β-Xylosidases and α-l-arabinofuranosidases: Accessory enzymes for arabinoxylan degradation

Biotechnology Advances 32 (2014) 316–332

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Research review paper

β-Xylosidases and α-L-arabinofuranosidases: Accessory enzymes forarabinoxylan degradation

Stijn Lagaert a, Annick Pollet b, Christophe M. Courtin b,⁎, Guido Volckaert a

a Division of Gene Technology, KU Leuven, Kasteelpark Arenberg 21—box 2462, 3001 Leuven, Belgiumb Laboratory of Food Chemistry and Biochemistry & Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven, Kasteelpark Arenberg 20—box 2463, 3001 Leuven, Belgium

⁎ Corresponding author. Tel.: +32 16 321917; fax: +3E-mail address: [email protected]

0734-9750/$ – see front matter © 2013 Elsevier Inc. All rihttp://dx.doi.org/10.1016/j.biotechadv.2013.11.005

a b s t r a c t
a r t i c l e i n f o
Article history:Received 24 July 2013Received in revised form 28 October 2013Accepted 9 November 2013Available online 15 November 2013

Keywords:HemicelluloseBiomassXylanolytic enzymesHemicellulasesGlycoside hydrolasesPolysaccharide degradation

Arabinoxylan (AX) is among the most abundant hemicelluloses on earth and one of the major componentsof feedstocks that are currently investigated as a source for advanced biofuels. As global research into thesesustainable biofuels is increasing, scientific knowledge about the enzymatic breakdownofAXadvanced significantlyover the last decade. This review focuses on the exo-acting AX hydrolases, such as α-arabinofuranosidases and β-xylosidases. It aims to provide a comprehensive overview of the diverse substrate specificities and correspondingstructural features found in the different glycoside hydrolase families. A careful review of the available literaturereveals a marked difference in activity between synthetically labeled and naturally occurring substrates, oftenleading to erroneous enzymatic annotations. Therefore, special attention is given to enzymes with experimentalevidence on the hydrolysis of natural polymers.

© 2013 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3172. The xylosidases and arabinofuranosidases of GH 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318

2.1. Substrate specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3182.2. Structural data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318

3. The reducing end xylose-releasing exo-oligoxylanases of GH 8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3193.1. Substrate specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3193.2. Structural data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320

4. The xylosidases of GH 39 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3204.1. Substrate specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3204.2. Structural data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320

5. The xylosidases and arabinofuranosidases of GH 43 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3225.1. Substrate specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3225.2. Structural data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322

6. The arabinofuranosidases of GH 51 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3246.1. Substrate specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3246.2. Structural data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

7. The xylosidases of GH 52 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3257.1. Substrate specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3257.2. Structural data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325



2 16 321997.(C.M. Courtin).

ghts reserved.

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317S. Lagaert et al. / Biotechnology Advances 32 (2014) 316–332

10. The xylosidases of GH 120 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32710.1. Substrate specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32710.2. Structural data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327

11. The enzymes of GH 30 and GH 116 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32711.1. Substrate specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327

12. Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329

1. Introduction

Increasing energy costs and environmental concerns have pushedthe global demand for sustainable renewable fuels. In the EuropeanUnion,Directive 2009/28/ECwas implemented in 2010 and setsmanda-tory goals to achieve a 10% share of renewable energy in the transportsector by 2020. So-called first generation biofuels are made fromsugar, starch or vegetable oil extracted from food crops, such as corn,sugarcane, soybeans and palms. However, these are under increasedscrutiny as they are considered to be responsible for a rise in food pricesand as the land conversion associatedwith their productionmay actual-ly increase carbon dioxide emissions (Fargione et al., 2008; Mitchell,2008). Therefore, the search for beneficial biofuels should focus onsustainable biomass feedstocks, such as waste biomass (e.g. wheatstraw) and biomass grown on degraded and abandoned agriculturallands planted with perennials (e.g. switchgrass) (Fargione et al., 2008;Tilman et al., 2009). This is also the aim of the European Commissionwhich, in their last proposal for amending the Directive 2009/28/EC,1

suggests to cap the share of conventional biofuels in the transport sectorto 5% and to increase the amount of advanced biofuels. Arabinoxylan(AX) is one of the major components of feedstocks that are currentlyinvestigated as a source for advanced biofuels (Fig. 1) (Pauly andKeegstra, 2008; Saha, 2003). Unsurprisingly, its enzymatic degradationis the subject of increasing research efforts. There is a need for moreaccurate information on existing and novel AX-degrading enzymesthat can be used in the production advanced biofuels (Dodd and Cann,2009; Saha, 2003). The properties of these enzymes, such as theirsubstrate specificity, activity and other biochemical properties need tobe studied and confronted with specific process requirements to evalu-ate their possible usefulness. In this review, the focus is on diversesubstrate specificities and corresponding structural features found inexo-acting AX hydrolases.

The AX backbone is composed of β-1,4-linked xylose residues(Darvill et al., 1980). Arabinose can be substituted at the C(O)2 and/orC(O)3 positions of the xylose residues (Perlin, 1951a, 1951b) and thedegree of substitution (DS) or arabinose to xylose ratio (A/X) is animportant parameter for AX properties. The arabinosyl residues canbe esterified with hydroxycinnamic acid derivatives, such as ferulicand p-coumaric acid (Kulkarni et al., 1999; Subramaniyan and Prema,2002). These hydroxycinnamic acids can from dimers to cross-linkarabinoxylan chains. In addition, ferulic acids can participate inheterocoupling with monolignols or lignin oligomers, therebycross-linking arabinoxylans to lignin. Ferulate cross-linking greatlyimpacts lignocellulosic polymer separation and hemicellulose fermen-tation in general (Grabber et al., 2009; Ishii, 1997).

AX degrading enzymes and other glycosidases hydrolyze the glyco-sidic bond in a stereoselective way, either with retention or inversion ofthe anomeric center (Koshland, 1953; Sinnott, 1990). Bothmechanismsdepend on two catalytic residues: a proton donor and a nucleophile/base. In inverting glycosidases, the catalytic residues are approximately

1 Proposal for a directive of the European parliament and of the council, COM (2012)595 (17 October sss).

10 Å apart (Rye andWithers, 2000; Wang et al., 1994) and the reactionproceeds through a single displacement mechanism. The general acidprotonates the glycosidic oxygen and the departure of the leavinggroup is accompanied by the nucleophilic attack of a water moleculethat has been deprotonated by the general base. In retaining glycosi-dases, the reaction occurs via a two-step double displacement mecha-nism and involves catalytic residues which are approximately 5.5 Åapart (Rye and Withers, 2000; Wang et al., 1994). In the first step, thegeneral acid protonates the glycosidic oxygenwhile the second catalyticresidue performs a nucleophilic attack at the anomeric carbon. Thisleads to the departure of the leaving group and the formation of a cova-lent intermediate. In the second step, the first catalytic residue now actsas a base and deprotonates an incoming water molecule, which hydro-lyzes the glycosyl-enzyme intermediate.

Different enzymes are needed for the degradation of AX.Endoxylanases (EC 3.2.1.8) hydrolyze the backbone in an endo-acting manner, but their activity is frequently hampered by the arabi-nose substitutions (Wong et al., 1988). α-L-Arabinofuranosidases(EC 3.2.1.55) cleave arabinose from the backbone and act in synergywith endoxylanases. To complete AX degradation, β-xylosidases(EC 3.2.1.37) are needed, which cleave xylose residues from the non-reducing end of the xylose chain in an exo-acting manner. β-xylosidases

Fig. 1. Cell-wall polymer (cellulose, hemicellulose and lignin) and hemicellulose composi-tion for a variety of feedstocks that are currently investigated as a source for advancedbiofuels. Reproduced with permission from Pauly and Keegstra (2008).

Fig. 2. Phylogenetic tree of the characterized glycoside hydrolase family 3 (GH 3) glycosi-dases. GenBank Accession numbers are shown succeeded by their activity (the ECnumber,excluding 3.2.1.), e.g. xylosidases: 37 and arabinofuranosidases: 55. For bifunctionalenzymes, the activities are separated by a slash. Arabinofuranosidase and xylosidase activ-ities are indicated with red branches.

318 S. Lagaert et al. / Biotechnology Advances 32 (2014) 316–332

and endoxylanases act in synergy, as endoxylanases generate more re-ducing ends for β-xylosidases to act on and β-xylosidases remove theend products that inhibit endoxylanases (Sunna and Antranikian, 1997).Recently, reducing end xylose-releasing exo-oligoxylanases (EC3.2.1.156) were discovered which act in an exo-acting manner on the re-ducing end of the xylose chain. Lately, several comprehensive reviews ap-peared on xylanases (Collins et al., 2005b; Pollet et al., 2010a). This reviewwill focus on the exo-acting enzymes.

Glycosidases are classified into glycoside hydrolase families (GH) inthe Carbohydrate Active Enzyme (CAZy) database (www.cazy.org) onthe basis of their amino acid sequence similarities (Cantarel et al.,2009). Hence, this classification reflects the structural features,evolutionary relationship and catalytic mechanism of the enzymes(Cantarel et al., 2009; Gebler et al., 1992). Arabinofuranosidases are pres-ent inGH3, 43, 51, 54 and 62,while xylosidases are found inGH3, 30, 39,43, 52, 54, 116 and 120. All these families performhydrolysis with reten-tion of the anomeric configuration, except for GH 43, which is aninverting GH. The reaction mechanism for GH 62 is not yet known. Thetwo characterized reducing end xylose-releasing exo-oligoxylanasesbelong to GH 8, which is known to invert the anomeric configuration.

