understanding the function of conserved variations in the catalytic loops of fungal glycoside...

Understanding the Function of ConservedVariations in the Catalytic Loops of FungalGlycoside Hydrolase Family 12

Andr�e R.L. Dam�asio,1 Marcelo V. Rubio,1,2 Leandro C. Oliveira,1,3 Fernando Segato,1

Bruno A. Dias,1,3 Ana P. Citadini,1 Douglas A. Paix~ao,1 Fabio M. Squina1

1Laboratorio Nacional de Ciencia e Tecnologia do Bioetanol (CTBE), Centro Nacional de

Pesquisa em Energia e Materiais (CNPEM), Campinas-SP, Brazil; telephone: þ55 19 3518

3111; fax: þ55 19 35183104; e-mail: [email protected] de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas-SP,

Brazil3Instituto de Biociencias, Letras e Ciencias Exatas, Universidade Estadual Paulista

(UNESP), S~ao Jos�e do Rio Preto, SP, Brazil

ABSTRACT: Enzymes that cleave the xyloglucan backbone atunbranched glucose residues have been identified in GHfamilies 5, 7, 12, 16, 44, and 74. Fungi produce enzymes thatpopulate 20 of 22 families that are considered critical for plantbiomass deconstruction. We searched for GH12-encodinggenes in 27 Eurotiomycetes genomes. After analyzing 50GH12-related sequences, the conserved variations of theamino acid sequences were examined. Compared to theendoglucanases, the endo-xyloglucanase-associated YSGdeletion at the negative subsites of the catalytic cleft with aSST insertion at the reducing end of the substrate-bindingcrevice is highly conserved. In addition, a highly conservedalanine residue was identified in all xyloglucan-specificenzymes, and this residue is substituted by arginine in morepromiscuous glucanases. To understand the basis for thexyloglucan specificity displayed by certain GH12 enzymes,two fungal GH12 endoglucanases were chosen for mutagen-esis and functional studies: an endo-xyloglucanase fromAspergillus clavatus (AclaXegA) and an endoglucanase from A.terreus (AtEglD). Comprehensive molecular docking studiesand biochemical analyses were performed, revealing thatmutations at the entrance of the catalytic cleft in AtEglDresult in a wider binding cleft and the alteration of thesubstrate-cleavage pattern, implying that a trio of residuescoordinates the interactions and binding to linear glycans.The loop insertion at the crevice-reducing end of AclaXegA is

critical for catalytic efficiency to hydrolyze xyloglucan.The understanding of the structural elements governingendo-xyloglucanase activity on linear and branched glucanswill facilitate future enzyme modifications with potentialapplications in industrial biotechnology.

Biotechnol. Bioeng. 2014;111: 1494–1505.

� 2014 Wiley Periodicals, Inc.

KEYWORDS: fungal endoglucanases; GH12; endo-xyloglucanases; xyloglucan specificity

Introduction

Xyloglucan (XyG) is the most abundant hemicellulose in themajority of land plants, reaching 20% of the primary cell walldry weight (Gilbert et al., 2008). XyG is also the primarystorage polysaccharide in certain seeds, such as Tamarindusand Hymenaea courbaril (jatob�a) (Buckeridge, 2010). Likecellulose, XyG consists of a linear backbone of b-1,4-glucanlinkages but is distinguished by having up to 75%ofb-D-Glcp(b-D-glucopyranose) residues that are covalently linked toa-D-Xylp (a-D-xylopyranose) at the O-6 position (Carpitaand McCann, 2000). Depending on the source of XyG, aportion of a-D-Xylp residues may be further linked to b-D-galactopyranose (b-D-Galp) or a-L-arabinofuranose (a-L-Araf), and a portion of galactose residues may be extended bya-L-fucopyranose (a-L-Fucp) (Carpita and McCann, 2000).The incubation of XyG with cellulases produces oligosaccha-ride (XGOs) fingerprints because of an enzyme-specificmode of action combined with the fine structure of thepolysaccharide (Buckeridge, 2010; Buckeridge et al., 1992).

Enzymes that cleave the XyG backbone at unbranched Glcresidues have been identified in GH families 5, 7, 12, 16, 44,and 74. Members of the first five families operate throughthe canonical double-displacement mechanism of glycosyl

Andr�e R.L. Dam�asio and Marcelo V. Rubio contributed equally to this work.

Correspondence to: F.M. Squina

Contract grant sponsor: CNPq

Grant numbers: 474022/2011-4; 310177/2011-1

Contract grant sponsor: FAPESP

Grant numbers: 2008/58037-9; 2011/02169-4; 2011/13242-7

Contract grant sponsor: FAPESP IC Fellow

Contract grant number: 2012/12859-3

Received 15 November 2013; Revision received 24 January 2014; Accepted 27 January

2014

Accepted manuscript online 6 February 2014;

Article first published online 27 February 2014 in Wiley Online Library

(http://onlinelibrary.wiley.com/doi/10.1002/bit.25209/abstract).

