric or ferulic acid (7, 8). Whereas glucuronoxylan is the predominant form of xylan in 54

hardwood trees, xylan from coniferous softwoods contains both arabinofuranosyl and MeGlcA 55

substituents (9); xylans from cereals and grasses resemble softwood xylan although have varying 56

arabinose and MeGlcA contents. Consistent with the molecular diversity of xylans, its complete 57

enzymatic hydrolysis typically requires the concerted action of several enzymes, including endo-58

xylanases and β-xylosidases that target glycosidic linkages within the xylan backbone, along with α-L-59

arabinofuranosidases, α-glucuronidases, acetyl xylan esterases, feruloyl esterases, as well as 60

glucuronoyl esterases, which target branching substituents in different xylan types (10-12). Of these 61

activities, arabinofuransidases are represented in a particularly high number of carbohydrate-active 62

enzyme (CAZy) families (www.cazy.org). 63

Based on amino acid sequence, α-L-arabinofuranosidases (EC 3.2.1.55) have been classified into 64

glycoside hydrolase (GH) families: GH3, GH43, GH51, GH54, and GH62. Moreover, family GH1 65

includes exo-acting arabinofuranosidases acting on p-nitrophenyl-α-arabinofuranoside (pNP-AraF) and 66

α-1,5 arabino-oligosaccharides (13); family GH30 includes activities towards 1,5- α- L-arabinobiose 67

(14), and family GH93 includes exo-acting enzymes that release arabinobiose from the non-reducing 68

end of α-1,5- L-arabinan (15). Several of these enzyme families are further grouped into glycoside 69

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hydrolase clans: GH-A (GH1, GH30 and GH51), GH-E (GH93) and GH-F (GH43 and GH62), 70

reflecting the structural diversity of α- L-arabinofuranosidases. For example, the catalytic domain of 71

enzymes belonging to Clan GH-A adopt a (β/α)8 TIM barrel fold as exemplified by the structure of the 72

GH51 α- L-arabinofuranosidase from Geobacillus stearothermophilus T-6 (16). By contrast, Clan GH-73

E enzymes adopt a six-bladed β-propeller architecture (15), GH43 enzymes in Clan-GH-F display a 74

'non-Velcroed' five-bladed-β-propeller arrangement (17, 18), and the catalytic domain of the GH54 α- 75

L-arabinofuranosidase from Aspergillus kawachiia adopts a β-sandwich fold (19). 76

α- L-Arabinofuranosidases also vary in terms of substrate preference and the linkages they 77

hydrolyze. For example, GH51 as well as GH54 enzymes include α- L-arabinofuranosidases able to 78

remove branching α-L-Araf moieties from arabinan and xylans, although in most cases so far, enzymes 79

from these families tend to prefer p-nitrophenyl-α-arabinofuranoside (pNP-AraF) over polymeric 80

substrates (20-23). GH3 arabinofuranosidases include bifunctional enzymes displaying both α-L-81

arabinofuranosidase and β-D-xylopyranosidase activity when using pNP substrates (24). By comparison, 82

family GH43 includes a broad range of activities, including hydrolysis of pNP-AraF (25), endo-α-L-83

arabinanases (26, 27), exo-α-1,5-L-arabinanases (28), and bifunctional α-L-arabinofuranosidase/β-D-84

xylosidases (29). Family GH43 also includes arabinoxylan α-L-arabinofuranohydrolases (AXH) that 85

remove α-L-Araf residues that are (1→2) and (1→3) linked to mono-substituted β-D-Xylp (AXH-m 2,3) 86

(30), and that cleave α-L-Araf residues that are (1→3) linked to di-substituted β-D-Xylp (AXH-d3) (31). 87

Recently, the first 3-D structures of GH62 enzymes were published, one from Ustilago maydis 88

(UmAbf62A), one from Podospora anserina (PaAbf62A), and one from Streptomyces coelicolor 89

(ScAraf62A) (32, 33). These studies confirmed that GH62 enzymes adopt a five-bladed β-propeller 90

fold common to GH43 enzymes (32). While these studies generated important insight into structure 91

and functional characteristics of GH62 enzymes, there are comparatively few examples of detailed 92

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biochemical and structural analyses of this GH family. Given the high activity of GH62 enzymes on 93

polymeric arabinoxylan over low molecular substrates including pNP-AraF (32-38), GH62 α-L-94

arabinofuranosidases are particularly relevant candidates for fine tuning xylan chemistry as well as 95

enabling the complete saccharification of xylan to sugars. 96

Accordingly, to deepen our understanding of GH62 enzymes, we report the biochemical and 97

structural characterization of a thermostable, bacterial GH62 α-L-arabinofuranosidase (SthAbf62A) 98

from Streptomyces thermoviolaceus subsp. pingens Henssen, which has 67%, 58% and 31% amino acid 99

sequence identity to ScAraf62A, UmAbf62A and PaAbf62A, respectively. Consistent with the first 100

report of SthAbf62A by Tsujibo et al (37), SthAbf62A characterized herein released reducing sugars 101

from polymeric arabinoxylan. The current analysis further demonstrated that SthAbf62A can cleave α-102

L-Araf substituents that are (1→2) and (1→3) linked to mono-substituted β-D-Xylp in arabinoxylan and 103

can also hydrolyze non-reducing terminal α-1,5 linkages in debranched arabinan as well as pNP-104

arabinofuranoside, albeit at much lower specific activities. Structural analysis of SthAbf62A 105

complexes with L-arabinose and xylotetraose revealed a “Velcroed” five-bladed-β-propeller, and 106

provided insight into the molecular basis of substrate selectivity for this enzyme. 107

108

MATERIALS AND METHODS 109

Chemicals. Pfx polymerase was purchased from Invitrogen. The Qiagen stool kit, QIAEX II 110

Gel Extraction Kit, and QIAquick PCR purification kit, were purchased from QIAGEN. Wheat 111

arabinoxylan [high viscosity (WAX-HV) and low viscosity (WAX-LV)], rye flour arabinoxylan (RAX), 112

arabinan from sugar beet, debranched arabinan, rhamnogalacturonan (soy bean), larch arabinogalactan 113

and α-L-arabinofuranosidase from Bifidobacterium sp (E-AFAM2, AXH-d3) were purchased from 114

Megazyme, whereas oat spelt xylan (OSX), birchwood xylan, beechwood xylan and p-nitrophenyl-115

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glycosides were from Sigma-Aldrich. Soluble and insoluble fractions of OSX were prepared as 116

previously reported (39). Streptomyces thermoviolaceus subsp. pingens Henssen (ATCC#19283) was 117

purchased from the American Type Culture Collection (ATCC). All other chemicals were analytical 118

grade and were obtained from Sigma-Aldrich or Fisher Scientific. 119

DNA Manipulations. Streptomyces thermoviolaceus subsp. pingens Henssen was grown in 5 120

mL Luria-Bertani medium at 37°C for 4 days. Genomic DNA was extracted using the Qiagen stool kit 121

according to the manufacturer’s instructions and including a 5 min incubation at 95°C followed by an 122

additional bead beating step using Garnet bead tubes (Mo Bio, 0.70 mm). The gene encoding the mature 123

form of SthAbf62A (Genbank ID: KF958299) without a signal peptide (aa residues 1-35) was 124

amplified using the forward and reverse primers shown in Table S1 and the Pfx DNA polymerase 125