2. The xylosidases and arabinofuranosidases of GH 3

2.1. Substrate specificities

With over 4000 sequences, GH 3 is one of the largest familiesaccording to the CAZy database and besides xylosidases andarabinofuranosidases, it includes β-glucosidases (EC 3.2.1.21), β-N-acetylhexosaminidases (EC 3.2.1.52), glucan 1,3-β-glucosidases (EC3.2.1.58), glucan 1,4-β-glucosidases (EC 3.2.1.74) and exo-1,3-1,4-glucanases (EC 3.2.1.-). A phylogenetic tree of the characterized GH 3enzymes produced with ClustalX (Larkin et al., 2007) (http://www.clustal.org/) reveals that, besides two bifunctional xylosidases/β-glucosidases, all xylosidases and arabinofuranosidases are present intwo clades, A and B (Fig. 2). Clade A is composed of three clusters: onecollecting all the fungal AX degrading enzymes, one all those fromplantsand a smaller cluster with bacterial sequences. Clade B gathers the otherbacterial enzymes and one archaeal xylosidase/arabinofuranosidase.SignalP and TargetP analyses (Emanuelsson et al., 2007) (http://www.cbs.dtu.dk/services/) indicate all eukaryotic AX hydrolases are secreted,while all bacterial and archaeal enzymes lack a signal peptide, except forthe enzymes from Prevotella (GenBank ID: ADD92014,2 ADD92015,ADD92016 and ACN78955) and from an unidentified rumen bacterium(CAP07659).

Of special interest in GH 3 is the presence of several enzymes report-ed to be bifunctional. However, this classification is most of the timebased on the ability of these enzymes to cleave several synthetic sub-strates, such as p-nitrophenylxyloside (pNP-Xyl), pNP-arabinoside(pNP-Ara) and pNP-glucoside (pNP-Glu). When tested on naturalsubstrates, most enzymes show only the release of one type of sugarand are thus from a biological and applied perspective monofunctional(Table 1). The only true bifunctional AX degrading enzymes, whichcan release both xylose and arabinose from natural substrates, areplant hydrolases from Arabidopsis thaliana (AAM53325), Hordeumvulgare (AAK38481) and Medicago sativa (ABQ45227).

When looking at the activity of the enzymes on natural substrates, itis clear that arabinofuranosidase activity is exclusively found in thecluster of plant glycosidases. These arabinofuranosidases are able tocleave arabinose from arabinan or arabino-oligosaccharides as well asfrom AX. Only the GH 3 arabinofuranosidase from Pyrus pyrifolia(BAD98523) is not active on AX and liberates only arabinose from

2 All protein ID's refer to GenBank Accession numbers for proteins.

arabinan, arabino-oligosaccharides and plant cell wall polysaccharides(Tateishi et al., 2005). All GH 3 AX hydrolases from microorganismsshow exclusively xylosidase activity on substrates such as xylo-oligosaccharides (XOS), xylan and AX. Furthermore, AAA80156, one ofthe two AX degrading enzymes that are found separated from the twolarge clades, does not show xylosidase activity on natural substrates.

Specificity constants of xylosidases towards XOS with a varyingdegree of polymerization (DP)were determined for the GH 3 xylosidasesfrom Hypocrea jecorina (presumably CAA93248) and Talaromycesemersonii (presumably AAL32053) (Rasmussen et al., 2006). kcat/Km

values increase from DP2 up to DP5 and show a slow decrease at DP6,suggesting these xylosidases may interact with the substrate at 5subsites (−1 and +1 to +4). Similar observations were made for thebarley exo-1,3-1,4-glucanases which show markedly higher specificityconstants towards polymeric substrates than towards disaccharides(Hrmova and Fincher, 1997).

2.2. Structural data

Despite the size of GH 3, little structural data is available. To date,the structures of only six enzymes have been deposited in the pro-tein databank (http://www.pdb.org/). No database information isavailable for a xylosidase or arabinofuranosidase, although the struc-ture of a H. jecorina xylosidase (CAA93248) was recently briefly pre-sented (Sandgren et al., 2009) and a xylosidase from Streptomycesthermoviolaceus (BAB61064) has been crystallized (Morioka et al.,2010).

The general fold of GH 3 enzymes is anN-terminal (α/β)8 TIM barrelfold that is connected through a short linker with an (α/β) sandwichdomain (Harvey et al., 2000). The size of this last domain is different

http://www.cazy.org

http://www.clustal.org/

http://www.clustal.org/

http://www.cbs.dtu.dk/services/

http://www.cbs.dtu.dk/services/

http://www.pdb.org/

image of Fig.�2

Table 1Activities of bifunctional arabinoxylan (AX) glycoside hydrolase family 3 (GH 3) enzymeswith AX degrading activities on natural substrates. AGP: arabinogalactan protein, AOS: arabinooligosaccharides, AXOS: arabinoxylan-oligosaccharides, CWP: cell wall polysaccharides, XOS: xylo-oligosaccharides.

Accession nr. Organism Activity (CAZy) Activity (natural substrates) Remarks Reference

ArchaeaAAK43134 Sulfolobus solfataricus P2 3.2.1.37

3.2.1.553.2.1.37 (XOS, xylan) Morana et al. (2007)

BacteriaAAA80156 Erwinia chrysanthemi D1 3.2.1.21

3.2.1.37No activity (xylan, cellobiose) Vroemen et al. (1995)

ACN78955 Prevotella ruminicola 23 3.2.1.373.2.1.55

3.2.1.37 (XOS, AX) Dodd et al. (2009)

CAA91219 Thermoanaerobacter brockii 3.2.1.213.2.1.37

None tested Breves et al. (1997)

AAB23220 Thermoanaerobacter ethanolicus JW200 3.2.1.373.2.1.55

None tested Mai et al. (2000)

Eukaryotes (Fungi)EAA67023 Aspergillus nidulans FGSC A4 3.2.1.37

3.2.1.553.2.1.37 (XOS) 3.2.1.55 (AX) stated, but not apparent

from dataBauer et al. (2006)

Eukaryotes (Plants)AAM53325 Arabidopsis thaliana 3.2.1.37

3.2.1.553.2.1.37 and 3.2.1.55 (AX, AXOS, arabinan) More release of arabinose Minic et al. (2004)

AAK96639 Arabidopsis thaliana 3.2.1.373.2.1.55

3.2.1.55 (AX, arabinan) Charles et al. (2006)

AAK38481 Hordeum vulgare 3.2.1.373.2.1.55

3.2.1.37 and 3.2.1.55 (AX, AXOS, XOS, AOS) More release of xylose from AXOS Lee et al. (2003)

ABQ45227 Medicago sativa subsp. × varia 3.2.1.373.2.1.55

3.2.1.37 and 3.2.1.55 (AX, XOS, AOS, CWP) Xiong et al. (2007)

BAE44362 Raphanus sativus 3.2.1.373.2.1.55

3.2.1.55 (AX, arabinan, AGP) Kotake et al. (2006)

UnidentifiedACY24766 Uncultured organism 3.2.1.21

3.2.1.373.2.1.37 (xylan, XOS) Beloqui et al. (2010)


among several enzymes, with an (α/β)5 sandwich domain in theThermotoga neapolitana β-glucosidase (ABI29899) (Pozzo et al., 2010)and an (α/β)6 domain in the Klyuveromyces marxianus β-glucosidase(ACY95404) (Yoshida et al., 2010) and barley β-glucan glucohydrolase(AAD23382) (Varghese et al., 1999). In the N-acetylglucosaminidasesit is often much shorter, e.g. AAA64351 from Bacillus subtiliswhich hasonly an αβα sandwich domain (Litzinger et al., 2010), or is evencompletely absent like in AAF93857 from Vibrio cholerae (Stubbs et al.,2007). In about one tenth of the GH 3 enzymes, a PA14 domain isinserted in the (α/β) sandwich domain (Yoshida et al., 2010). Thisdomain was shown to bind glucose and to play a critical role in thesubstrate specificity of the K. marxianus β-glucosidase (Yoshida et al.,2010). However, Pfam analysis (http://pfam.sanger.ac.uk/) shows thisPA14 domain is not widespreadwithin the characterized AX hydrolasesand is limited to β-xylosidases from Prevotella ruminocola (ACN78955),Prevotella bryantii (ACN78955) and an unidentified organism(ACY24766). The (α/β)8 TIM barrel and (α/β) sandwich domains canbe further complemented with a third domain at the C-terminus(Harvey et al., 2000) and the β-glucosidases of T. neapolitana andK. marxianus both have a C-terminal fibronectin type III domain ofunknown function (Pozzo et al., 2010; Yoshida et al., 2010).

The GH 3 catalytic nucleophile is an aspartate, which is conservedamong all family members and is located in the N-terminal (α/β)8TIM barrel domain. The catalytic acid is less conserved and difficult ifnot impossible to predict from simple alignments (Harvey et al.,2000). In studied β-glucosidases, a β-glucan glucohydrolase and aglucosylceramidase, the catalytic acid is a glutamate that is positionedin the (α/β) sandwich domain and the active site is located at theinterface of the two domains (Chir et al., 2002; Paal et al., 2004; Pozzoet al., 2010; Varghese et al., 1999; Yoshida et al., 2010). In N-acetylglucosaminidases, where the (α/β) sandwich domain is oftenmuch reduced, a conserved histidine in the (α/β)8 TIM barrel domainis suggested to perform the general acid/base function (Litzinger et al.,2010). The active site of barley β-glucan glucohydrolase and the β-

-

glucosidases from K. marxianus and T. neopolitana forms a pocket with a−1 and+1 subsite, with most of the interactions taking place at the−1 subsite (Pozzo et al., 2010; Yoshida et al., 2010).

Currently, the little structural information that is available of a GH 3AX hydrolase stems from a β -xylosidase from H. jecorina. This enzymewas shown to have three domains, the typical GH 3 N-terminal (α/β)8TIM barrel and (α/β)6 sandwich domain, followed by a third domainwith unspecified fold (Rojas et al., 2005; Sandgren et al., 2009). Amore detailed publication of this structure and the structure of thecrystallized β-xylosidase from S. thermoviolaceus should provide moreinformation in the near future (Morioka et al., 2010).