DOI 10.1002/bit.25209

ARTICLE

1494 Biotechnology and Bioengineering, Vol. 111, No. 8, August, 2014 � 2014 Wiley Periodicals, Inc.

transfer, which involves a covalent glycosyl-enzyme interme-diate and results in the net retention of the anomericconfiguration. Enzymes from GH74 operate by a single-displacement, anomeric configuration-inverting mechanisminvolving the direct attack of water on the sugar ring (Gilbertet al., 2008). In addition to endo-acting xyloglucanases,debranching enzymes are needed to complete XyG depo-lymerization. The XGOs side chains are removed by alpha-1,2-L-fucosidases (GH95, EC 3.2.1.63) and glucosidases(GH1, 2, 35, 42, and 43; EC 3.2.1.23), generating xylosylatedXGOs (Iglesias et al., 2006), which are further hydrolyzed intomonosaccharides by the concerted and sequential actions ofxylosidases (GH31; EC 3.2.1.X) and glucosidases (GH1 andGH3; EC 3.2.1.21) (Buckeridge et al., 2000).The basis of endoglucanase specificity to XyG and the

ability of certain glucanases to hydrolyze both branched andlinear polysaccharides has long been recognized. However,the question posed in 1997 by Vincken et al., that is, “Whatdetermines xyloglucanase activity?” has not been fullyaddressed to date. Conserved sequence variations amongthe GH12 endo-xyloglucanases and endoglucanases werepreviously reported (Master et al., 2008). The GH12 endo-xyloglucanases have an YSG deletion at the non-reducing-end of the catalytic cleft (loop 1) and a SST insertion at thereducing end of the substrate-binding crevice (loop 2). Inaddition, there is a highly conserved alanine residue in loop2 in all endo-xyloglucanases that is replaced by arginine inmore promiscuous GH12 glucanases.This study aimed to elucidate the structural role of

conserved sequence variations in loops at the entry and exit ofthe catalytic cleft that define fungal GH12 xyloglucan-specificenzymes. The GH12-encoding genes derived from 27Eurotiomycetes genomes support the hypothesis that theconserved sequence deletion and insertion variations arestructural determinants. To understand the basis of thexyloglucan specificity displayed by certain GH12 enzymes,two fungal GH12 endoglucanases were chosen for mutagen-esis and functional studies: the endo-xyloglucanase derivedfrom Aspergillus clavatus (AclaXegA) and the promiscuousendoglucanase from A. terreus (AtEglD). Molecular dockingstudies and biochemical analysis of the GH12 mutantscomprehensively described the role of the loops at thexyloglucan/b-glucan interaction site in the catalytic cleft andduring hydrolysis.

Materials and Methods

Cloning and Expression of AtEglD and AclaXegA

PCR-amplified gene fragments were digested with NotI andXbaI, after which they were isolated by excising a thin slicefrom a 0.8%-agarose electrophoresis gel and purified witha QIAquick Gel Extraction kit (Qiagen, Venlo, NL). Thefragments were then ligated into NotI/XbaI-digested pEXPYRplasmid with T4-fast ligase (Promega, Fitchburg, WI) andtransformed into Caþ-competent DH5a Escherichia coli(Promega). Random ampicillin-resistant colonies were

selected and grown in 5-mL LB-ampicillin broth, theplasmids were purified (Sambrook et al., 1988) and digestedwith NotI/XbaI, and the insert size was verified by 1% agarose-gel electrophoresis (Sambrook et al., 1988). Plasmids with thecorrect insert size were fully sequenced, and clones with thecorrect DNA sequence were used for the transformation ofAspergillus nidulans strain A773 (pyrG89; wA3; pyroA4), aspreviously described (Segato et al., 2012).Next, 107–108 spores/mL were inoculated in liquid mini-

mal medium supplemented with 5% maltose, distributedonto dishes and incubated without shaking at 37�C for 2–3 days. The mycelial mat was lifted with a spatula anddiscarded, and the medium was collected by filtration,centrifuged at 10,000g for 10min prior to concentration byultra-filtration (10,000-Da cutoff, Millipore, Billerica, MA),quantified by the Bradford method (Bradford, 1976),validated for purity by SDS–PAGE (Shapiro et al., 1967)and used in biochemical studies.

Site-Directed Mutagenesis

Site-directed mutagenesis was carried out by the standardPCR-based method using AtEglD and AclaXegA parentalgenes as templates. The amplified fragments were used astemplates for the overlap-extension PCR technique (Heck-man and Pease, 2007) to fuse the two genes in a single ORF.The fused fragment was digested with the restriction enzymesNotI and XbaI and cloned into pEXPYR (Segato et al., 2012).

Protein Purification

The target proteins were purified in two steps. Theconcentrated and dialyzed protein samples were applied toan ion-exchange Resource Q column equilibrated with20mM sodium phosphate buffer, pH 7.4, and the proteinswere eluted with a linear 0-to-1M sodium chloride gradient(Äkta Purifier, GE, Little Chalfont, UK). Fractions active onbeta-glucan or xyloglucan were collected and loaded onto aSuperdex G-75 (10mm� 30mm) gel-filtration column andequilibrated with 50mM ammonium acetate buffer, pH 5.0,and eluted fractions showing enzymatic activity wereanalyzed by SDS–PAGE. Single-band fractions were com-bined, concentrated and used for further biochemicalanalysis. The flow rate used for both chromatographic stepswas 0.5mLmin�1. Purified fractions were validated bySDS–PAGE.