(Invitrogen). The infusion cloning kit from Clontech (California, USA) was used to transfer purified 126

PCR products to the p15Tv-L expression plasmid (GenBank accession EF456736), generating p15Tv-127

L_SthAbf62A. Site-specific mutagenesis was carried out according to a modified QuikChangeTM 128

(Stratagene) method that uses partially overlapping primers (Table S1) (40). All constructs were 129

verified by DNA sequencing at the Center of Applied Genomics at the SickKids Hospital in Toronto. 130

Purification of wild-type and mutant α-L-arabinofuranosidase. Recombinant Escherichia 131

coli BL21(λDE3) codon plus harboring p15Tv-L_SthAbf62A was propagated at 37 °C in 1 L of Luria-132

Bertani medium supplemented with 0.5 M d-sorbitol, 2.5 mM glycine betaine, 34 μg/mL 133

chloramphenicol and 100 μg/mL ampicillin. At OD600nm of 0.6, the cultivation temperature was 134

reduced to 15 °C and recombinant protein expression was induced with 0.5 mM isopropyl β-d-135

thiogalactopyranoside. 136

The next day, induced cultures were harvested by centrifugation at 6000 × g for 10 min. Cell pellets 137

(approx. 4.5 g fresh weight) were suspended in binding buffer [300 mM NaCl, 50 mM 4-(2-138

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hydroxyethyl)-1-piperazinepropane sulfonic acid (HEPES), pH 7.0, 5% glycerol, 5 mM imidazole] and 139

disrupted by sonication. Cell debris was removed by centrifugation (17,500 × g, 20 min) and 140

supernatants were passed through a 0.45-μm filter before being incubated with 5.0 mL bed volume of 141

Ni-NTA resin (Ni2+-nitrilotriacetate, Qiagen) for 45 min at 4°C. Resin samples were then washed with 142

200 mL of washing buffer (300 mM NaCl, 50 mM HEPES, pH 7.0, 5% (v/v) glycerol, 50 mM 143

imidazole) and bound protein was eluted with approximately 30 mL of elution buffer (300 mM NaCl, 144

50 mM HEPES, pH 7.0, 5% v/v glycerol, 250 mM imidazole). Active fractions were pooled, 145

exchanged to 20 mM HEPES buffer (pH 7.0) using a Bio-Gel® P10 column, and then further purified 146

by anion chromatography using the Uno-Q column (5.0 mL) from BioRad. A BIOSHOP Duoflow 147

(Bio-Rad, Canada) system was used to pass 20 mM HEPES buffer (pH 7.0) with a NaCl gradient from 148

0 to 0.5 M through the Uno-Q column over 20 column volumes, and SthAbf62A wild-type as well as 149

mutant enzymes were eluted at 0.15 M NaCl. A Bio-Gel P10 column was then used to transfer the 150

purified protein to 20 mM HEPES buffer (pH 7.0) containing 300 mM NaCl, and centrifugal filter units 151

(10K; Millipore) were used for concentrating protein. Protein aliquots were flash frozen in liquid 152

nitrogen and then stored at -80°C. 153

Protein concentrations were determined using the Bradford assay (41) and bovine serum albumin as 154

a standard. SDS-PAGE was performed and stained with Coomassie Blue according to established 155

procedures. 156

Enzyme activity assays. Hydrolysis of arabinoxylans was monitored by measuring released 157

reducing sugars using the Nelson- Somogyi procedure (42). The standard assay solution contained 0.5% 158

(w/v) (5.0 mg/mL) polysaccharide in 0.5 mL of 100 mM HEPES buffer (pH 7.0). The reaction was 159

initiated by adding an amount of enzyme determined to release products in a linear relation to time 160

when incubated at 45°C for up to 30 min, which were also the temperature and incubation time used to 161

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measure enzyme activities. One unit of the activity was defined as the amount of enzyme releasing 1 162

μmoL of L-(+)-arabinose equivalent per minute. L-(+)-Arabinose was used to generate a standard curve 163

(0.05-0.6 mg/mL). 164

SthAbf62A activity on 4-nitrophenyl (pNP) glycosides was determined by measuring pNP release. 165

The typical reaction mixture contained 2.0 mM substrate in 0.30 mL of 100 mM HEPES buffer (pH 166

7.0). After initiating the reaction by adding enzyme, the reaction mixture was incubated for 20 min at 167

45°C. The reaction was terminated by adding 0.30 mL of 4% (w/v) Na2CO3. The amount of pNP 168

formed was measured at A405nm (extinction coefficient ε 405 = 17,600 M−1 cm−1) and pNP was used to 169

generate a standard curve. As also indicated above, the enzyme added to each assay was optimized to 170

measure initial reaction rates. All enzyme assays were carried out in triplicate. 171

Kinetic parameters were determined using 0.1 mM to 25 mM pNP-AraF; apparent kinetic 172

parameters were also obtained with 0.05% to 1.5% (0.5 to 15 mg/mL) WAX-HV, and 0.1% to 15% (1.0 173

to 150 mg/mL) sugar beet arabinan. Initial rates were obtained by measuring reaction products formed 174

after 30 min at 45°C and pH 7.0 as described in the standard assay. Kinetic parameters were then 175

calculated using the Michaelis-Menten equation or conventional substrate inhibition equation (Eq.1) 176

using GraphPad Prism 5.0 (GraphPad Software, USA): 177

178

(Eq. 1) 179

180

Where v is the initial velocity at various substrate concentrations and Km(app) is the apparent Michaelis 181

constant for substrate. Ksi (app) represents the apparent dissociation constant for the inhibitory SES 182

ternary complex (43). 183

Optimum reaction conditions, enzyme stability, and effects of divalent ions. All enzyme 184

)][

1(][

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max

appsiappm K

SSK

SVv

+⋅+

⋅=

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assays were performed using the standard activity assay with 0.5% (w/v) WAX-HV and incubation at 185

45°C for 30 min; reaction products were then detected using the Nelson-Somogyi method. The effect 186

of pH on SthAbf62A activity was determined by performing the activity assay at pH 3.5 to 10.5 with 187

increments of 0.5 pH units; a universal buffer (100 mM acetic acid, 100 mM boric acid and 100 mM 188

phosphoric acid, adjusted to the target pH using sodium hydroxide solution) was used for this analysis. 189