3. The reducing end xylose-releasing exo-oligoxylanases of GH 8


GH 8 contains almost 600 sequences, mostly coding for cellulases(EC 3.2.1.4) and chitosanases (EC 3.2.1.132), but there are alsolicheninases (EC 3.2.1.73), xylanases (EC 3.2.1.8) and reducing endxylose-releasing exo-oligoxylanases (EC 3.2.1.156, further called rexhydrolases) present. A phylogenetic analysis shows that xylanasesform a distinct clade and rex hydrolases constitute a separate branchwithin this clade, suggesting rex hydrolases evolved from xylanases(Lagaert et al., 2007). For the moment, only two rex hydrolases havebeen characterized, Rex from Bacillus halodurans (BAB05824) andRexA from Bifidobacterium adolescentis (BAF39081) (Honda andKitaoka, 2004; Lagaert et al., 2007). As their names imply, they constitu-tively release a xylose residue from the reducing end of XOS. They don'thydrolyze xylobiose, have a preference for xylotriose and their specific-ity constants decrease as theDP of the XOS increases.While RexA showslow activity on polymeric xylan, Rex is not active on this substrate. Theyare not able to hydrolyze xylose or XOS labeledwith pNP at the reducingend (Honda and Kitaoka, 2004; Lagaert et al., 2007, 2011). GH 8 en-zymes are known to act with inversion of the anomeric configuration

http://pfam.sanger.ac.uk/

http://BAF39081

ncbi-n:AB758288

ncbi-p:CAA99595

ncbi-p:BAF40305

ncbi-p:AAO84266

ncbi-p:ABP67153

ncbi-p:AAC28125

ncbi-p:AAC38457

ncbi-p:ABI34800

ncbi-p:AAD45520

ncbi-p:BAA90771

ncbi-p:AAA61708

ncbi-p:AAD35369


and for RexA, it was shown that XOS hydrolysis leads to the releaseof the β-anomer of xylose and the α-anomer of XOS (Honda andKitaoka, 2004). Furthermore, RexA exclusively hydrolyzes XOS in theβ-anomer configuration and the produced α-anomeric XOS must thusundergo a spontaneous mutarotation before the next xylose residuecan be cleaved off (Honda and Kitaoka, 2004).


The overall topology of GH 8 is an (α/α)6 barrel fold, also present inthe structures of the Pseudoaltermonas haloplanktis xylanase andB. halodurans rex hydrolase (Fushinobu et al., 2005; Van Petegemet al., 2003). In the P. haloplanktis xylanase, the active site is situatedin a cleft that accommodates the substrate with six subsites: −3to −1 binds three glycon xyloses, while +1 to +3 accommodatesthree aglycon xyloses. Modeling and substrate specificity analysis indi-cates that subsite +3 is partially blocked by a small loop in a GH 8xylanase from an uncultured bacterium (ABB71891) (Pollet et al.,2010b). In the B. halodurans rex hydrolase, this loop has a far more pro-found effect as its Leu318 and His319 completely block subsite +2(Fig. 3) (Fushinobu et al., 2005). His319 is also involved in a directhydrogen bond with the β-hydroxyl of the xylose at subsite +1, there-by contributing to the discrimination of the anomeric configuration ofthe XOS substrate. These structural features support the descent of rexhydrolases from GH 8 xylanases.

The proton donor is a glutamate (Glu70 in B. halodurans rex hydro-lase numbering) that is completely conserved in GH 8. The generalbase, however, is not conserved and, depending on its position, GH 8is divided in three subfamilies (GH 8a, GH 8b and GH 8c) (Adachiet al., 2004). The GH 8 endoxylanases and rex hydrolases belong tosubfamily 8a and have an aspartate as a catalytic base (Asp263)(Collins et al., 2005a; Honda and Kitaoka, 2004). A third conserved cat-alytically important residue is another aspartate (Asp128) that is in-volved in sugar-ring distortion and transition-state stabilization(Collins et al., 2005a; De Vos et al., 2006; Honda and Kitaoka, 2004).

Fig. 3. The substrate binding cleft of glycoside hydrolase family 8 (GH 8) B. halodurans rexhydrolase. Cartoon representation of B. halodurans rex hydrolase in complex withxylobiose (yellow) in subsites−1 and−2 (PDB ID: 1WU6), the xylose residue in subsite+1 is from PDB ID: 1WU5. The Leu and His from the loop that blocks subsite +2 arecolored magenta.

4. The xylosidases of GH 39


GH 39 is a relatively small family (less than 400 sequences) andthe 12 characterized enzymes are mammalian α-L-iduronidases (EC3.2.1.76) and bacterial xylosidases. Few β-xylosidases were tested onnatural substrates. A B. halodurans xylosidase (BAB04787) releasesxylose from XOS, arabinoxylan-oligosaccharides (AXOS) andAX, but hydrolysis of xylobiose is barely detectable (Smaaliet al., 2006; Wagschal et al., 2008). The GH 39 xylosidasesfrom Thermoanaerobacterium saccharolyticum and Clostridiumstercorarium (AAA27369 and AAA23063) also exhibit only limitedactivity on xylobiose (Adelsberger et al., 2004; Lee and Zeikus,1993; Wagschal et al., 2005) and the T. saccharolyticum xylosidasehas a much higher activity towards xylotriose. Neither enzymeshows any activity towards polymeric AX, but the C. stercorariumxylosidase binds xylan with enough strength to be used for affinitypurification (Adelsberger et al., 2004; Lee and Zeikus, 1993).

When incubated with xylobiose, the wild type xylosidases fromB. halodurans and T. saccharolyticum both produce detectable amountsof xylotriose (Lee and Zeikus, 1993; Muzard et al., 2009; Smaali et al.,2006).When pNP-Xyl is used as a substrate, the enzymes produce a va-riety pNP-xylobiosides and pNP-xylotriosides (Armand et al., 1996;Muzard et al., 2009). However, these condensation reactions proceedwith poor regioselectivity, as the products contain β-1,2, β-1,3 and β-1,4 linkages. In the presence of methanol, the B. halodurans xylosidasegenerates methyl D-xylosides from pNP-Xyl, xylobiose and xylotriose(Muzard et al., 2009). With xylotriose as a donor, the xylosidaseshows a clear preference towards primary alcohols with a low chainlength (C1–C5) as an acceptor.


Several GH 39 xylosidases exist as tetramers in solution (Czjzeket al., 2004b; Wagschal et al., 2008; Yang et al., 2004). This tetramericform is also observed in the crystal structures of the xylosidases fromG. stearothermophilus and T. saccharolyticum (Czjzek et al., 2005; Yanget al., 2004). Both enzymes have a three domain structure: a catalytic(β/α)8 barrel domain is linked to a β-sandwich domainwhich is follow-ed by a small α-helical domain. The role of the two non-catalyticdomains is unknown, but they may be involved in multimer formationor carbohydrate binding. All characterized GH 39 xylosidases from ther-mophilic bacteria contain these three domains (Czjzek et al., 2005).Catalysis takes place in a pocket at the bottom of a deep cleft (Fig. 4).

Fig. 4. Molecular surface of the glycoside hydrolase family 39 (GH 39) xylosidase fromG. stearothermophilus in complex with 2,5-dinitro-phenyl-β-D-xyloside (PDB ID: 2BFG,chain A).

image of Fig.�3

image of Fig.�4


Although the largest complexed substrate is a 2,5-dinitro-phenyl-β-D-xyloside that shows interactions at the −1 and +1 subsite, the depthof the cleft suggests more aglycon subsites (Fig. 4). At least a+2 subsiteis to be expected based on the lower activity on xylobiose compared toxylotriose.

Glu277 was identified as the catalytic nucleophile in theT. saccharolyticum xylosidase, while Glu160 was found to be the

Table 2Activities of all glycoside hydrolase family 43 (GH 43) enzymes that were tested on naturoligosaccharides, debr. arabinan: debranched arabinan, GOS: galacto-oligosaccharides, XOS: xy

Accession nr. Organism Activ

Exo-α-1,5-arabinofuranosidaseADB43999 Uncultured bacterium 3.2.1BAA90772 Streptomyces chartreusis GS901 3.2.1BAC68753 Streptomyces avermitilisMA-4680 3.2.1

β-1,3-xylosidaseBAF98235 Vibrio sp. XY-214 3.2.1

XylanaseAAB95326 Caldicellulosiruptor sp. Rt69B.1 3.2.1AAD30363 Caldicellulosiruptor sp. Tok7B.1 3.2.1ACZ98594 Cellulosilyticum ruminicola CGMCC 1.5065 3.2.1

Galactan 1,3-β-galactosidaseABN51896 Clostridium thermocellum ATCC 27405 3.2.1BAC69820 Streptomyces avermitilisMA-4680 3.2.1BAH29957 Irpex lacteus NBRC 5367 3.2.1BAD98241 Phanerochaete chrysosporium 3.2.1

β-xylosidaseABR49445 Alkaliphilus metalliredigens QYMF 3.2.1BAB07402 Bacillus halodurans C-125 3.2.1CAA29235 Bacillus pumilis IPO 3.2.1AAC97375 Bacillus pumilis PLS 3.2.1AAB08024 Bacteroides ovatus V975 3.2.1BAF39209 Bifidobacterium adolescentis ATCC 15703 3.2.1ADC85541 Bifidobacterium animalis subsp. lactis BB-12 3.2.1AAA63610 Butyrivibrio fibrisolvens GS 113 3.2.1AAT98625 Geobacillus stearothermophilus T-6; NCIMB 40222 3.2.1ABC75004 Geobacillus thermoleovorans IT-08 3.2.1CAA89208 Prevotella bryantii B14 3.2.1AAB97967 Selenomonas ruminantium GA192 3.2.1XP_391644 Gibberella zeae PH-1 3.2.1BAC75546 Penicillium herquei IFO 4674 3.2.1

α-L-arabinofuranosidaseCAB13699 Bacillus subtilis subsp. subtilis str. 168 3.2.1BAF39204 Bifidobacterium adolescentis ATCC 15703 3.2.1AAO67499 Bifidobacterium adolescentis DSM 20083 3.2.1XP_391670 Gibberella zeae PH-1 3.2.1CAA40378 Paenibacillus polymyxa ATCC 842 3.2.1ABB92159 Uncultured bacterium 3.2.1CAL81199 Humicola insolens DSM 18000 3.2.1

Bifunctional β-xylosidase/α-L-arabinofuranosidaseACF39706 Uncultured bacterium 3.2.1