Enzymatic Properties

Reducing sugars were determined using 3,5-dinitrosalicylicacid (DNS) and monitored colorimetrically at 540 nm(Miller, 1959) using an Infinite1 200 PRO microplate reader(TECAN, Mannedorf, CH). One unit of enzyme was definedas the quantity of enzyme needed to release reducing sugars atrate of 1mmol/min under standard conditions. The standardassay was conducted for 10min in 50mMMcIlvaine glycine-added buffer at pH 5.5, 50�C, with substrates at 2.0mgmL�1

and 1mg of purified enzymes.

Dam�asio et al.: Structural Determinants That Define Fungal GH12 Specificity 1495

Biotechnology and Bioengineering

The optimal pH and temperature were determined forAtEglD and AclaXegA activities using barley b-glucan andxyloglucan from tamarind (XyG), respectively, as substratesunder standard conditions. The assays for substratespecificity were evaluated in various substrates, includingarabinan from the sugar beet, debranched arabinan, lineararabinan, rye arabinoxylan, larch arabinogalactan, galacto-manan, XyG, oat-spelt xylan, wheat arabinoxylan, barleyb-glucan, carboxymethylcellulose (CMC) and xylan frombeechwood. The polysaccharides were purchased from SigmaAldrich (St. Louis, MO) or Megazyme, Co. (Wicklow, IE).

The kinetic parameters were estimated for all enzymesfrom initial rates at 11 substrate concentrations of 1–14.4 and1–10.8mgmL�1 for b-glucan and XyG, respectively. Theassays were carried out under standard conditions to assessVmax, Km, and Kcat.

Capillary Electrophoresis (CE)

Oligosaccharides (Megazyme) were derivatized with 8-aminopyreno-1,3,6-trisulfonic acid (APTS) by reductiveamination (Naran et al., 2007). Enzymatic hydrolysis oflabeled substrates was performed at 50�C. To analyze thecleavage patterns, capillary-zone electrophoresis (CZE)of substrate-breakdown products was performed using aP/ACE MQD instrument (Beckman Coulter, Pasadena, CA)equipped with a laser-induced fluorescence detector. A fused-silica capillary (TSP 050375, Polymicro Technologies,Phoenix, AZ) with an internal diameter of 50mm and totallength 31 cm was used as separation column for oligosac-charides. The electrophoresis conditions were as follows:30 kV/70–100mA at 20�C using sodium phosphate buffer(40mM, pH 2.5). Because of the small volumes of capillaryelectrophoresis combined with the small variations in bufferstrength, retention times vary slightly when comparingseparate electrophoresis runs.

Circular Dichroism

Spectra of far-UV circular dichroism (CD) were taken on aJASCO J-810 spectropolarimeter (Jasco, Inc., Tokyo, Japan)equipped with a Peltier temperature-control unit using awavelength range of 195–240 nm, and a 0.1-cm-path quartzcuvet, and the solvent spectra were subtracted in allexperiments to eliminate background effects. CD spectrawere the average of eight accumulations taken using ascanning speed of 100 nmmin�1, a spectral bandwidth of1 nm, and a response time of 0.5 s. The protein concen-trations were 0.2mgmL�1 in 50mM sodium phosphatebuffer, pH 7.4. Thermal denaturation was characterized bymeasuring the changes in ellipticity at 218.5 nm induced byan increase in temperature from 20 to 100�C at 1�Cmin�1

(Cota et al., 2011).

Homology Molecular Modeling and Analysis of Wild andMutant Types

The initial structure was modeled using homologousenzymes extracted from the protein data bank (PDB). The

atomic coordinates from Aspergillus niger endoglucanase (PDBid: 1KS5) (Khademi et al., 2002) and Aspergillus aculeatusxyloglucanase (PDB id: 3VL8) (Yoshizawa et al., 2012) wereused as templates to generate structural models for AtEglD byrestraint-based modeling, as implemented in the MODEL-LER program (Sali and Blundell, 1993) in the HHpredServer (Soding et al., 2005). The mutants were constructedby removing the target regions, connecting the extremitieswhere the region was removed and applying the Dunbrackrotamer libraries (Dunbrack, 2002) in the UCSF Chimerapackage, version 1.7 (Pettersen et al., 2004). Next, theconformation energy was minimized using the Chimerainterface with the AMBER ff12SB force field employed to5000 steepest descent steps and 5000 conjugate gradientsteps, both of which had a size of 0.02 A

�.

The changes in the dynamic behavior of all enzymes wereanalyzed using Normal Modes (NMA) (Hollup et al., 2005).The NMA approach models the residues using beads in thealpha-carbon position. The application uses a harmonicpotential to define the residue interactions and analyze thelow frequency normal modes. The fluctuations are calculatedusing the atomic displacement (Di), given by

Di ¼ d2iPn1 d

2i

� �

where n is the total number of residues in the protein and di isthe component of the eigenvector corresponding to the iresidue. The fluctuations are normalized to have a maximumpeak at 1.0.