The effect of reaction temperature on enzyme activity was examined by performing the standard assay 190

at temperatures ranging from 5°C to 85°C. To determine pH and temperature stability, the enzyme was 191

pre-incubated in the universal buffer (pH 4 to 10.5) at 4°C for 24 h, or else at 25°C to 80°C for 60 min 192

in 100 mM HEPES buffer (pH 7.0), before being adjusted to standard assay conditions to measure 193

residual SthAbf62A activity. The effect of metal ions was determined by adding 2.0 mM of different 194

metal chloride salts to the standard activity assay. 195

High pressure anion exchange chromatography (HPAEC) detection of reaction products. 196

To confirm the selective hydrolysis of arabinose substituents, products of WAX-HV and sugar beet 197

arabinan from treatment with SthAraf62A were analyzed by HPAEC (Dionex, ICS-5000+ DC) 198

equipped with a pulsed amperometric detector (PAD), Carbopac™ PA1 column (250×2 mm, Dionex) 199

and CarboPac™ PA20 guard column (30×3 mm, Dionex) (44). Reactions (0.5 mL) contained 0.5% 200

(w/v) of WAX-HV or arabinan and SthAbf62A (2.5 μg for WAX-HV and 12.0 μg for arabinan), as 201

described in the standard activity assay. After 15 min, 30 min and 45 min of incubation, reactions were 202

stopped by boiling for 10 min; 100 µL samples were then mixed with 2 volumes of acetone to 203

precipitate polymeric arabinoxylan, and centrifuged at 13,000 x g for 10 min at 4°C. Supernatants 204

containing the liberated sugars were transferred to a clean tube and the acetone was removed using a 205

Speedvac (Thermo). The remaining supernatant was lyophilized, dissolved in 100 µL ultra-pure water 206

(MilliQ), and then filtered through 0.22 µm centrifuge tube filters (Corning). Samples (10 µL) were 207

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injected on the Carbopac™ PA1 column kept at 30°C, and eluted with 50 mM NaOH at 0.25 mL/ min 208

for over 30 min. L-Arabinose and D-xylose (40 µg/ ml) were used as standards. 209

Preparation of wheat arabinoxylan with singly substituted L-arabinose. Arabinoxylan with 210

singly substituted α-1,2 and 1,3-arabinose was generated by selective removal of α-1,3-arabinose from 211

doubly substituted xylose residues using the arabinofuranosidase from Bifidobacterium sp. (AXH-d3) 212

(36). Briefly, 0.2% (w/v) of WAX-HV was treated with 5 U of AXH-d3 in 50 mL 20 mM potassium-213

phosphate buffer (pH 6.0) at 40°C for 24 h; an additional 2 U of AXH-d3 was then added and the 214

reaction was incubated for another 24 h to ensure near complete removal of the α-1,3-arabinose from 215

doubly substituted positions. After boiling for 10 min, arabinoxylan containing singly substituted α-1,2 216

and 1,3-arabinose was precipitated by adding two volumes of 95% ethanol followed by centrifugation 217

(15,000 × g, 10 min). The pellet was dissolved in 50 mL water and again precipitated with 95% ethanol. 218

The precipitate was then dissolved in 20 mL water and lyophilized. 219

1H NMR analysis for the action mode of SthAbf62A towards arabinoxylan. To 220

qualitatively evaluate the regioselectivity of SthAbf62A on arabinoxylan, one-dimensional 1H NMR 221

spectra were collected for intact and AXH-d3 treated WAX-HV before and after treatment with 222

SthAbf62A. Briefly, 1% (w/v) arabinoxylan (intact or singly substituted) was digested for 2 h or 24 h at 223

45°C using 3.0 µg of SthAbf62A in a 0.6 mL reaction solution containing 100 mM HEPES buffer (pH 224

7.0). Reactions were terminated by boiling for 10 min. The treated arabinoxylan was then precipitated 225

by adding 1.2 mL of 95% ethanol and separated by centrifugation (15,000 × g, 10 min). The pellet was 226

suspended in 0.6 mL water and again precipitated with 95% ethanol. To overcome the precipitation of 227

arabinoxylan following treatment with arabinofuranosidases (45, 46), a GH11 endoxylanase from 228

Thermobifida fusca, TfxA(47), was used to hydrolyze the debranched arabinoxylan into arabino-229

xylooligosaccharides (48). The pellet was suspended in 0.6 mL of 10 mM 3-(N-morpholino) 230

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propanesulfonic acid (MOPS) buffer (pH 7.0) and then treated with 1.2 µg TfxA at 50°C. After 24 h, 231

an additional 1.2 µg TfxA was added and the reaction was incubated for another 24 h. Reaction 232

mixtures were subsequently boiled for 15 min to inactivate the endoxylanse, then lyophilized and 233

dissolved in 0.6 mL D2O two times before NMR analysis. 1H NMR spectra were obtained at 25°C in 5.0 234

mm NMR tubes (Norell) using an Agilent DD2 600 MHz spectrometer, with a scan number of 16, and 235

relaxation delay of 10 s. The data were recorded using VnmrJ 4.0 (Agilent) and processed using 236

MestReNova 8.1.0 software (Mestrelab Research). The methylene protons at position 2 of MOPS 237

(2.035 ppm) were used as the internal reference. The NMR signal assignment was based on previously 238

published reports (36, 49). 239

Protein crystallization, data collection and structure determination. SthAbf62A was 240

concentrated to 18 mg/mL in 10 mM HEPES (pH 7.5) containing 500 mM NaCl. The crystallization 241

screen was performed by mixing 0.5 μL of protein with 0.5 μL of reservoir buffer using a Mosquito 242

crystallization robot. The SthAbf62A crystals were obtained by a sitting-drop vapour diffusion 243

technique at 25°C with a condition containing 0.1 M Bis-tris propane (pH 6.5) and 20% polyethylene 244

glycol monomethyl ether (5 K). For the co-crystallization, SthAbf62A (14 mg/mL) was first incubated 245

overnight at 4°C with xylotetraose and L-arabinose at a final concentrations of 5 mg/mL and 200 mM, 246

respectively. The co-crystallization reservoir buffer for xylotetraose contained 0.2 M sodium formate 247

and 20 % polyethylene glycol 3350, and that for L-arabinose contained 0.1 M HEPES (pH 7.5), 0.2 M 248

ammonium acetate, and 25% polyethylene glycol 3350. The crystals were grown at 25°C using the 249

sitting-drop vapour diffusion technique. The crystals were then flash frozen in a 100 K liquid nitrogen 250

stream using paratone-N oil as a cryoprotectant. Diffraction data for SthAbf62A apoenzyme was 251

collected at 100 K on a Rigaku HF-007 rotating anode with a Rigaku R-AXIS IV++ detector. 252

Diffractor data for SthAbf62A-arabinose and SthAbf62A-xylotetraose complexes were collected at the 253

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Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source, beamline 19-254

ID with an ADSC Quantum 315r detector. All diffraction data was processed in HKL3000 (50). The 255