3.2.1

ArabinanaseBAA20372 Bacillus subtilis IFO3134 3.2.1CAB15969 Bacillus subtilis subsp. subtilis str. 168 3.2.1CAA99586 Bacillus subtilis subsp. subtilis str. 168 3.2.1BAB64339 Bacillus thermodenitrificans TS-3 3.2.1ACE84667 Cellvibrio japonicus Ueda 107 3.2.1ACE73676 Geobacillus stearothermophilus T-6 3.2.1ABQ46657 Thermotoga petrophila RKU-1 3.2.1ADB43998 Uncultured bacterium 3.2.1AAG27441 Aspergillus aculeatus CBS 101.43 3.2.1EAA58736 Aspergillus nidulans FGSC A4 3.2.1EAA58810 Aspergillus nidulans FGSC A4 3.2.1AAA32682 Aspergillus niger CBS120.49/N400 3.2.1BAD15018 Penicillium chrysogenum 31B 3.2.1

acid/base catalyst in the xylosidases of T. saccharolyticum andB. stearothermophilus (Bravman et al., 2001a; Vocadlo et al., 1998,2002). These catalytic residues are strictly conserved in GH 39 and thedistance of 5 Å between them is consistent with the retaining mecha-nism of the enzyme family (Armand et al., 1996; Yang et al., 2004).The Glu160Ala xylosidase from T. saccharolyticum, but not fromG. stearothermophilus, displays an unusual bell-shaped pH profile,

al substrates. AOS: arabino-oligosaccharides, AX: arabinoxylan, AXOS: arabinoxylan-lo-oligosaccharides.

ity (natural substrates) Reference

.- (debr. arabinan) Wong et al. (2008)

.- (debr. arabinan) Matsuo et al. (2000)

.- (debr. arabinan) Ichinose et al. (2008)

.- (β-1,3-XOS) Umemoto et al. (2008)

.8 (xylan) Morris et al. (1999)

.8 (xylan) Gibbs et al. (2000)

.8 (xylan) Cai et al. (2010)

45 (GOS, galactan) Ichinose et al. (2006b)45 (galactan) Ichinose et al. (2006a)45 (GOS, galactan) Kotake et al. (2009)45 (GOS, galactan) Ichinose et al. (2005)

.37 (XOS) Jordan et al. (2013)

.37 (AXOS) Smaali et al. (2006); Wagschal et al. (2012)

.37 (XOS) Panbangred et al. (1984); Xu et al. (1991)

.37 (xylan) La Grange et al. (2000)

.37 (XOS) Whitehead and Hespell (1990)

.37 (XOS, AXOS) Lagaert et al. (2011)

.37 (XOS) Gilad et al. (2010)

.37 (XOS) Sewell et al. (1989)

.37 (XOS) Shallom et al. (2005)

.37 (XOS, xylan) Wagschal et al. (2009b)

.37 (XOS, xylan) Gasparic et al. (1995)

.37 (XOS, xylan) Whitehead and Cotta (2001)

.37 (AXOS) Carapito et al. (2009)

.37 (AXOS) Ito et al. (2003)

.55 (AX, AXOS) Bourgois et al. (2007)

.55 (AX, AXOS) Lagaert et al. (2010)

.55 (AX, AXOS, arabinan) Lagaert et al. (2010); van den Broek et al. (2005)

.55 (AXOS) Carapito et al. (2009)

.55 (AX) Morales et al. (1995)

.55 (AX, arabinan) Wagschal et al. (2007)

.55 (AX, AXOS) McKee et al. (2012); Sørensen et al. (2006);Sørensen et al. (2007)

.37 (XOS, xylan) and

.55 (AX, AOS, arabinan)Wagschal et al. (2009a)

.99 (arabinan) Sakamoto et al. (1997)

.99 (arabinan) Inácio and de Sá-Nogueira (2008)

.99 (arabinan) Proctor et al. (2005)

.99 (arabinan) Takao et al. (2002)

.99 (arabinan) McKie et al. (1997)

.99 (arabinan) Alhassid et al. (2009)

.99 (arabinan) Squina et al. (2010)

.99 (arabinan) Wong et al. (2009)

.99 (debr. arabinan) Skjøt et al. (2001)

.99 (debr. arabinan) Bauer et al. (2006)

.99 (debr. arabinan) Bauer et al. (2006)

.99 (arabinan) Flipphi et al. (1993a); Veen et al. (1991)

.99 (AOS) Sakamoto et al. (2005)

ncbi-n:AB758288

ncbi-p:ABC55452

ncbi-p:CAA99576

ncbi-p:AAO84266

ncbi-p:AAN05450

ncbi-p:AAC28125

nif-antibody:ZP_00503782

ncbi-p:ABI34800

ncbi-p:AAD45520

ncbi-p:BAA90771

ncbi-p:AAA61708

ncbi-p:AAD35369

ncbi-p:AAF19575

ncbi-p:BAB21568

ncbi-p:EAA65870

ncbi-p:AAC41644

ncbi-p:ADZ98861

ncbi-p:AAK21879

ncbi-p:CAL81200

ncbi-p:BAG71680

ncbi-p:ABO93602

ncbi-p:ABO93602

ncbi-p:AAB97967

ncbi-p:XP_391644

ncbi-p:BAC75546

ncbi-p:CAB13699

ncbi-p:BAF39204

ncbi-p:AAO67499

ncbi-p:XP_391670

ncbi-p:CAA40378

ncbi-p:ABB92159

ncbi-p:CAL81199

ncbi-p:ACF39706

ncbi-p:BAA20372

ncbi-p:CAB15969

ncbi-p:CAA99586

ncbi-p:BAB64339

ncbi-p:ACE84667

ncbi-p:ACE73676

ncbi-p:ABQ46657

ncbi-p:ADB43998

ncbi-p:AAG27441

ncbi-p:EAA58736

http://EAA58810

ncbi-p:AAA32682

ncbi-p:BAD15018


suggesting an auxiliary acid/base (Vocadlo et al., 2002). Likely, Glu165 isresponsible for this behavior, as its rotameric forms can be withinhydrogen bonding distance from Glu160 (Czjzek et al., 2005).

5. The xylosidases and arabinofuranosidases of GH 43


Besides xylosidases and arabinofuranosidases, GH 43 containsarabinanases (EC 3.2.1.99), galactan 1,3-β-galactosidases (EC 3.2.1.145),xylanases (EC 3.2.1.8), exo-α-1,5-L-arabinofuranosidases (EC 3.2.1.-)and a β-1,3-xylosidase (EC 3.2.1.72). All characterized enzymes arefrom fungal or bacterial origin, although the CAZy database also presentsGH 43 sequences from archeae and higher plants (A. thaliana andZ. mays). Like GH 3, this family is known for its many bifunctionalenzymes and a phylogenetic tree shows a quasi random distribution ofarabinofuranosidase, xylosidase and bifunctional arabino-furanosidase/xylosidase activities (not shown). However, as is also the case in GH 3,this is mostly the result of the broad substrate specificity towardssynthetic pNP- or methylumbelliferyl (MUF)-linked monosaccha-rides. When looking at the experimental evidence, all except onecleave only a single type of sugar from natural substrates (Table 2). Theone enzyme that displays bifunctional xylosidase/arabinofuranosidaseactivity (ACF39706) releases xylose from XOS and xylan and arabinosefrom arabino-oligosaccharides, arabinan and AX (Wagschal et al.,2009a). A phylogenetic tree of GH43 sequences with known enzymeactivities on natural substrates show 5 clades with a much less randomdistribution (Fig. 5). Xylosidases and arabinofuranosidases are bothpresent in clades A and C, while exo-α-1,5-L-arabinofuranosidases,galactan 1,3-β-galactosidases and arabinanases are found in clades B, Dand E, respectively.

Clade A contains two clusters, one with β-xylosidases (cluster A1)and one with arabinofuranosidases and xylanases (cluster A2), althoughthe occurrence of xylanase activities in GH 43 should be considered withsome reservations. Twoof the four xylanases are fromCaldicellulosiruptorsp. Rt69B.1 and Tok7B.1 (AAB95326 and AAD30363, respectively). Bothenzymes contain a GH 10 and GH 43 domain (Gibbs et al., 2000;Morris et al., 1999). As GH 10 is a family that exclusively holds xylanases,the reported xylanase activity most likely originates from this domain. A

Fig. 5. Phylogenetic tree of the glycoside hydrolase family 43 (GH 43) enzymeswith char-acterized activity on natural substrates. Nomenclature as in Fig. 2.

third enzyme (CAA40378 from Paenibacillus polymyxa) showed xylanaseactivity using a zymogram analysis (Gosalbes et al., 1991). However, alater study could not detect xylanase activity and observed only arabi-nose release from AX (Morales et al., 1995). The last GH 43 xylanase isonly described recently and limited experimental information is avail-able (Cai et al., 2010). In this case, xylanase activitywas also only demon-strated with zymogram analysis. To add to the confusion, CAZy lists thisenzyme as a xylosidase instead of a xylanase and suggests the sequenceis only a fragment of the mature protein, although we could not find aliterature reference for this hypothesis. In conclusion, we believe moreexperimental evidence is needed to firmly state xylanase activity linkedto a GH 43 domain.

In clade C three clusters can be detected. Two arabinofuranosidasesform cluster 1 (C1), cluster 2 (C2) contains both arabinofuranosidasesand xylosidases and cluster 3 (C3) groups xylosidases. Interestingly,the bifunctional xylosidase/arabinofuranosidase (ACF39706) in cluster2 is positioned between xylosidases and arabinofuranosidases, suggest-ing itmay be an intermediate in the evolution of the former to the latter.In the same regard, the β-1,3-xylosidase (BAF98235 from Vibrio sp. XY-214) is located at the base of cluster 3, where it branches of fromarabinofuranosidases. Combinedwith rotation of the substrate betweenGH 43 arabinofuranosidases and xylosidases (see Section 5.2), this maysuggest that the β-1,3-xylosidase is an intermediate form in the evolu-tion of these two activities.