Molecular Docking

Cellohexaose and xyloglucan oligosaccharide structureswere built using GLYCAM04 (Basma et al., 2001; Kirschnerand Woods, 2001a,b). Docking was performed using theprocedure described by Sagermann (Sagermann andMatthews, 2002) through UCSF Chimera and UCSF dock(Lang et al., 2009; Moustakas et al., 2006; Pettersen et al.,2004). The enzymes and ligands were initially prepared usingthe Prep Dock tool in the Chimera interface (AMBER ss12SBforce field for proteins and AM1-BCC force fields for othercases). The cavity of the binding site was suggested by thedocking program and is consistent with the results of recentwork (Gloster et al., 2007). The spatial conformations of theenzymes were aligned, and the grid box was defined similarlyfor all of the conformations. Three hundred docking runswere performed for each substrate. The ligand, receptor,ligand orientations and overlap bins were set to 0.2 A

�, and the

distance tolerance for matching between the atoms and thereceptor was set to 0.75 A

�.

The conformations obtained from the docking runs wereanalyzed for the distance between the central oxygen (O4) ofthe ligand and the C-alpha position of the residue ALA:102 inAclaXegA and AclaXegADSST and between the central oxygenand the C-alpha position of the residue ALA:97 in AtEglD and

1496 Biotechnology and Bioengineering, Vol. 111, No. 8, August, 2014

AtEglDDYSG. These distances were organized in histogramsusing bins of 2 A

�, emphasizing the differences in substrate

accommodation and displacement.

Results and Discussion

The Majority of the Eurotiomycetes Genomes Contain aSingle Gene for Endo-Xyloglucanase

The comparative analysis of the filamentous fungal genomesrevealed a large number of CAZy genes, ranging from 171 to285 per genome. For example, Trichoderma reesei containsapproximately 200 GH enzymes and A. niger encodesapproximately 250 GHs (Jovanovic et al., 2009). Accordingto Jovanovic et al. (2009), although 115 GH families arerecognized by the CAZY database, only 22 families containenzymes critical for plant biomass deconstruction. Fungiproduce enzymes in 20 of these families, covering all activitiesneeded for the efficient conversion of natural biomass(Jovanovic et al., 2009).The class Eurotiomycetes (Ascomycota, Pezizomycotina)

is a monophyletic group comprising two major clades ofvery different ascomycetous fungi: (i) the subclass Euro-tiomycetidae; and (ii) the subclass Chaetothyriomycetidae.Eurotiomycetidae includes producers of toxic and usefulsecondary metabolites, fermentation agents used to makefood products and enzymes, xerophiles and psychrophiles,and the important genetic model A. nidulans (Geiser et al.,2006).We searched for GH12-encoding genes in 27 Euro-

tiomycetes genomes using the JGIMycoCosm tool (Grigorievet al., 2012), revealing 50 related GH12 sequences. Thegenetic model Neurospora crassa (Znameroski and Glass, 2013)

and the model for biomass deconstruction, T. reesei, werechosen as non-Eurotiomycetes external groups. Apart fromA. brasiliensis and A. flavus, all analyzed Eurotiomyceteshave one copy of a GH12 endo-xyloglucanase-encodinggene (Supplementary Table SI). A. carbonarius, A. glaucus,A. nidulans, and A. zonatus have no copies of GH12endoglucanase-encoding genes. Interesting, N. crassa carriesno GH12 enzymes. Moreover, we did not find any GH12endo-xyloglucanase-encoding genes in the T. reesei genome(Supplementary Table SI).The phylogenetic tree of Aspergilli GH12 enzymes revealed

two well-defined clades and a common ancestor of GH12endoglucanases and endo-xyloglucanases (Fig. 1). Sequencesin the same clade exhibited high similarity, ranging from 52%to 70% amino acid identity.

Eurotiomycetes GH12 Endo-Xyloglucanases (3.2.1.151)Have Conserved Deletions and Insertions Compared toGH12 Endoglucanases

After careful analysis of 50 GH12-related sequences from 27Eurotiomycetes, the conserved variations of endo-xyloglu-canase and endoglucanase amino acid sequences werenoteworthy. Compared to the endoglucanase amino acidsequences, the endo-xyloglucanases have two highly con-served variations located in loop 1 and loop 2 (Fig. 2A), andthese variations can be observed across all the analyzedEurotiomycetes (Supplementary Fig. S1).The predicted molecular models for AclaXegA and AtEglD

exhibited a typical b-jelly roll tertiary structure described forGH12 (Fig. 2) (Gloster et al., 2007). These two structuresshare the same topology, with mainly b-strands in theconcave region (Fig. 2B and C). These models of AclaXegA

Figure 1. Phylogenetic tree of fungal endoglucanases from glycoside hydrolase, family 12. All of these sequences are derived from eukaryotes (except the root sequence) and

previously characterized enzymes from the Carbohydrate-Active Enzymes (CAZy) database (www.cazy.org). The scale bar indicates branch length. For the phylogenetic analysis,

the amino acid sequences were aligned using ClustalX 1.83 software. The phylogenetic tree was built using Mega 4 software. The highlighted symbols represent the GH12 enzymes

studied in this work: AclaXegA (ACLA_029940) and AtEglD (ATEG_09894).



http://www.cazy.org/

and AtEglD allowed the depiction of conserved amino acidvariations among GH12-related sequences. Specifically, theGH12 endo-xyloglucanases show a deletion event at the non-reducing end of the catalytic cleft (loop 1) and an insertionevent at the reducing end of the substrate-binding crevice(loop 2). Accordingly, AclaXegA has an YSG deletion in theloop between B5 and B6 (loop 1) (Fig. 2B) and a SSTinsertion in the loop between B6 and B8 (loop 2) (Fig. 2C). Inaddition to these conserved variations, there is a highlyconserved alanine (A) residue in loop 2 in all GH12 endo-xyloglucanases (Supplementary Figs. S1 and S2A) that isreplaced by arginine (AtEglD arginine 123 (R123)) in morepromiscuous GH12 glucanases (Fig. 2A).