SthAbf62A apoenzyme structure was solved by molecular replacement using the structure of a GH62 256

family α-L-arabinofuranosidase from Scytalidium thermophilum (unpublished) as the search model and 257

PHENIX.phaser (51). All models were refined using PHENIX and Coot (52) with 5% of observed 258

reflections set aside for Rfree calculations. Ligands were built into residual positive Fo–Fc density after 259

building the protein atoms. All geometries were verified with PHENIX.refine and the RCSB PDB 260

Validation server. Conformation of L-arabinose and nucleophilic water was verified by calculation of a 261

simulated annealing omit map, where these atoms were removed, followed by Cartesian simulating 262

annealing using Phenix.refine (default parameters). Average B-factor and bond angle/bond length rmsd 263

values were calculated using PHENIX. Table 3 lists data collection and final model refinement statistics. 264

Structure figures were prepared using PyMol (The PyMOL Molecular Graphics System. DeLano 265

Scientific; San Carlos, CA, USA: 2002). Structures were deposited to the Protein Databank under 266

accession codes 4O8N (SthAbf62A), 4O8O (SthAbf62A complexed with L-arabinose) and 4O8P 267

(SthAbf62A complexed with xylotetraose). 268

269

RESULTS AND DISCUSSION 270

General enzyme properties of SthAbf62A. The recombinant SthAbf62A was functionally 271

expressed in E. coli with an N-terminal His6-tag and purified to homogeneity with a typical protein 272

yield of ~12 mg protein per liter of bacterial culture. The molecular weight of recombinant SthAbf62A 273

estimated by SDS–PAGE was 36.5 kDa (Fig. S1), which is consistent with the predicted molecular 274

mass (38.3 kDa). The optimal pH of SthAbf62A activity on wheat arabinoxylan was approximately pH 275

7.0 (Fig. 1A), which is 2.0 pH units higher than previously reported for the identical α- L-276

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arabinofuranosidase from Streptomyces thermoviolaceus OPC-520 (AB110643.1) (37). Though 277

initially surprising, this difference in pH optima could be explained by the comparatively long 278

incubation time (2 h) used in earlier reports to measure the pH optimum, together with the pH stability 279

of SthAbf62A (see below). For comparison, reported pH optima of other characterized GH62 α- L-280

arabinofuranosidases are: pH 6.0 for SliAraf62 from Streptomyces lividans (34), pH 5.5 for ScAraf62A 281

from S. coelicolor (33), pH 5.0 for PchAraf62 from Penicillium chrysogenum (36), pH 7.0 for 282

CcAraf62A from Coprinopsis cinerea (38), pH 5.0 for UmAbf62A from Ustilago maydis, and pH 5.0 283

for PaAbf62A from Podospora anserina (32). 284

Stability studies showed that SthAbf62A retained over 70% activity after 24 h at 4°C between pH 285

from 6.0 to 9.5 (Fig. 1B). By comparison, CcAra62A retains 80% activity after 1 h at 20°C between pH 286

7.0 to 8.5 (38), and ScAraf62A is stable for 1 h at 35°C between pH 7.0 and 9.0 (33). Further, 287

SthAbf62A showed highest activity at 55°C in a 30 min enzyme assay (Fig. 1C), and retained over 70% 288

and 45% of activity after 60 min at 55°C and 60°C, respectively (Fig. 1D). By contrast, PchAra62, 289

SliAra62, and ScAraf62A lost all activity after 60 min at 60°C (33, 34, 36), and the optimal reaction 290

temperature for both UmAbf62A and PaAbf62A is 37°C and 55°C respectively (32, 53). 291

Consistent with earlier reports (37), the presence of 2.0 mM Zn2+, Cd2+, Hg2+ and Cu2+ reduced 292

sugar release from WAX-HV by SthAbf62A by more than 70%, while 2.0 mM Ca2+ and Mg2+ activated 293

SthAbf62A by at least 60% (Fig. S2). However, addition of 2.0 mM ethylenediaminetetraacetic acid 294

(EDTA) did not significantly affect SthAbf62A activity, suggesting that the enzyme does not strictly 295

depend on ion supplementation despite the coordinated calcium ion observed in the SthAbf62A 296

structure (see below). 297

Substrate specificity of SthAbf62A. SthAbf62A activity was highest on WAX-HV and RAX, 298

followed by water-soluble OSX and WAX-LV (Table 1). This result is consistent with the considerably 299

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higher arabinose-to-xylose ratio in WAX-HV and RAX (ranging from 0.5 to 0.7) (54, 55), compared to 300

WAX-LV (approx. 0.3) (54), and OSX (approx. 0.12) (56, 57). Notably however, when normalizing 301

wheat, rye and water soluble OSX to arabinose content, approximately 8 % (WAX-HV), 6 % ( WAX-302

LV and RAX) and 17 % (OSX) of the total arabinose present was released after 30 min by 0.4 μg 303

SthAbf62A in standard assay conditions. This suggests that arabinose in water-soluble OSX is 304

comparatively accessible to hydrolysis by SthAbf62A, which likely reflects the particular solubility of 305

this OSX preparation. More interestingly, similar extents of arabinose released from wheat and rye 306

arabinoxylan after 30 min suggests that the differences in occurrence of α-L-Araf (1→3) and α- L-Araf 307

(1→2) substituents in WAX-HV, WAX-LV and RAW (54) does not significantly affect initial rates of 308

SthAbf62A activity. 309

Though not detected during the characterization of α- L-arabinofuranosidase from Streptomyces 310

thermoviolaceus OPC-520 (37), SthAbf62A also released arabinose from sugar beet arabinan, which 311

consists of an α-1,5 linked L-arabinofuranose backbone with ca. 60% α-1,3 linked L-arabinofuranose 312

substitutions. HPAEC analyses further confirmed that L-arabinose was the only monosaccharide 313

released from arabinoxylan and arabinan (Fig. S3). Given the polydispersity of the arabinoxylan and 314

arabinan substrates, molar values of these substrates could not be accurately calculated, and so only 315

apparent kinetic parameters could be obtained to evaluate SthAbf62A activity in greater detail. These 316

analyses revealed that the apparent catalytic efficiency (kcat/KM) of SthAbf62A was nearly 70 times 317

higher on wheat arabinoxylan than sugar beet arabinan (Table 2). At the same time, the kinetic data 318

were best explained by the conventional substrate inhibition model (Eq.1), and apparent substrate 319

inhibition constants (Ksi (app)) were lower for arabinoxylan than arabinan (Table 2; Fig. S4). Since 320

sample viscosities did not perceptibly differ at maximum substrate concentrations, the lower Ksi(app) 321