Specificity constants towards XOS with different lengths weredetermined for the xylosidases from B. adolescentis (BAF39209),S. ruminantium (AAB97967) and G. thermoleovorans (ABC75004). kcat/Km values are similar for DP 2–6 for the B. adolescentis enzyme, whilethe S. ruminantium xylosidase shows a preference towards xylobioseand specificity constants decrease with larger chain lengths (DP 2–6)(Jordan et al., 2007; Lagaert et al., 2011). The activity of theG. thermoleovorans xylosidase was only evaluated on xylobioseand xylotriose, but this enzyme also showed a higher substratepreference towards xylobiose than xylotriose (Wagschal et al.,2009b). These observations indicate that XOS interact with GH 43xylosidases at two subsites (−1 and +1).

The substrate specificities towards specific arabinose linkages in AXare characterized for a number of GH 43 arabinofuranosidases. The twoenzymes that form cluster C1 (CAL81199 and AAO67499) both cleavethe arabinose at the C(O)3 position from xyloses with substitutions atthe C(O)2 as well as the C(O)3 position (Lagaert et al., 2010; McKeeet al., 2012; Sørensen et al., 2006, 2007; van den Broek et al., 2005).On the other hand, BAF39204 (part of cluster C2) and CAB13699 (partof A2) exclusively hydrolyze arabinose linkages from xyloses that haveonly one arabinose residue linked to their C(O)2 or C(O)3 positions(Bourgois et al., 2007; Lagaert et al., 2010). These two types ofarabinofuranosidaseswork in synergy, as removal of C(O)3-linked arab-inoses from disubstituted xyloses by the first type of enzymes, such asbelonging to cluster C1, makes more C(O)2-linked arabinoses availablefor hydrolysis by the second type of enzymes, such as those belongingto cluster C2 or A2 (Lagaert et al., 2010; Sørensen et al., 2006; VanLaere et al., 1999).


Three enzymes from cluster C3 (BAF98235, AAB97967 andAAT98625) exist as a tetramer in solution (Brunzelle et al., 2008; Brüxet al., 2006; Umemoto et al., 2008). AAB97967 and AAT98625 wereshown to be dimers of dimers with stronger interactions between themonomers composing the dimers than between the dimers that formthe tetramers. Another enzyme from this cluster (BAB07402) appearsas a dimer (Smaali et al., 2006), while enzymes from other clustersand clades are monomeric (Alhassid et al., 2009; Squina et al., 2010;Vandermarliere et al., 2009;Wagschal et al., 2007, 2009a). Amultimericquaternary structure may therefore be limited to the enzymes ofcluster C3.

image of Fig.�5


The signature structure of GH 43 enzymes is a catalytic domainwith afive-bladed β-propeller fold (Nurizzo et al., 2002). The crystal structureof two xylosidases (AAB97967 from S. ruminantium and AAT98625from G. stearothermophilus, both part of cluster C3) and one arab-inofuranosidase (CAB13699 from B. subtilis, cluster A2, and CAL81199from H. insolence, cluster C1) have been published (Brunzelle et al.,2008; Brüx et al., 2006; McKee et al., 2012; Vandermarliere et al.,2009). All four enzymes contain an additional C-terminal β-sandwichdomain. Multiple alignment of characterized GH 43 enzymes indicatesthat this second domain is present in the whole family except in theclusters A1, E1 and clade B (not shown). In cluster A2 this domain is clas-sified in the CAZy database as a carbohydrate bindingmodule of family 6(CBM 6). CBM 6modules have two binding sites that can interact with avariety of different substrates, like xylan and XOS, cellulose and cello-oligosaccharides, lamarin and glucans (Fernandes et al., 1999; Henshawet al., 2004). At least in the structure of the GH 43 arabinofuranosidaseof B. subtilis, aromatic residues of CBM 6 that play a crucial role in carbo-hydrate binding are lacking in the first binding site and a loop blocks thesecond site, suggesting a loss of substrate binding during evolution(Vandermarliere et al., 2009). However, not all GH 43 enzymes havelost the binding capabilities of this module. The β-sandwich domainfrom the exo-1,5-α-L-arabinofuranosidase from Streptomyces avermitilis(BAC68753 from clade B) binds branched arabinan and AX and its re-moval leads to a decreased activity against debranched arabinan(Ichinose et al., 2008). The crystal structure of this enzyme shows arabi-nose bound at three sites in this C-terminal domain that belongs to CBM42.

The different nature of the activities found in GH 43, e.g. arab-inanases (endo-hydrolysis of a linear backbone), xylosidases and exo-1,5-α-L-arabinofuranosidases (exo-hydrolysis of a linear backbone)and arabinofuranosidases (exo-hydrolysis of backbone substituents)

Fig. 6.Different orientations of substrates in glycoside hydrolase family 43 (GH 43) enzymes. A.residues are shown in green.Orange: arabinohexaose bound to C. japonicus arabinanase (PDB IDStreptomyces avermitilis exo-1,5-α-arabinofuranosidase (PDB ID: 3AKH), blue: xylobiose from Garabinofuranosidase (PDB ID: 3C7O), this substrate does not contain the glycon arabinose tha(1), the B. substilis arabinofuranosidase (2) and the G. stearothermophilus xylosidase (3).

requires different substrate orientations in the active site (Fig. 6). Inxylosidases and exo-1,5-α-L-arabinofuranosidases, the active site is apocket that harbors the sugar residue at the non-reducing end and therest of the substrate backbone is positioned at right angles to theenzyme surface. The substrate is positioned similarly in arabinanases,but here the active site doesn't form a pocket but a cleft and allowsthe substrate to continue along −2 and −3 subsites. In contrast, inarabinofuranosidases, the xylose backbone is positioned perpendicularto the previous substrates and is bound in a long groove on the enzymessurface and the active site is a pocket at the bottom of this groove(Vandermarliere et al., 2009). The structure of the H. insolensarabinofuranosidase (CAL81199) was recently published and revealsthe structural determinants for the specificity towards C(O)3-linkedarabinose on disubstituted xyloses (McKee et al., 2012). In addition toa deep pocket that houses the active site, the arabinofuranosidase hous-es a shallower adjacent pocket that binds to the C(O)2-linked arabinoseof these xylose residues.

Three residues are completely conserved in family 43, two aspar-tates and a glutamate. Despite the different activities and substrateorientations, these residues are similarly positioned in the active site.In the xylosidase from G. stearothermophilus Asp15 and Glu187 weredemonstrated to be the general base and general acid, respectively(Shallom et al., 2005). The third catalytic residue, Asp128, is thoughtto be involved in pKa modulation and the orientation of the catalyticacid and the substrate (Brüx et al., 2006; Nurizzo et al., 2002). It hasbeen noted that glycoside hydrolases with five-bladed β-propellerfolds never conform to the general distances between the catalyticresidues of inverting and retaining enzymes (Brunzelle et al., 2008).Indeed, although GH 43 enzymes act with inversion of the anomericconfiguration (Jordan et al., 2007; Kersters-Hilderson et al., 1976; Pitsonet al., 1996), the distance between the general acid and general base is

Superposition of different substrates bound in the active site of GH 43 enzymes. Active site: 1GYE),magenta: arabinose in the−1 subsite and arabinobiose in the+1 to+2 subsite of. stearothermophilus β-xylosidase (PDB ID: 2EXJ), yellow: xylotetraose bound to B. substilist would be located in the −1 subsite. B. Molecular surface of the C. japonicus arabinanase

image of Fig.�6

Fig. 7. Phylogenetic tree of characterized enzymes from glycoside hydrolase family 51 (GH51). Nomenclature as in Fig. 2.


approximately 7 Å, significantly less than the 10 Ånormally observed ininverting enzymes (Brüx et al., 2006; Rye and Withers, 2000; Wanget al., 1994). Of particular interest is the study of McKee et al. (2012),which showed that the conversion of Tyr166, which neighbors the cat-alytic Asp167 in the H. insolens arabinofuranosidase, to alanine intro-duces endo-xylanase activity while the enzyme keeps its, albeitlowered, arabinofuranosidase activity. The Tyr166Alamutation disruptsthe lip of the active site pocket, thereby allowing the xylan backboneinto the active site.

6. The arabinofuranosidases of GH 51


With the exception of four characterized endoglucanases (3.2.1.4),GH 51 exclusively holds arabinofuranosidases. Despite the relativelymoderate size of this family (over 700 sequences), it contains the largestnumber of studied arabinofuranosidases (35 enzymes) and most ofthese enzymes were characterized on natural substrates (Table 3). Afew arabinofuranosidases cleave a variety of monosaccharides linkedto pNP or MUF, but in contrast to GH 43, no bifunctionality is claimedon this basis. There is, however, one bifunctional enzyme fromA. thaliana (AAF19575) that releases both xylose and arabinose fromAX and AXOS (Minic et al., 2004). A phylogenetic tree puts all GH 51sequences in three groups: clade A, clade B and group C (Fig. 7). CladeA is further divided into four clusters: A1, A2, A3 and A4. Two bacterialenzymes are present in cluster A1, while all plant and fungal GH 51arabinofuranosidases are grouped in clusters A2 and A3, respectively.The GH 51 endoglucanases form cluster A4. Cluster B and group C gatherthe remaining bacterial arabinofuranosidases and the fungal arab-inofuranosidase from Aspergillus nidulans (EAA65870). SignalP andTargetP analysis indicates that all plant and fungal arabinofuranosidasesare secreted with the exception of the arabinofuranosidase fromA. nidulans. In contrast, based on the same analysis, the bacterial enzymes

Table 3Activities of glycoside hydrolase family 51 (GH 51) arabinofuranosidases on natural substrates

Accession nr. Organism Active on

ACB54691 Anoxybacillus kestanbolensis AC26SARI AX, AXOS, arabinaAB758288 Aureobasidium pullulans ATCC 20524 AXABC55452 Bacillus pumilus ARA AXCAA99576 Bacillus subtilis subsp. subtilis str. 168 AX, AXOS, arabinaCAA99595 Bacillus subtilis subsp. subtilis str. 168 AXOS, arabinan, AO

BAF40305 Bifidobacterium adolescentis ATCC 15703 AX, AXOS, arabinaAAO84266 Bifidobacterium longum B667 AX,AXOS, arabinan