The YSG AclaXegA deletion shortens loop 1 at the non-reducing end of the catalytic cleft, subtracting an aromaticinteraction described as critical for cellopentaose substratebinding in T. harzianum GH12 endoglucanase (Prateset al., 2013). In addition, loop 2 is longer in fungal GH12endo-xyloglucanases than in endoglucanases. This insertion

in AclaXegA loop 2 is adjacent to the so-called “cord” region(P131, I132), which is conserved in all analyzed fungal GH12amino acid sequences (Supplementary Fig. S1). The cordregion was previously described and contributes with aminoacid residues to the substrate-binding cleft that are likelyinvolved in binding the reducing end of the substrate(Sandgren et al., 2001).

As shown in Figure 2D, the residues of the catalytic cleft arehighly conserved between fungal GH12s. Previous elegantstructural studies of fungal and bacterial GH12 enzymes haveprovided the basis to define substrate binding to the cordregion, as well as the core residues of the cleft that are likely tobe involved in binding the reducing end of the substrate (þ1and þ2 subsites) (Gloster et al., 2007; Sandgren et al., 2001).All these characteristics can indicate the substrate bindingproperties of xyloglucan-specific enzymes relative to morepromiscuous endoglucanases, as well as the inability of fungalGH12 endo-xyloglucanases to hydrolyze unbranched sub-strates (Sandgren et al., 2001; Master et al., 2008).

Figure 2. Comparison of the AclaXegA and AtEglD structures. A: Alignment of the primary sequences of AclaXegA and AtEglD. Endoglucanase 3 from Trichoderma harzianum

(ThEG3) was adopted as a reference. B and C: The b-jellyroll structure of AclaXegA (template PDB: 3VL8; C-score 1.76) and AtEglD (template PDB: 1KS5A; C-score 1.88). The

highlighted blue (Panel B) and orange (Panel C) regions indicate the deletions. The asterisk indicates an arginine that is highly conserved in endoglucanases and substituted by

alanine in xyloglucanases. D: Close-up of the substrate-binding cleft of fungal glycoside GH12. The side chains of some of the most important residues for AclaXegA (blue) and

AtEglD (red) are drawn. The surfaces on the right side highlight the same residues in the cleft. The asterisks denote the deletion (YSG) and insertion (SST) in AclaXegA.


AclaXegA and AtEglD Are Typical Fungal GlycosideHydrolases From Family 12

To understand the structural basis of xyloglucan specificitydisplayed by certain GH12 enzymes, we chose two fungalGH12 endoglucanases to conduct mutagenesis and func-tional studies: the endoglucanase-encoding gene from A.terreus (ATEG_09894/AtEglD) and the endo-xyloglucanase-encoding gene from A. clavatus (ACLA_029940/AclaXegA).The pattern of hydrolysis of the unbranched XyG

backbone by fungal GH12 enzymes has been examinedpreviously (Damasio et al., 2012; Master et al., 2008; Songet al., 2013), including comprehensive studies on substraterecognition, as in the description of the A. niger xyloglucan-specific GH12 (AnXEG12A), which prefers xyloglucan-oligosaccharides containing more than six glucose units,and a study of the importance of the xylose substitution at the�3 and þ1 positions (Powlowski et al., 2009).AclaXegA is a strict xyloglucan-specific enzyme that

hydrolyzes xyloglucan at unbranched glucose residues(Fig. 3A and C). Moreover, AtEglD is a promiscuous endo-glucanase that preferentially hydrolyzes undecorated glucanbackbones (b-glucan) over xyloglucan (Fig. 3A). AtEglD andAclaXegA share the same mode of operation, releasingXXXG, XLXG, and XLLG from xyloglucan as the finalproducts (data not shown). The hydrolysis of b-glucan byAtEglD released cellotetraose, cellotriose, and cellobiose asmajor products (Fig. 3B).

Protein Secretion and Folding

To ensure proper post-translational modifications the genesdescribed in this work were cloned into the pEXPYR shuttlevector and were transformed in A. nidulans to reach high levelsof target protein secretion (Segato et al., 2012).The target proteins (AclaXegA, AclaXegADSST, AtEglD,

AtEglDDYSG, AtEglDR123A) were purified by two chro-matographic-purification steps. The secondary structureswere evaluated by circular-dichroism (CD) (SupplementaryFig. S2), and the data were analyzed using the DichroMatchdatabase (Supplementary Table SII). AtEglD and AclaXegAshowed a predominance of b-strand secondary structures.Despite AtEglDDYSG, the mutants (AtEglDR123A and AclaX-egADSST) had similar CD profiles compared to the parentalenzymes. Although AtEglDDYSG exhibited a change on theCD profile, the b-strand was also the predominant secondarystructure. The Dichromatch analyses suggest a possiblestructural rearrangement with b-strand loss, and the gainof irregular structure for AtEglDDYSG (SupplementaryTable SII).