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value for arabinoxylan suggests preferred binding by SthAbf62A of xylan over arabinan backbone 322

structures. 323

Low but detectable activity was also observed towards rhamnogalacturonan, debranched 324

arabinan, as well as pNP-α- L-arabinofuranoside and pNP-α- L-arabinopyranoside (Table 1). Notably, 325

the catalytic efficiency (kcat/KM) of SthAbf62A on pNP-α- L-arabinofuranoside was ~330 times higher 326

than pNP-α- L-arabinopyranoside, which was mainly explained by differences in kcat and reveals the 327

high preference for furanose forms of α- L-arabinose. HPAEC analyses confirmed that L-arabinose was 328

the only product released from debranched α-1,5 linked arabinan (data not shown), and kinetic analyses 329

determined that the kcat for arabinan and pNP-α- L-arabinofuranoside were 30 and 140 times lower than 330

the apparent kcat for WAX-HV, respectively (Table 2). The clear preference of SthAbf62A for 331

polymeric substrates (i.e. arabinoxylan) is consistent with other GH62 members (32-36, 38), and 332

continues to distinguish GH62 enzymes from arabinofuranosidases identified in other GH families. 333

Regio-selectivity of SthAbf62A. 1H NMR analysis was used to then investigate the 334

regiospecificity of SthAbf62A towards α-L-Araf (1→2) and α-L-Araf (1→3) linkages in arabinoxylan. 335

Selective removal of α-L-Araf (1→3) linked to mono-substituted β-D-Xylp by SthAbf62A was shown 336

by the disappearance of the 1H signal at 5.42 ppm (red line; Fig. 2B) (49, 54, 57). To clarify whether 337

SthAbf62A activity was restricted to single α-1,3 linkages or else singly substituted xylose, 338

arabinoxylan with only single α-1,3 and α-1,2 arabinose substitutions was generated using 339

arabinofuranosidase (AXH-d3) from Bifidobacterium sp, which selectively removes α-L-Araf (1→3) 340

linked to di-substituted β-D-Xylp (36, 46). The effect of AXH-d3 treatment was confirmed by the 341

disappearance of signals near 5.28 ppm and 5.22 ppm corresponding the (1→3)-linked and (1→2)-342

linked α-L-Araf substituents of doubly substituted β-D-Xylp, and consequent increase in the signal near 343

5.30 ppm, which corresponds to α-L-Araf (1→2) linked to mono-substituted β-D-Xylp (Fig. 2A)(54). 344

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Subsequent treatment with SthAbf62A significantly reduced signals at both 5.42 ppm and 5.30 ppm 345

(Fig. 2C), consistent with hydrolysis of both of α-1,3 and α-1,2 L-arabinofuranosyl linkages to singly 346

substituted xylose (i.e. AXH-m2,3 activity). This analysis shows that SthAbf62A alone selectively 347

removes singly substituted α-1,3 arabinose from wheat arabinoxylan, but similar to PchAraf62 (36) and 348

a GH43 AXH-m 2,3 from B. subtilis (30), can also target singly substituted α-1,2 linked arabinose after 349

enzymatic removal of α-1,3 L-arabinofuranosyl substituents from di-substituted xylopyranosyl residues. 350

Overall structure. Three structures of SthAbf62A were determined by molecular replacement 351

(Table 3), which included one enzyme structure and two enzyme complex structures co-crystallized 352

with reaction products of xylotetraose and L-arabinose respectively (Protein Data Bank code: 4O8N, 353

4O8O and 4O8P) . The SthAbf62A enzyme adopts a β-propeller fold with five blades (marked I–V) 354

twisted and radially arranged around the pseudo five-fold axis. The blades are linked through loops 355

with variant length, and each blade was made up of four antiparallel β-strands in a typical W-like 356

topology with the first strand being the innermost (Fig. 3A). The only exception is blade IV, which was 357

formed by a typical four antiparallel β-strand arrangement (β14, β15, β16 and β17) plus the small strand 358

of β19 (Fig. 3C). This additional strand closes up one end of the β-propeller together with its flanking 359

loop region. 360

Structure similarity searches of the Protein Data Bank with the Dali Server showed that the 361

overall structure of SthAbf62A resembled that of enzymes in GH43, GH32 and GH68 families, which 362

similarly adopt a five-bladed β-propeller fold (Table S2) (18, 57-62). The overall structure of 363

SthAbf62A superimposed well with the catalytic domain of these enzymes (Table S2), which is 364

consistent with divergent evolution of enzymes from families GH32, 43, 62 and 68 (58). 365

Superimposition of SthAbf62A with enzymes from these families also facilitated the prediction of the 366

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two catalytic residues (Asp53 and Glu213), and of a third residue (Asp161) believed to modulate the 367

pKa of the catalytic general acid (Fig. S5) (18) . 368

While we were completing our analysis of SthAbf62A, the first structures were published for 369

GH62 α-arabinofuranosidases, including UmAbf62A from Ustilago maydis, PaAbf62A from 370

Podospora anserina, and ScAraf62A from Streptomyces coelicolor (32, 33). In line with these first 371

structures of GH62 α-arabinofuranosidases, the five-bladed β-propeller fold of SthAbf62A is stabilized 372

through a molecular Velcro between antiparallel β-strands 1 and 21 in blade V formed through five 373

hydrogen bonds and several hydrophobic interactions (Fig. S6). Belonging to subfamily GH62_2, 374

SthAbf62A shared additional similarities to UmAbf62A and ScAraf62A. For instance, SthAbf62A 375

contains a disulfide bond between Cys27 and Cys297, which constrains the N-terminus of the enzyme 376

in the vicinity of the loop connecting strands 19 and 20 (Fig. 3). Additionally, a calcium ion in the 377

central channel of the fold was buried right under the L-arabinose binding pocket (Fig. 3, 4), displaying 378

a hepta-coordination with His280, Gln216 and five water molecules. 379

Since SthAbf62A shares over 65% sequence identity with ScAraf62A, but demonstrates 380

considerably higher optimal reaction temperature and temperature stability, these enzymes were further 381

compared in terms of 18 sequence characteristics thought to confer thermostability. Relative to 382

ScArab62A, SthAbf62A scored higher in 11 of these 18 parameters, including higher number of 383

predicted hydrogen bonding interactions and salt bridges, and increased core hydrophobicity (Table S3). 384

Though main determinants of thermostability are still difficult to predict, the growing number of GH62 385

enzyme structures, including those from temperature stable members, will undoubtedly facilitate future 386

protein engineering efforts aimed at improving applications of GH62 enzymes. 387

The active site of SthAbf62A. To understand the structural basis for catalysis and substrate 388

binding, the structure of SthAbf62A complexes with its reaction products was obtained by separate co-389