ABP67153 Caldicellulosiruptor saccharolyticus DSM 8903 AOSACE86344 Cellvibrio japonicus Ueda107 AX, AXOS, arabinaAAN05450 Clostridium cellulovorans AX, AXOS, arabinaAAC28125 Clostridium stercorarium NCIMB 11754 AX, AXOSZP_00503782 Clostridium thermocellum ATCC 27405 AX, AXOS, arabinaAAC38456 Cytophaga xylanolytica AX, arabinanAAC38457 Cytophaga xylanolytica AX, arabinanABI34800 Geobacillus caldoxylosilyticus TK4 arabinan, AOSAAD45520 Geobacillus stearothermophilus T-6 NCIMB 40222 arabinan, AX (loweBAA90771 Streptomyces chartreusis GS901 AX, arabinan, AOSAAA61708 Streptomyces lividans 66 AX, AXOS, arabinaACY69989 Streptomyces sp. S9 ACCC 41168 AXCAA76421 Thermobacillus xylanilyticus D3 AX, AXOSAAD35369 Thermotoga maritima MSB8 arabinanAAF19575 Arabidopsis thaliana AX, AXOS, arabinaBAB21568 Aspergillus awamori IFO4033 AX, AXOS, arabinaEAA65870 Aspergillus nidulans FGSC A4 AOSAAC41644 Aspergillus niger CBS 120.49/N400 AOSADZ98861 Chrysosporium lucknowense C1 AOSAAK21879 Hordeum vulgare AX, AXOS, arabinaCAL81200 Meripilus giganteus CBS 521.91 AX, AXOSBAG71680 Penicillium chrysogenum 31B AX, arabinan, AOSABO93602 Penicillium purpurogenum MYA-38 AX, arabinan, AOS

remain in the cytoplasm, except in Streptomyces lividans (AAA50393),Streptomyces chartreusis (BAA90771), Cytophaga xylanolytica (AAC38456and AAC38457) and Cellvibrio japonicus (ACE86344).

Most GH 51 arabinofuranosidases release arabinose from AX, AXOS,arabinan and arabino-oligosaccharides (Table 3). Nonetheless, a num-ber of the enzymes from clade B show specificity towards arabinan(oligosaccharides) and are not active on AX(OS) (ABP67153, ABI34800

. AOS: arabino-oligosaccharides, AX: arabinoxylan, AXOS: arabinoxylan-oligosaccharides.

Inactive on References

n, AOS Canakci et al. (2008)Ohta et al. (2013)Pei and Shao (2008)

n, AOS Bourgois (2008); Inacio et al. (2008)S AX Bourgois (2008); Hoffmam et al. (2013);

Inacio et al. (2008)n Lagaert et al. (2010), AOS Lagaert (2013); Margolles and de los

Reyes-Gavilán (2003)AX, arabinan Lim et al. (2010)

n, AOS Beylot et al. (2001)n Kosugi et al. (2002)

Adelsberger et al. (2004)n, AOS Taylor et al. (2006)

Kim et al. (1998); Renner and Breznak (1998)Kim et al. (1998)

AX Canakci et al. (2007)r) Gilead and Shoham (1995)

AXOS Matsuo et al. (2000)n Manin et al. (1994)

Shi et al. (2010)Debeche et al. (2000); Rémond et al. (2008)

AX Miyazaki (2005)n Minic et al. (2004)n, AOS Kaneko et al. (1998b)

Bauer et al. (2006)AX Flipphi et al. (1993b)

Kühnel et al. (2011)n, AOS Lee et al. (2001)

Sørensen et al. (2006)Sakamoto and Kawasaki (2003); Sakamoto et al. (2013)Fritz et al. (2008)

ncbi-p:ACB54691

ncbi-n:AB758288

ncbi-p:ABC55452

ncbi-p:CAA99576

ncbi-p:CAA99595

ncbi-p:BAF40305

ncbi-p:AAO84266

ncbi-p:ABP67153

ncbi-p:ACE86344

ncbi-p:AAN05450

ncbi-p:AAC28125

nif-antibody:ZP_00503782

ncbi-p:AAC38456

ncbi-p:AAC38457

ncbi-p:ABI34800

ncbi-p:AAD45520

ncbi-p:BAA90771

ncbi-p:AAA61708

ncbi-p:ACY69989

ncbi-p:CAA76421

ncbi-p:AAD35369

ncbi-p:AAF19575

ncbi-p:BAB21568

ncbi-p:EAA65870

ncbi-p:AAC41644

ncbi-p:ADZ98861

ncbi-p:AAK21879

ncbi-p:CAL81200

ncbi-p:BAG71680

ncbi-p:ABO93602

image of Fig.�7

Fig. 8. Different loop conformations in glycoside hydrolase family 51 (GH 51)arabinofuranosidases. Cartoon representation of the T. xylanilyticus arabinofuranosidasewith β2α2 in the open (green, PDB ID: 2VRQ, chain A) and the closed conformation(blue, only loop shown, PDB ID: 2VRQ, chain C). The cartoon representation of theB. longum arabinofuranosidase is shown in gray and shows the insertion in loop β7α7that forms an α-helix and bends over the substrate binding cleft (PDB ID: 2Y2W). Thesubstrate (yellow, PDB ID: 2VRQ, chain A) and Trp99 from T. xylanilyticus are shown insticks.


and AAD35369) (Canakci et al., 2007; Lim et al., 2010; Miyazaki, 2005)or show a much lower activity towards AX(OS) (AAD45520 andCAA99595) (Bourgois, 2008; Gilead and Shoham, 1995). In general,when comparing activity on branched arabinan and debranchedarabinan, activity on the branched form is much higher, suggestinghydrolysis of the C(O)2- and C(O)3-linkages between arabinose substit-uents and backbone residues (Beylot et al., 2001; Lagaert et al., 2010;Matsuo et al., 2000; Miyazaki, 2005; Sakamoto and Kawasaki, 2003;Taylor et al., 2006). Specific cleavage of arabinose substituents wasdemonstrated with H1-NMR for CAA99576 from B. subtilis (Bourgois,2008). The Aspergillus enzymes are the only GH 51 enzymes we foundwhich show a higher activity on the α-1,5-linked arabinose mainchain (Bauer et al., 2006; de Vries and Visser, 2001; Kaneko et al.,1998b).

Like in GH 43, the substrate specificity towards specific arabinosesubstitutions in AX or AXOS is determined for a number of GH 51enzymes. Arabinofuranosidases fromMeripilus giganteus and Penicilliumchrysogenum (CAL81200 and BAG71680, respectively, both cluster A3)and B. subtilis (CAA76421, group C) exclusively hydrolyze arabinoselinked to the C(O)2 or C(O)3 of monosubstituted xylose residues(Bourgois, 2008; Sakamoto et al., 2013; Sørensen et al., 2006), whileH. vulgare AAK21879 (cluster A2) and C. japonicus ACE86344 (group C)also show a preference towards these linkages, but are able to release asmall amount of arabinose from disubstituted xyloses (Beylot et al.,2001; Lee et al., 2001). Furthermore, the crystal structures of B. longumAAO84266,Clostridium thermocellum ZP_00503782,G. stearothermophilusAAD45520 (all clade B) and Thermobacillus xylanilyticus CAA76421(group C) suggest the size of the active site can only harbor the arabinosefrom monosubstituted xyloses (see Section 6.2). GH 51 enzymes thatspecifically cleave arabinose fromdisubstituted xylose are limited to clus-ter A1. Similar to the GH 43 enzymes with activity towards disubstitutedxylose, B. adolescentis BAF40305 exclusively cleaves the arabinose onposition C(O)3 of a disubstituted xylose (Lagaert et al., 2010). No directevidence towards linkage preference is present for the other enzyme ofcluster A1, but S. chartreusis BAA90771 releases arabinose from AX andnot from two tested AXOS samples (Matsuo et al., 2000). As both samplesonly contained monosubstituted xyloses, it is not unlikely that thisenzyme is also specific towards arabinose on disubstituted xyloseresidues.


GH 51 arabinofuranosidases are found as hexamers in solution(Hövel et al., 2003b; Inacio et al., 2008; Miyazaki, 2005; Taylor et al.,2006). Thismultimeric configuration is also seen in the crystal structuresof B. longum AAO84266, Clostridium thermocellum ZP_00503782,G. stearothermophilus AAD45520, Thermobacillus xylanilyticusCAA76421, Thermotoga maritime AAD35369 and Thermotoga petrophilaABQ46651 (Hövel et al., 2003a; Im et al., 2012; Lagaert, 2013; Paës et al.,2008; Souza et al., 2011; Taylor et al., 2006). GH 51 is a member of thesame glycoside hydrolase clan (A) as GH 39 and shares a similar fold.The catalytic domain is a N-terminal (β/α)8 TIM barrel, followed by aβ-sandwich domain of unknown function. One of the strands of thisC-terminal domain is formed by approximately 10 N-terminal aminoacids. Multiple alignment of the studied arabinofuranosidases indicatesthis two domain architecture is present in all arabinofuranosidases,although the S. lividans AAA61708 may contain an additional thirddomain at its C-terminus.

The catalytic acid/base and nucleophile of the arabinofuranosidasefrom G. stearothermophilus are a conserved Glu175 and Glu294, respec-tively (Shallom et al., 2002a, 2002b). They are separated by approxi-mately 5 Å, typical for the retaining mechanism displayed in thisfamily (Hövel et al., 2003a; Pitson et al., 1996). These catalytic residuesline a pocket shaped active site at the bottom of a large, non-lineargroove. The structure with the largest complexed substrate is that ofT. xylanilyticus arabinofuranosidase with arabinose linked to the C(O)3

of the middle xylose in xylotriose (Paës et al., 2008). It reveals interac-tions at four subsites: −1 (the pocket that harbors the arabinose),+1, +2 and +2′ (the groove that binds the middle, reducing end andnon-reducing end xylose, respectively). Substrate specificity of GH 51enzymes seems to be influenced by the variation of the length and flex-ibility of several loops that surround the active site (Lagaert, 2013). Theβ7α7 loop varies considerably in length and in the structure ofB. longum AAO84266, an insertion in this loop forms an α-helix andbends over subsite +2′, hindering large and substituted chains(Fig. 8) (Lagaert, 2013). In the T. xylanilyticus arabinofuranosidase, theβ2α2 loop can adopt an open and closed conformation (Fig. 8) (Paëset al., 2008). In the closed conformation, Trp99 provides an importantinteraction with the arabinosyl moiety. Due to the larger sizes of theβ7α7 loop, the open conformation is sterically impossible in thearabinofuranosidases from G. stearothermophilus, C. thermocellum andB. longum (Lagaert, 2013; Paës et al., 2008).