The SST Deletion in AclaXegA Did Not Affect theEnzymes Affinity for the Substrate But Did Reduce theCatalytic Efficiency

The characterization by Normal-Mode Analysis (NMA)revealed discrete fluctuation changes in the deleted loop

region (S133, S134, and T135) (Fig. 4A and B). These datacorroborate the CD results, as the denaturation midpoint(Tm) was exactly the same for AclaXegA and AclaXegADSST

(Fig. 4C). Additionally, almost no changes were observed inprotein secondary structures (Supplementary Fig. S2). Themode of operation of AclaXegADSST was the same as thatobserved for the parental enzyme, releasing XXXG, XLXG,and XLLG as major products from xyloglucan (Fig. 4D) aswell as the pH and temperature optimum (SupplementaryFig. S3).The best fit of the substrate in the catalytic cleft was reached

after three hundred molecular docking attempts; however,the substrate positioning in the catalytic crevice was exactlythe same in all attempts for the parental AclaXegA (Fig. 6A).

Figure 3. Substrate specificity and mode of operation of AclaXegA and AtEglD on

polysaccharides. A: AclaXegA was highly specific to xyloglucan hydrolysis. B:

Capillary electrophoresis of APTS-labeled oligosaccharides after the hydrolysis of b-

glucan by AtEglD and of xyloglucan from tamarind by AclaXegA (C). APTS-labeled

glucose (C1), cellotetraose (C4), cellopentaose (C5) and cellohexaose (C6). The APTS-

labeled xyloglucan oligosaccharides (XXXG, XLXG, and XLLG). XyG, xyloglucan from

tamarind; CMC, carboxymethylcellulose. The segments of the xyloglucan polymer are

named based on a one-letter unambiguous system. Unsubstituted D-Glcp is designated

as G; the a-D-Xylp-(1! 6)-b-D-Glcp residue is designated as X; the b-D-Galp-(1! 2)-

a-D-Xylp-(1! 6)-b-D-Glcp is designated as L. The assay was carried out using AtEglD

or AclaXegA at 0.086mM and the substrate at 10mg/mL in 50 mM ammonium acetate

buffer, pH 5.5 for 5 h at 50�C.



The docking analysis for AclaXegADSST revealed that thesubstrate fit poorly in the catalytic cleft due to a loss ofinteraction at the cord region (Fig. 6B), mainly at theþ2 andþ3 subsites. Conversely, the interactions in the negativesubsites were maintained, highlighting the importance ofW8 andW23, which are critical to substrate placement in thecrevice. According to previous studies, the a-D-Xylpsubstitution at the �3 position is necessary for efficienthydrolytic activity, and substrate occupation of the hydro-phobic �4 position improves substrate binding 30-fold(Powlowski et al., 2009).

The Km for AclaXegADSST on XyG was unchanged, unlikethe Vmax and the turnover number (Kcat), which weresignificantly reduced. Thus, the catalytic efficiency ofAclaXegADSST for xyloglucan hydrolysis decreased approxi-mately eightfold (Table I). This result is in agreement withthe docking data. The SST deletion changed the enzyme-substrate binding interactions, most notably at the AclaXegApositive subsites, resulting in substrate misfitting and

instability at the catalytic cleft and changes in hydrolyticperformance.

According to our findings, after the loop 2 truncation inAclaXegA (AclaXegADSST), the cord region residues, inparticular Pro129 and Ile130 which are likely to form thebottom of the þ2 and þ3 subsites (Sandgren et al., 2005),were displaced (Fig. 6A and B). These findings underline therole of positive subsites in substrate interaction with thecatalytic crevice, along with the xylosyl substitution of XyG attheþ1 site. Indeed, the xylosyl substitution at theþ1 positionis essential for substrate binding and hydrolysis, and morethan one positive subsite is required for efficient hydrolysis(Powlowski et al., 2009).

The histogram (Fig. 7A) illustrates the distributionof docking populations obtained for AclaXegA andAclaXegADSST. The parental enzyme showed a narrowdistribution compared to AclaXegADSST. These differencescorroborate the variations in substrate accommodation at thenegative subsites, as well as the inferior substrate-conversion

Figure 4. Dynamic behavior of AclaXegA and AclaXegADSST evaluated by normal mode analysis (NMA). A: The mobility profile along the primary sequence of AclaXegA,

expressed via the fluctuations normalized of the carbon alpha atoms using the 7–12 low-frequency normal modes. The black box highlights themutated region.B: The protein regions

that the displacement changed during the analysis are highlighted by red balls. The asterisk correlates the mutated region in the graph to that in the predicted structure. C: Thermal

denaturation curve at pH 7.4. The thermal denaturation curve was obtained by monitoring at 217.6 nm.D: Capillary electrophoresis of APTS-labeled oligosaccharides after hydrolysis

of xyloglucan from tamarind (XyG) by AclaXegA and AclaXegADSST for 2 h. The xyloglucan oligosaccharide nomenclature is described in Figure 3.


rate of AclaXegADSST, which is caused by the spatialrestrictions needed to adjust the substrate in the catalyticcleft.