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crystallizations with L-arabinose and xylotetraose. A bound L-arabinose molecule was observed in a 390

narrow and deep pocket (-1 subsite) composed of relatively conserved amino acids, which was open 391

toward the molecular surface (Fig. 3; Fig. S7). It was formed mainly by the five first and innermost β-392

strands of each blade (β2, β6, β10, β14 and β18) and lined by about 12 amino acids (within 4Å distance 393

from L-arabinose), 3 of which were acidic amino acid residues, making this pocket mainly negative 394

charged (Fig. S7). The two predicted catalytic residues (Asp53 and Glu213), along with the putative 395

pKa modulator (Asp161), were pointing into this pocket (Fig. 3A,B), and were donated by the β2 sheet 396

in blade I, β14 in blade IV and β10 in blade III, respectively (Fig. 3C). The Asp53Ala and Glu213Ala 397

substitutions led to complete loss of enzyme activity, whereas the Asp161Ala substitution retained 398

detectable, albeit low activity on WAX-HV (Table S4); the lost activity could not be rescued by NaN3. 399

The site-directed mutagenesis results reported herein support the catalytic importance of Asp53, Glu213 400

and Asp161, and are consistent with mutagenesis studies of ScAraf62A, which similarly implicate 401

corresponding Asp202, Glu361 and Asp309 residues in catalysis (33). 402

The L-arabinofuranose molecule was modeled with an α-configuration (Fig. 4). The ligand was 403

anchored in the L-arabinose binding pocket mainly through several polar interactions (Fig. 4), such as 404

hydrogen bonds between sugar ring O4 and Nζ of Lys52 (3.4 Å) along with Oηof Tyr314, O2 and Nδ1 of 405

His280 (2.9 Å) as well as Oδ1 and Oδ2 of Asp161 (2.7 Å), and O3 with Oδ1 of Asp161 (2.6 Å) and Nζ2 406

of Gln121 (2.8 Å). In particular, the contacts between O5 of L-arabinofuranose and Nζ of Lys52(2.8 Å) 407

as well as Oδ2 of Asp53 (2.6 Å) might be important for discriminating between furanose and pyranose 408

forms of L-arabinose as observed for pNP substrates (Table 2). The catalytic Glu213 side chain was 409

proximal to O1 of L-arabinose in a distance of 2.8 Å. All the residues involved in the above hydrogen 410

bonds are contributed by β sheets and conserved across the sequences in the GH62 family (Fig. S8). In 411

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contrast, residues for hydrophobic interactions (i.e. Trp76, Tyr314, Ile160 and Thr68) were not highly 412

conserved, except Tyr314. Notably, in SthAbf62A, Trp76 along with Tyr314 appeared to contact the L-413

arabinose ring through edge on edge apolar interactions (59) that were further stabilized through 414

interactions with the side chains of Ile160 and Thr68. By comparison, stacking interactions in 415

UmAbf62A were predicted between arabinose and Tyr58 (32). Accordingly, binding of arabinose by 416

SthAbf62A appears more similar to ScAraf62A, which interacts with the flat surface of arabinose 417

through hydrophobic interactions with Ile308 (33). 418

The structure of SthAbf62A in complex with L-arabinofuranose revealed insights into the catalytic 419

mechanism of this enzyme. There was a water molecule closely positioned near the anomeric carbon 420

(2.8 Å) that also formed a hydrogen bond with the expected general base, Asp53 (Fig. 4). We also 421

observed electron density shared between this water molecule and the anomeric carbon (Fig. 4). Finally, 422

the distance between Asp53 and the expected general acid, Glu213, was 8 Å. These observations are all 423

consistent with an inverting mechanism for this enzyme, with the described water molecule acting as 424

the nucleophile for attack on the anomeric carbon. 425

The co-crystallization of SthAbf62A with xylotetraose, a reaction product, revealed the binding 426

mode of the xylan backbone within an open cleft situated above the catalytic pocket. Four xylose 427

binding subsites were observed along the cleft; the xylotetraose chain fit the electron density features 428

equally well in two opposite orientations, which is similar to the model proposed for acetylxylan 429

esterases (60). The nomenclature described in McKee et al (61) was followed herein to name the 430

subsites that bound xylotetraose. In Orientation 1 of xylotetraose, the reducing end xylose at subsite 431

+2R situated above the space between blade IV and V, whereas the non-reducing end xylose at subsite 432

+3NR situated above the space between blade I and II (Fig. 5). Orientation 1 would allow hydrolysis of 433

α-1,3 linked arabinose since O3 of xylose points towards the active site pocket, while the reverse 434

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orientation (Orientation 2) would favor the hydrolysis of α-1,2 linked arabinose. In both orientations, 435

xylose at subsite +1 was stabilized through hydrophobic stacking interactions with Tyr314 and 436

hydrogen bonds with Glu213 (Fig. 5; Table S5). The non-reducing sugar unit at subsite +3NR in 437

Orientation 1, and corresponding reducing end sugar at subsite +3R in Orientation 2, did not form any 438

hydrogen bonds with the enzyme and instead was stabilized via a stacking interaction with Trp122, 439

which is conserved in most sequences with occasional substitution to Gln (Fig. S8). Hydrogen-bonding 440

interactions observed between the xylotetraose backbone and the enzyme are listed in Table S5. 441

Notably, while the +2NR subsite in Orientation 1 and Orientation 2 is characterized by hydrogen bond 442

interactions with Trp76 and Arg208 respectively, Orientation 1 lacks the additional hydrogen bond 443

observed in Orientation 2 between Arg239 and the endocyclic oxygen of the nonreducing end xylose. 444

A cation, modeled as potassium, was bound in the arabinose-binding site (-1 subsite) in the SthAbf62A-445

xylotetraose complex and accordingly this ion could interact with the C3-OH (Orientation 1) or the C2-446

OH (Orientation 2) of xylose at subsite +1(Fig. 5). Taken together, the SthAbf62A substrate binding 447

cleft and active site adopt a “T-like” architecture (Fig. S9), reminiscent of that observed in two GH43 448

side chain-cleaving α-L-arabinofuranosidases (BsAXH-m2,3 (18) and HiAXH-d3 (61)), suggesting the 449

“T-like” architecture is a common feature of debranching glycoside hydrolases. 450

451

Conformation changes following ligand binding. Besides the aromatic residues described 452

above, the xylan binding cleft of SthAbf62A was mainly lined by non-conserved amino acids from loop 453

regions, which was also noted in BsAXH-m2,3, UmAbf62A, PaAbf62A and ScAraf62A (18, 32, 33), 454

suggesting that enzymes from this clan adopt a dynamic substrate binding cleft. Consistent with this 455

prediction, xylotetraose and L-arabinose binding induced small local conformation changes in 456

SthAbf62A (Fig. 6). Superimpositions between the SthAbf62A structure and enzyme complexes 457

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showed pairwise root-mean-square deviation (RMSD) values of 0.362 Å (303 Cα atoms) and 0.173 Å 458

(302 Cα atoms) for xylotetraose and L-arabinose complex structures, respectively. 459

In the SthAbf62A-xylotetraose complex structure, the difference was attributed largely to 460

conformational differences between the five loop regions (72-75,121-126, 148-160, 235-238 and 308-461