GH 52 is a small family, with only 32 bacterial sequences, fromwhich six enzyme products were characterized. Exclusively xylosidaseactivity is observed. The GH 52 enzymes from Aeromonas caviae andG. stearothermophilus 21 hydrolyze XOS to xylose (Nanmori et al.,1990; Suzuki et al., 2001). The xylosidase from A. caviae showstransglycosylation activity and incubation with xylotriose leads initiallyto the production of xylotetraose and xylopentaose (Suzuki et al., 2001).Incubation of the xylosidase from G. stearothermophilus T-6 with pNP-Xyl leads to the release of pNP and the formation of higher order XOS,while no free xylose is observed (Bravman et al., 2003).


Although the crystallization and 2 Å data collection of theG. stearothermophilus T-6 xylosidase was already reported in 2004, nocrystal structures of GH 52 enzymes are available yet (Czjzek et al.,2004a). The xylosidases from A. caviae and G. stearothermophilus (strain21 aswell as T-6) form dimers in solution, although the xylosidase fromGeobacillus pallidus is suggested to be a trimer (Contreras et al., 2008;Nanmori et al., 1990; Quintero et al., 2007; Suzuki et al., 2001). Interest-ingly, isothermal titration calorimetry indicates that the dimeric form of

image of Fig.�8

Fig. 9. Structure of the glycoside hydrolase family 54 (GH 54) arabinofuranosidase ofA. awamori. Cartoon representation of the catalytic and CBM 42 domain (orange andpurple, respectively) of the A. awamori arabinofuranosidase in complex with arabinose(yellow stick representation). The catalytic residues are shown in red, the arabinose bind-ing residues of CBM 42 in blue (PDB ID: 1WD4).


the G. stearothermophilus T-6 xylosidase only contains a single activesite, formed by the residues of both monomers (Contreras et al., 2008).This enzyme was shown to perform hydrolysis with retention of theanomeric configuration using the likely catalytic residues Glu337 andGlu413 (Bravman et al., 2001b). The enzyme from G. pallidus is formedby α-helices (44%) and β-sheets (40%), while these values are 30% forboth secondary structures in the G. stearothermophilus T-6 xylosidase(Contreras et al., 2008; Quintero et al., 2007).



GH 54 is a relatively small family with less than 100 sequences, splitequally amongst bacteria and eukaryota. All 22 currently characterizedenzymes are highly conserved fungal proteins that exclusively displayarabinofuranosidase activity on natural substrates. Solely based on thehydrolysis of pNP-Xyl and pNP-Ara, the Hypocrea koningii GH 54enzyme (AAA81024) is called a bifunctional arabinofuranosidase/xylosidase (Wan et al., 2007a). All enzymes that were tested on AXand/or arabinan release only arabinose from both substrates (de Wetet al., 2008; Guais et al., 2010; Kaneko et al., 1998a, 1998b; Kormelinket al., 1993; Sakamoto et al., 2013; Sakamoto and Kawasaki, 2003;Takata et al., 2010). When comparing hydrolysis of debranched andbranched arabinan, activity is always lower or non-existent on thedebranched form (Kaneko et al., 1998a, 1998b; Sakamoto andKawasaki, 2003; Sakamoto et al., 2013; Takata et al., 2010). Several en-zymes also cleave arabinose from arabinogalactan (Kaneko et al.,1998b; Kormelink et al., 1993; Sakamoto and Kawasaki, 2003; Takataet al., 2010), although the Aurobasidium pullulans arabinofuranosidase(AAR87863) does not (de Wet et al., 2008). Aspergillus niger AbfB(AAB53944) was shown to release arabinose from AXOS onlywhen the arabinose was bound to a singly substituted xylose residueat the non-reducing end (Kormelink et al., 1993). Similarly, thearabinofuranosidases from Aspergillus awamori (BAB21567) and H.jecorina (CAA93243) readily cleave arabinose linked to the C(O)3 posi-tion of the non-reducing end xylose in xylobiose, while the activity isabsent ormuch reduced towards arabinose substituted at the C(O)3 po-sition of the middle xylose in xylotriose (Kaneko et al., 1998a, 1998b).On the other hand, the GH 54 arabinofuranosidase from P. chrysogenum(BAG71681) is able to release arabinose from single as well as doublesubstituted xylose in wheat AX (Sakamoto et al., 2013).


ArfB from A. awamori is the only GH 54 enzyme with a determinedstructure. The protein is monomeric in the asymmetric unit of thecrystals, similar to the natural occurrence of other GH 54 arab-inofuranosidases in solution (De Ioannes et al., 2000; de Wet et al.,2008;Miyanaga et al., 2004). It comprises a catalytic C-terminal domainwith a β-sandwich fold, linked to a domain with a β-trefoil fold whichbelongs to CBM 42 (Fig. 9) (Miyanaga et al., 2004). Pfam analysisshows this two domain architecture is present in all characterized GH54 arabinofuranosidases, with the exception of EAA29123 from Neuros-pora crassa and CAL85369 from Penicillium funiculosum. In EAA29123the second domain is absent, while it is replaced with a CBM 1 domainthat specifically binds cellulose in CAL85369 (Guais et al., 2010).

Based on their position in relation to the arabinose in the catalyticsite of the A. awamori arabinofuranosidase structure and the drop inactivity of mutants, Glu221 and Asp297 are the suggested catalyticnucleophile and acid/base, respectively (Miyanaga et al., 2004). Anextensive mutagenesis study of the H. koningii arabinofuranosidasesupports this hypothesis and points to a third catalytic residue, mostlikely involved in substrate binding (Asp219 in A. awamori numbering)(Wan et al., 2007b). In the A. awamori crystal structure, Glu221 andAsp297 are separated by 5.6 Å, in accordance with the retaining

mechanism displayed by GH 54 enzymes (Miyanaga et al., 2004;Pitson et al., 1996). They line a concave, negatively charged pocketthat forms the −1 catalytic subsite (Miyanaga et al., 2004). Eightconserved cysteine residues are responsible for three disulfide bridgesin the catalytic domain and one in the CBM 42 domain (Miyanagaet al., 2004).

The CBM 42 domain of the A. awamori arabinofuranosidase containsthree subdomains (α, β and γ) of about 50 amino acid residues andstructures in complex with arabinose, arabinofuranosyl-xylobiose andarabinotriose are published (Miyanaga et al., 2004, 2006). In all threecases, binding is observed in the pockets of the β and γ, but not the αsubdomain. Interestingly, CBM 42 binds only the non-reducing endarabinose side chains and not the arabinose or xylose backbone(Miyanaga et al., 2006). Arabinose is recognized in the same mannerin both binding pockets. The side chain sugar forms hydrogen bondswith an aspartate and histidine, while stacked between two tyrosineresidues (Miyanaga et al., 2006). Mutation of these aspartates in the A.awamori arabinofuranosidase or removal of the whole CBM domain inthe H. jecorina enzyme leads to a strong decrease in affinity for AX(Miyanaga et al., 2006; Nogawa et al., 1999). Furthermore, while theactivity of thesemutants towards pNP-Ara does not change, the activitytowards AX decreases to 1–10% (Miyanaga et al., 2006; Nogawa et al.,1999).



GH 62 is a small family of highly conserved bacterial and eukaryoticsequences (71 and 36, respectively), which exclusively comprisesarabinofuranosidases. All GH 62 enzymes release arabinose from AX(Bauer et al., 2006; Beylot et al., 2001; Kimura et al., 2000; Lange et al.,2006; Madrid et al., 1996; Sakamoto et al., 2011; Tsujibo et al., 2002;Vincent et al., 1997). When tested on arabinan, they release arabinosefrom the branched, but not from the debranched form (Bauer et al.,2006; Kimura et al., 2000; Madrid et al., 1996; Tsujibo et al., 2002;Vincent et al., 1997). Notable exceptions are ACE85320 from C. japonicusand BAG71682 from P. chrysogenum, which are also inactive on branchedarabinan (Beylot et al., 2001; Sakamoto et al., 2011). While pNP-Ara isoften, if not always, the best substrate for arabinofuranosidases of otherGHs, several GH 62 enzymes display a remarkably low activity on thissubstrate (Beylot et al., 2001; Sakamoto et al., 2011; Tsujibo et al.,2002; Vincent et al., 1997).

image of Fig.�9


Substrate specificities of GH 62 enzymes towards specific arabinosesubstitutions in AXOS and AX were determined for ACE85320from C. japonicus, CAM07245 from Penicillium capsulatum andBAG71682 from P. chrysogenum. All three enzymes are able to cleavearabinose from monosubstituted, but not from disubstitutedxyloses in AX or AXOS (Beylot et al., 2001; Lange et al., 2006;Sakamoto et al., 2011). Furthermore, the C. japonicus and P. capsulatumarabinofuranosidase show a preference towards C(O)3-linked arabi-nose compared to C(O)2-linked residues (Beylot et al., 2001; Langeet al., 2006).


Although the arabinofuranosidase from A. niger 3 M43 has beencrystallized a long time ago, no crystal structure from a GH 62 enzymeis available (Scott et al., 1997). However, as GH 62 belongs to GH clanF together with GH 43, the general fold is expected to be a 5-bladed β-propeller. On the same basis, GH 62 enzymes may be the secondarabinofuranosidase family which acts with inversion of the anomericcenter. Multiple alignment of GH 62 with sequences from GHs 32, 43and 68 points to the same three catalytic residues as found in GH 43(Pons et al., 2004). Glu and Asp are thought to be the general acid andbase, respectively, and a secondAsp is likely involved in pKamodulationand orientation of the catalytic acid and the substrate.