The YSG Deletion in AtEglD Altered Catalysis andReduced Thermal Stability

The trio of residues that contribute important hydrophobicinteractions (Y7, W22, Y111; using the residue numbering ofAtEglD) at the non-reducing end of the catalytic cleft is highlyconserved in fungal GH12 endoglucanases (EC 3.2.1.4). Y7,

W28, and Y111 are conserved in 100%, 100%, and 40% of theendoglucanase sequences, respectively, in 27 Eurotiomycetesgenomes analyzed in this study. While Y7 can be substitutedby tryptophan in some endo-xyloglucanases; Y111 is absentin all endo-xyloglucanases and substituted by serine in 60%of the endoglucanases analyzed in this study (SupplementaryFig. S1).The characterization of AtEglD and mutants using NMA

indicated that several protein regions displayed differences inmobility after YSG deletion (AtEglDDYSG), most notablyin the truncated loop 1 region (Fig. 5A and B). This result is

Figure 5. Evaluation of the dynamic behavior of AtEglD, AtEglDDYSG, and AtEglDR123A using NMA. A: The NMA evaluation of the fluctuations, normalized as described in

Figure 4A. The black box highlights the YSG deletion region. B: The protein regions changed by the displacement during AtEglDDYSG analysis are highlighted with red balls. The

asterisks indicate the YSG deletion in the graph and in the predicted structure. C: Thermal-denaturation curve at pH 7.4. The thermal-denaturation curve was obtained by monitoring

at 217.6 nm. Capillary electrophoresis of APTS-labeled oligosaccharides after hydrolysis of xyloglucan from tamarind (D) and APTS-labeled cellohexaose for 2 h (E).

Table I. Wild-type and mutant kinetic parameters.

Protein Substrate Km (mg/mL) Vmax (mmol product/min/mMenzyme) Kcat (s�1) Kcat/Km

AtEglD b-Glucan 5.39� 1.3 463� 45 77.1 14.3XyGa 4.74� 2.9 219� 25 36.5 7.70

AtEglDR123A b-Glucan 5.04� 1.1 240� 20 40.0 7.94XyG 5.51� 1.8 115� 16 19.2 3.48

AtEglDDYSG b-Glucan 5.41� 1.2 10.6� 1.1 1.74 0.322XyG 1.53� 0.14 1.39� 0.037 0.230 0.150

AclaXegA XyG 2.38� 0.82 218� 24 36.3 15.3AclaXegADSST XyG 1.89� 0.49 22.5� 1.5 3.70 1.95

ND, not detected.aXyloglucan from tamarind.



in accordance with CD analysis, as higher-mobility AtEglD-DYSG can explain the reduction of mutant Tm at over 10�

(Fig. 5C). The pH and temperature optima were unalteredafter YSG deletion (Supplementary Fig. S4).

The catalytic efficiency of AtEglDDYSG in the xyloglucanandb-glucan degradationwas significantly reduced (Table I).The Y111 residue is absent in the AtEglDDYSG loop 1, aspreviously mentioned. Thus, the lack of this key residue inAtEglDDYSG was expected to have meaningful implicationsfor substrate–enzyme interactions. After the AtEglDDYSG

deletion, cellohexaose was misfitted at the catalytic cleft(Fig. 6C and D).

The mode of operation of AtEglDDYSG was unchanged forxyloglucan hydrolysis (Fig. 5D). In the other hand, the modeof operation on b-glucan hydrolysis was changed, producingcellotriose as a major product after cellohexaose hydrolysis(Fig. 5E). The conformations obtained after docking runsshowed a single distribution for the parental enzyme andcellohexaose fit into the binding site (Fig. 7B). Conversely, thecellohexaose swings away the catalytic cleft of AtEglDDYSG,and the correct alignment between the catalytic triad and the

substrate is not reached most of the time (Fig. 7B) due to theYSG deletion at the cleft entrance (Fig. 6D).

Our results provide a biochemical basis for the MDsimulations reported by Prates et al. (2013). According toPrates et al., an inspection of the trajectories for a GH12endoglucanase from T. harzianum (ThEG3) cellotetraose andcellopentaose models reveals that after approximately 10 ns,the substrate swings away from residues Y7 and W23 and isnot oriented along the crevice but remains connected to theenzyme by the Y112 hydrophobic contact. After 20–30 ns,the substrate fits back into the crevice in a conformationthat resembles that of ThEG3 (Prates et al., 2013).