320), which were all involved in forming the xylan backbone binding cleft. Indeed, the pairwise RMSD 462

value of Cα atoms computed using only these residues increased to 0.682 Å and 0.411 Å for 463

xylotetraose and L-arabinose complex structures, respectively, also indicating larger conformational 464

differences induced by xylotetraose binding. Movement within these loop regions shifted the side 465

chains of several amino acid residues, including Trp122, Asp315 and Tyr314 along with Trp76 and 466

Arg208 from β-sheets, all of which were involved in xylotetraose binding. Furthermore, the side chain 467

of Arg75 from loop 72-75 shifted towards the ligand by 2.3 Å upon xylotetraose binding (Fig. 6). Such 468

movement within the substrate binding cleft could explain the accommodation of arabinan substrates, 469

and non-catalytic interactions between GH62 arabinofuranosidases and various oligosaccharides, 470

including cello-oligosaccharides that can inhibit enzyme activity (32). 471

Binding of L-arabinose caused similar, albeit smaller, shifts within these mentioned regions. 472

However, the conformation of the L-arabinose binding pocket barely changed (pairwise Cα atoms 473

RMSD values of 0.277 and 0.198 Å for xylotetraose and L-arabinose complex structures, respectively). 474

475

CONCLUSIONS 476

In this study, we conducted a function/structure characterization of a thermostable, bacterial α-477

arabinofuranosidase from family GH62, namely SthAbf62A from Streptomyces thermoviolaceus. In 478

particular, new insights regarding the molecular basis for substrate selectivity among GH62 enzymes 479

were gained by studying complexes of SthAbf62A with L-arabinose and xylotetraose. All structural 480

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features of SthAbf62A underpin the strict specificity for arabinofuranosyl groups bound to backbone 481

moieties through an alpha glycosidic configuration. Additionally, similar to BsAXH-m2,3 (18), the 482

narrowness of the arabinose-binding pocket likely prevents acceptance of α-L-Araf residues that are 483

(1→2) and (1→3) linked to di-substituted β-D-Xylp, which is also consistent with the 1H NMR 484

analyses of SthAbf62A reaction products (Fig. 2B). The complex structure with xylotetraose predicts at 485

least four subsites that bind the xylan backbone, which likely contributes to the clear preference of 486

SthAbf62A and other GH62 arabinofuranosidases for hydrolysis of polymeric arabinoxylan. Although 487

it is unclear how neighboring arabinose substituents may influence the orientation of arabinoxylan 488

substrates, the potential to bind xylo-oligosaccharides in opposite orientations explains the activity of 489

SthAbf62A on both L-Araf substituents that are (1→2) and (1→3) linked to mono-substituted β-D-Xylp 490

in arabinoxylan. 491

In summary, the recent increase in structure and functional characterization of GH62 492

arabinofuranosidases reflects the particular relevance of this enzyme family for modification and 493

characterization of polymeric and oligomeric arabinoxylans. In addition to predicting amino acid 494

residues important to overall enzyme stability, a compelling prospect of these recent insights is to fine-495

tune GH62 activities through better predictions of amino acids that govern regio-selectivity and 496

accommodation of singly versus doubly substituted substrates. 497

498

Acknowledgements 499

We thank Prof. Alexander Yakunin, Greg Brown and Rosa Di Leo from University of Toronto 500

for technical support; Prof. Wilson at Cornell University and Dr. T. Vuong at University of Toronto for 501

providing endoxylanase TfxA from Thermomonospora fusca; Dr. J. Tang and Dr. M. Nejad for NMR 502

spectra acquisition; M. Gudmundsson and Dr. M. Couturier for insightful discussions. We thank B. 503

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Nocek at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source for 504

x-ray diffraction data collection. Funding for this research was provided by the Government of Ontario 505

for the project "Forest FAB: Applied Genomics for Functionalized Fibre and Biochemicals" (ORF-RE-506

05-005), the Natural Sciences and Engineering Research Council of Canada, and the Finnish Funding 507

Agency for Technology and Innovation (TEKES). 508

509

510

511

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properties of arabinoxylan films. Carbohydr. Res. 343:753-7. 634

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109:6537-42. 676

677 678 679 680

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FIGURE LEGENDS 1

Figure 1. Effect of pH and temperature on SthAbf62A activity and stability. (A) The effect 2

of pH on enzyme activity; (B) pH stability of purified SthAbf62A; (C) The effect of temperature 3

on SthAbf62A activity; (D) Thermostability of purified SthAbf62A. Activity assays were 4

performed using 0.5% wheat arabinoxylan (high viscosity) and 0.4 μg SthAbf62A. 5

6

Figure 2. 1H NMR analysis of enzyme treated wheat arabinoxylan. m-1,2 and m-1,3-α-L-Ara 7

refer to mono-substituted β-D-Xylp at positions C2 and C3, respectively; d-1,2 and d-1,3-α-L-8

Ara refer to arabinose attached to double-substituted β-D-Xylp. (A) Wheat arabinoxylan (high 9

viscosity) before (black line) and after (red line) treatment with AXH-d3 from Bifidobacterium 10

sp. for 48 h; the effect of AXH-d3 treatment was confirmed by the disappearance of the signals 11

near 5.28 ppm and 5.22 ppm and concomitant increase in the signal near 5.30 ppm (54). (B) 12

Wheat arabinoxylan (high viscosity) before (black line) and after (red line) treatment with 13

SthAbf62A (3.0 μg) in 100 mM HEPES buffer (pH 7.0) at 45°C for 2 h; removal of α-L-Araf 14

(1→3) linked to mono-substituted β-D-Xylp was indicated by the disappearance of the H1 signal 15

at 5.42 ppm (red line); signals at 5.22 ppm and 5.28 ppm for (1→3)-linked and (1→2)-linked α-16

L-Araf substituents of doubly substituted β-D-Xylp remained after treatment for up to 24 h (data 17

not shown). (C) AXH-d3 treated arabinoxylan before (black line) and after (red line) treatment 18

with SthAbf62A (3.0 μg) in 100 mM HEPES buffer (pH 7.0) at 45°C for 2 h; removal of (1→3)-19

linked and (1→2)-linked α-L-Araf substituents of singly substituted β-D-Xylp was indicated by 20

the disappearance of the H1 signal at both of 5.42 ppm and 5.28 ppm (red line). The methylene 21

protons at position 2 of MOPS (2.035 ppm) were used as the internal reference. 22

23

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Figure 3. The overall structure of SthAbf62A revealing a five-bladed β-propeller fold. 24

(A) Top view showing the numbering of 5 blades (I–V), three predicted catalytic residues, a 25

disulfide bond between Cys297 and Cys27, a molecular Velcro boxed in grey dash line and a 26

calcium ion coordinated in the central channel of the fold. (B) Side view showing the active site 27

surrounded by loops. (C) Overall topology of SthAbf62A highlighting the molecular Velcro 28

between β1 and β21 by an orange dashed line, and the position of three predicted catalytic 29

residues. β-strands are indicated by arrows and helices are indicated by cylinders. 30