Multiple sequence alignment and pfamanalysis shows that the char-acterized members typically contain a single catalytic domain, with theexception of three bacterial enzymes which contain additional N-terminal GH and/or CBM domains. ACE85320 from C. japonicus has atotal of three domains, from the N-terminus a CBM 2, CBM 35 and GH62 domain. Deletion and fusion experiments with the CBM 2 domainof ACE85320 showed that this domain binds crystalline cellulose(Kellett et al., 1990). On the other hand, the C. japonicus CBM35 domaininteracts in a calcium-dependentmanner with unsubstituted xylan, butnot with the substituted form or XOS (Bolam et al., 2004). AAC26524from S. lividans contains one N-terminal domain that belongs to CBM13 and binds AX (Dupont et al., 1998; Vincent et al., 1997). AAD32559from Streptomyces chattanoogensis has three modules, a GH 10 domainlinked through a CBM 13 module to the GH 62 domain. No substratespecificities or binding characteristics have been determined, but asGH 10 and GH 62 are families with exclusively xylanases andarabinofuranosidases, respectively, the enzyme is thought to haveboth activities (Hernandez et al., 2001).



Recently, GH 120was introduced. It contains 40 bacterial sequences,from which two enzymes were characterized. T. saccharolyticumxylosidase ABM68042 actswith retention of the anomeric configurationand cleaves xylose from both xylobiose and xylotriose (Huang et al.,2012; Shao et al., 2011). In contrast, B. adolescentis xylosidaseBAF39080 is almost inactive on xylobiose, but readily hydrolyzes XOSwith a higher DP (Lagaert et al., 2011). Furthermore, it releases up to30% of xylose from AXOS, but hydrolysis of AX is below 1%.


Crystal structures of the T. saccharolyticum xylosidase have beenpublished recently (Huang et al., 2012). They show that the enzyme isassembled as a tetramer and contains two domains, folded as a right-handed β-helix and a β-sandwich domain. Although xylobiose isthe largest complexed substrate in the crystal structures, an opencarbohydrate-binding cleft suggests that the enzyme is able to accom-modate larger substrates as well. Hydrolysis of substrates is performedby the retaining mechanisms, accomplished by Asp382 as the

nucleophile and a Glu405 as the general acid/base. Interestingly, outsideof the catalytic cleft, the enzyme displays nine xylose-binding sites at itssurface (Huang et al., 2012). It would be worthwhile to compare withstructural data on the B. adolescentis xylosidase to get more insight inthe structural determinants that prevent it to hydrolyze of xylobiose.

11. The enzymes of GH 30 and GH 116


According to the CAZy database, GH 30 contains three xylosidases.AAK19754 from Phytophthora infestans hydrolyzes pNP-Xyl and pNP-Glu and is therefore named a bifunctional glucosidase/xylosidase, butit does not act on any tested natural substrate, such as xylan, AX andxyloglucan (Brunner et al., 2002). The characterization of ABC55722from B. adolescentis Int57 and ABX45137 from Bifidobacterium brevehas not been published, but their suggested activities stem from thecomment in their sequence files in the NCBI database. It is currentlyunknown on which substrates the xylosidase activities were observed,although the B. breve enzyme is suggested to hydrolyze kakkalide andginsenoside according to deposition ABX45137. We ourselves re-combinantly expressed an enzyme from B. adolescentis ATTC15703which has 94 and 93% identity with the GH 30 enzymes from B. breveand B. adolescentis Int57, respectively. Although it hydrolyzes pNP-Xyland pNP-Ara, it does not release xylose, arabinose or glucose fromxylan, AX, XOS, debranched and branched arabinan and xyloglucanand it lacks xylanase and cellulase activity (unpublished).

Similarly, the GH 116 enzyme from Sulfolobus solfataricus is a sug-gested bifunctional glucosidase/xylosidase because it hydrolyzes pNP-and MUF-linked glucosides and xylosides (Cobucci-Ponzano et al.,2010). However, it lacks activity on any tested oligosaccharide sub-strate, like XOS and glucooligosaccharides. All these results suggest GH30 and GH 116 may not contain true xylosidases which are able tohydrolyze XOS or xylan.

12. Synthesis

In spite of the fact that synthetic substrates come in handy forscreening, determining optimal conditions, etc., the above literaturereview suggests that hydrolysis of them may be insufficient to claim acertain enzyme activity. A number of xylosidases that were only testedon pNP-linked substrates are most likely arabinofuranosidases and viceversa. This review therefore focuses on results obtained with naturalsubstrates, such as XOS, AX and xylan.

An important difference regarding the substrate specificities ofxylosidases is their discriminations between substrates with varyinglength. While limited, data on GH 3 xylosidases suggest they display apreference towards polymeric substrates, although XOS are in the endcompletely degraded to xylose. There are indications that the prefer-ence of these xylosidases is due to a relatively large number of subsites.On the other hand, two GH 43 enzymes show a preference towardsxylobiose, while a third enzyme is indifferent towards XOS with DP2–DP6. Likely, GH 43 xylosidases have only one or two substrate bindingsubsites. In contrast to GH 3 and GH 43, GH 39 xylosidases and at leastone GH 120 enzyme display very limited activity towards xylobiose,but readily cleave larger substrates, indicating that these enzymeshave at least three subsites that are responsible for these properties. Itwould be interesting to see if combinations of these xylosidases withdifferent properties can lead to higher yields in AX degradation process-es. Other interesting candidates in this respect are the rex hydrolases,which hydrolyze the xylan backbone from the reducing end.

Although only a limited amount of arabinofuranosidases have beentested for their substrate specificities towards specific arabinose substi-tutions in AX, all of them fall in two separate classes. Those that are ableto hydrolyze arabinoses from monosubstituted xyloses, both C(O)2- orC(O)3-linked, and those that cleave the arabinose from the C(O)3

igsn:ATTC15703


position of disubstituted xyloses (Fig. 10). The arabinofuranosidasesfrom the first group seem to be most widespread, with 2 GH 43, 4GH 51, 3 GH 54 and 2 GH 62 enzymes. Until now, only threearabinofuranosidases show hydrolysis of arabinoses from disubstitutedxyloses, two from GH 43 and one from GH 51, while the properties of asecondGH51 enzyme suggest the same specificity. Both GH families arevery distinct, in structure as well as in hydrolysis mechanism. Since theGH classification is widely assumed to show evolutionary relationships,it is likely that the presence of substrate specificities towards C(O)3-linked arabinose on disubstituted xylose in both families is a result ofconvergent evolution. It remains to be seen if all arabinofuranosidasesare part of these two classes, or if other enzymes will be found withother specificities, e.g. towards C(O)3-linked arabinoses, regardless ofmono- or disubstituted xyloses, or towards C(O)2-linked arabinose ondisubstituted xylose. Nonetheless, for now, all current evidencesuggests that for processes where an intensive enzymatic degradation

AX

AXOS (mono-and

AXOS (monos

XOS

β-xylosidases GH 3, 39, 43, 52, 120

xylos

Fig. 10. Schematic overview of arabinoxylan (AX) degradation. Arrows indicate cleavage sites. ECollins et al. (2005b).

of AX is wanted, such as in the production of biofuels, the synergisticaction of enzymes from both classes is needed (Fig. 10). Of particularinterest in this regard is the study of Rasmussen et al. (2012), whereinsynergistic AX degradation between several different types of enzymeswas examined. In addition to the observed synergy between the twotypes of arabinofuranosidases, arabinofuranosidases also act synergisti-cally with β-xylosidases. Optimal hydrolysis required the combinedaction of an endoxylanase, the two types of arabinofuranosidases anda β-xylosidase (Rasmussen et al., 2012).

Since arabinofuranosidases and xylosidases both are crucialenzymes for an intensive AX degradation, enzymes displaying bothactivities have a clear advantage. Hence, it is no surprise that significantinterest went to these bifunctional enzymes and that authors are eagerto claim this property. However, when carefully looking at literaturedata on the 23 bifunctional xylosidases/arabinofuranosidases from theCAZy database, it is clear that most of these display the dual activity

EndoxylanasesGH 5,8, 10 and 11

disubstituted)

di(3)-specific arabinofuranosidasesGH 43, 51, 54 and 62

ubstituted)

mono-specific arabinofuranosidasesGH 43 and 51

Rex hydrolasesGH 8

e

ndoxylanases cleave the xylan backbone randomly, endoxylanase families are taken from

image of Fig.�10


only on synthetic substrates. If incubated with natural substrates, ingeneral, they release either xylose or arabinose, but not both. We onlyfound five exceptions: three plant GH 3, one bacterial GH 43 and oneplant GH 51 enzymes release both arabinose and xylose from naturalsubstrates. Even in these cases, when a comparison between the twodifferent activities was made, the enzymes showed a preferencetowards the hydrolysis of one type of sugar. For example, the GH 3and GH 51 bifunctional enzymes from A. thaliana release xylose lessefficiently than arabinose from natural substrates, in contrast, the GH3 enzyme from H. vulgare releases more xylose than arabinose fromAXOS (Lee et al., 2003; Minic et al., 2004). Crystal structures of thesebifunctional enzymes would greatly aid in explaining why theseenzymes, and not the others, are able to release both xylose and arabi-nose from natural substrates.

Abbreviations
A/X arabinose to xylose rationAX arabinoxylanAXOS arabinoxylan-oligosaccharidesCAZy Carbohydrate Active EnzymeCBM 6 carbohydrate binding module of family 6DP degree of polymerizationDS degree of substitutionGH glycoside hydrolase familiesMUF methylumbelliferylpNP-Ara pNP-arabinosidepNP-Glu pNP-glucosidepNP-Xyl p-nitrophenylxylosideXOS xylo-oligosaccharides
Acknowledgments

Financial support from the European Commission in the Communi-ties 7th Framework Programme (FP7/2007–2013) for the Biocore Pro-ject (grant agreement n°FP7-241566) is gratefully appreciated. Thispublication reflects only author's views and the Community is not liablefor any use that may be made of the information contained in thispublication. We further acknowledge the financial support from the‘Instituut voor de aanmoediging van Innovatie door Wetenschap enTechnologie in Vlaanderen’ (IWT) for SBO project funding (IMPAXOS,IWT/040073/SBO).

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β-Xylosidases and α-l-arabinofuranosidases: Accessory enzymes for arabinoxylan degradation

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