31

Figure 4. Interactions between SthAbf62A and L-arabinofuranose bound in the active site 32

pocket. 33

(A) L-Arabinofuranose is shown in pink and amino acid residues in grey; polar interactions are 34

indicated by black dotted lines. A calcium ion shown as an orange sphere was buried under the 35

L-arabinose binding pocket, displaying a hepta-coordination with His280, Gln216 and five water 36

molecules indicated as red spheres. (B) Focus on a nucleophilic water molecule, which shows 37

shared electron density with C1 of L-arabinose, as indicated by the black box. The electron 38

density shown is simulated annealing Fo-Fc omit density contoured at 2.0 σ. 39

40

Figure 5. Interactions between SthAbf62A and xylotetraose bound in the substrate binding 41

cleft. Xylotetraose is shown in yellow, amino acids predicted to interact with the bound 42

oligosaccharide are shown in grey, and a potassium ion bound in the arabinose-binding site (-1 43

subsite) is shown as a purple sphere. Black dotted lines indicate polar interactions; all contacts 44

are listed in Table S4. The xylotetraose is shown in Orientation 1. All contacts remained in 45

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Orientation 2, with an extra hydrogen bond between Arg239 and the endocyclic oxygen of 46

xylose unit at +2NR subsite. 47

48

Figure 6. Main conformational changes in SthAbf62A upon ligand binding. 49

Structural elements of SthAbf62A are shown in orange; corresponding regions of the enzyme-50

xylotetraose complex are shown in grey and enzyme-L-arabinose complex are shown in green. 51

Xylotetraose is shown in yellow and the enzyme surface shown in grey corresponds to the 52

enzyme-xylotetraose complex. Superimposition of the three structures was conducted using 53

DaliLite. 54

55

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Table 1. Specific activity of SthAbf62A on polymeric xylans and pNP synthetic substrates

Polymeric substrate (μg SthAbf62A)a Specific Activity

(μmol product /min/

µmol enzyme)

Relative activity

(%)

High viscosity wheat arabinoxylan

(WAX-HV; 0.4 μg)

1535 ± 92 100.0

Low viscosity wheat arabinoxylan

(WAX-LV; 0.4 μg)

930 ± 55 60.6

Rye arabinoxylan (RAX; 0.4 μg) 1533 ± 118 99.9

Soluble oat spelt xylan

(soluble OSX; 0.4 μg)

1082 ± 66 70.5

Insoluble oat spelt xylan

(insoluble OSX; 24 μg)

80 ± 23 5.2

Arabinan from sugar beet (24 μg) 47± 6 3.0

Debranched arabinan (60 μg) 1 ± 0.4 0.07

Rhamnogalacturonan (60 μg) 0.4 ± 0.1 0.02

pNP synthetic substrate (μg SthAbf62A)a Specific Activity

(μmol product /min/

µmol enzyme)

Relative activity

(%)

pNP-α-L-arabinofuranoside

(2 μg)

51 ± 2 100

pNP-α-L-arabinopyranoside

(78 μg)

0.3 ± 0.02 0.5

pNP-β-D-xylopyranoside

(224 μg)

0.02 ± 0.003 0.05

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aAmounts of purified SthAbf62A added to each reaction to measure initial reaction velocities.

Activities obtained with WAX-HV and pNP-α-L-arabinofuranoside were taken as 100% in

respective comparisons. No activity was detected towards birchwood xylan, beechwood xylan,

apple pectin, larch arabinogalactan, pNP-β-L-arabinopyranoside, pNP-α-D-mannopyranoside,

pNP-α-D-galactopyranoside, pNP-α-L-fucopyranoside and pNP-α-D-glucopyranoside.

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Table 2. Kinetics of SthAbf62A on selected substrates

Substrate

(μg SthAbf62A)a

kcat (app)

(s-1)

Km(app)

(mg/ml)

Ksi(app)

(mg/ml)

kcat/Km

(s-1·mg-1·ml)

WAX-HV

(0.5 μg) b

180 ± 65 12 ± 5 1.5 ± 0.5 15

Sugar beet arabinan

(4 μg) b

6 ± 0.5 32 ± 4.3 106 ± 18 0.2

kcat

(s-1)

Km

(mM)

kcat/Km

(s-1·mM-1)

pNP-α-L-arabinofuranoside

(2 μg) c

1.3 ± 0.003 3 ± 0.1

0.5

pNP-α-L-

arabinopyranoside

(78 μg) c

0.01 ± 0.0002 8.2 ± 0.4 0.001

`

aAmounts of purified SthAbf62A added to each reaction to measure initial reaction velocities.

b Conventional substrate inhibition equation (Eq.1) was used for data fitting.

c The Michaelis-Menten equation was used for data fitting

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Table 3. X-ray diffraction data collection and refinement statistics

Ligand

PDB Code

apoenzyme

4O8N

Xylotetraose

4O8P

L-α-arabinose

4O8O

Data collection

Space group P212121 P212121 P212121

Cell dimensions

a, b, c, Å 59.81, 65.68, 84.95 42.49, 64.64, 121.30 60.05, 65.74, 85.20

α, β, γ, ° 90, 90, 90 90, 90, 90 90, 90, 90

Resolution, Å 25.00 – 1.65 25.00 – 1.56 18.00 – 1.18

Rmergea 0.081 (0.587)a 0.086 (0.517)b 0.050 (0.508)c

I / σ(I) 29.96 (4.0) 21.77 (3.4) 31.45 (3.2)

Completeness, % 96.2 (92.7) 99.6 (99.5) 98.1 (96.1)

Redundancy 5.9 (6.0) 4.6 (4.7) 4.9 (4.8)

Refinement

Resolution, Å 24.45 – 1.65 23.68 – 1.56 17.47 – 1.21

No. of reflections 38657 48371 101459

R-factor/free R-factord 12.7/16.3 (18.0/24.0)d 13.0/15.3 (17.2/19.9)e 13.9/15.6 (19.6/21.67)f

No. of refined atoms

Protein

Ca2+

Ligand

Solvent

Water

2391

1

N/A

39

540

2444

1

35

39

474

2405

1

10

0

516

B-factors

Protein

Ca2+

Ligand

15.3

10.8

N/A

13.0

6.1

19.8

14.3

7.8

15.0

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Solvent

Water

35.9

33.1

53.2

31.6

N/A

33.2

R.m.s.d.

Bond lengths, Å

Bond angles, °

0.012

1.333

0.019

1.808

0.012

1.384

Ramachandran

Most favoured (%)

Additionally fav.

Generously fav.

Disallowed

84.9

15.1

0

0

87.4

12.6

0

0

85.8

14.2

0

0

Values in parentheses refer to high resolution shells: a1.65-1.68, b1.56-1.59, c1.18-1.20, d1.65-

1.68, e1.56-1.59, f1.21-1.24

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A

B

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