The AtEglDDYSG model also indicates that the catalytic-cleft volume is increased (Fig. 6D), thus validating theprevious insights of Powlowski et al. (2009); the more openconformation of the binding cleft predicted for AnXEG12A(endo-xyloglucanase) compared to HgGH12 (endogluca-nase) may provide interactions with linear polymericsubstrates. Conversely, the narrow substrate-binding cleftof HgGH12 compared to AnXEG12A could explain the

Figure 6. Molecular docking analysis. AclaXegA (A) and AclaXegADSST (B) in

complex with XXXG/XXX. Red: conserved aromatic residues (W8 and W23) at the non-

reducing end of the catalytic cleft; Light blue: residues of the cord region (P131, I132);

Yellow: the deleted residues (SST) in AclaXegADSST. AtEglD (C) and AtEglDDYSG (D) in

complex with cellohexaose (C6). Red: conserved aromatic residues (Y7,W22, and Y111)

at the non-reducing end of the catalytic cleft. The Y111 residue was deleted in

AtEglDDYSG.

Figure 7. Histograms generated by molecular docking. Histograms of the

distance distributions of the 300 molecular docking experiments run for wild-type and

mutants to AclaXegA (A) and AtEglD (B).


reduced activity of HgGH12 on branched polysaccharides(Powlowski et al., 2009).Here, the wider binding cleft observed for AclaXegA was

correlated with the lack of activity on b-glucan. Similarly, thenarrower cleft of AtEglD was related to a lower efficiency inxyloglucan hydrolysis. Accordingly, the YSG deletion causedfurther enlargement of the volume of the crevice, followed bythe reduction of b-glucan-hydrolysis capacity (Table I).

The R123A Mutation in AtEglD Led to Vibration ModeVariations and Reduced the Catalytic Efficiency

The R123 in AtEglD is a highly conserved residue in fungalGH12 endoglucanases and is replaced by alanine in all theGH12 fungal endo-xyloglucanases analyzed here (Supple-mentary Fig. S1). Although the mode of operation andthe pH and temperature optima were unchanged (Fig. 5Dand E and Supplementary Fig. S4), the catalytic efficiency ofAtEglDR123A was reduced twofold for b-glucan and xyloglu-can hydrolysis (Table I).Prates et al. (2013) suggested, based on MD simulations,

that the interaction of the R124 in ThEG3 (equivalent to R123in AtEglD) with I128 is critical for B9 strand stabilization.Accordingly, this stabilization was missed in the AtEglDR123A

mutant.

The dynamic behavior, evaluated by NMA, revealed thatseveral regions displayed differences in the vibration mode(Fig. 5A), such as the residues in the B9 strand and itsadjacent loop (residues from 138 to 147, Fig. 2). Inaccordance with the CD analysis, this higher mobilitycompared to the parental enzyme reduced the mutantprotein Tm by over 10�.The AtEglD R123 is located in loop 2 and is also adjacent to

the cord region previously discussed for the AclaXegADSST

mutant. Accordingly, the AtEglDR123A mutation also alteredsubstrate–enzyme interactions at the positive subsites,reducing catalytic efficiency but not affecting the enzyme’saffinity for the substrate (Table I).

The Loop 1 Extension in Fungal GH12 EndoglucanasesGoverns Linear Glucan Hydrolysis

The loop 1 extension (YSG) at the non-reducing end of thecatalytic cleft is essential to the interactions at the�4 and�3subsites and hydrolysis of linear b-glucan. The CE analysis ofthe AtEglD mode of operation indicated that cellobiose wasthe major product after APTS-labeled cellopentaose hydro-lysis and that cellobiose and cellotetraose were producedfrom APTS-labeled cellohexaose, suggesting the key role ofthe �3 and �4 GH12 subsites for the hydrolysis of linearglucans (Fig. 8). Moreover, AtEglD did not hydrolyzecellotetraose (data not shown), as this substrate can onlyinteract with the subsite region from �2 to þ2. Indeed,the inefficient substrate interaction at the �3 subsite byAtEglDDYSG resulted in reduced catalytic efficiency; after a12-h hydrolysis, cellopentaose (C5) was only partiallyhydrolyzed (Fig. 8A). Consequently, AtEglDDYSG interactionsat the negative subsites were drastically affected (Figs. 6Dand 7B), causing substrate misfitting in the catalytic creviceand a significant reduction in the efficiency of linear b-glucandegradation (Table I). Furthermore, the absence of YSGextension in AclaXegA resulted in a wider crevice than that ofAtEglD (Fig. 6).To summarize, our reports describe the role of structural

determinants in fungal GH12 enzymes. All fungal GH12endo-xyloglucanases have conserved variations at loop 1 andloop 2 compared to GH12 endoglucanase counterparts.The conserved deletion (YSG) shortens loop 1 at the non-reducing end of the catalytic cleft, contributing to a moreopen binding cleft compared to fungal GH12 endogluca-nases. Conversely, the GH12 endo-xyloglucanase SSTinsertion at loop 2, which is located in the positive subsites,is essential for the correct binding of xyloglucan and thus forthe catalytic efficiency of endo-xyloglucanase. Lastly, thearginine residue (R123 for AtEglD) that is conserved in allthe fungal GH12 endoglucanases is important for loop 2stabilization; loop 2, in turn, coordinates the substrateinteractions at the positive subsites.These conclusions not only contribute to a better

understanding of the specificity of fungal GH12 endo-xyloglucanases but can also help to direct efforts for therational design of site-directed mutagenesis and targeting

Figure 8. Oligosaccharide hydrolysis by AtEglD and AtEglDDYSG. Capillary

electrophoresis after hydrolysis of APTS-labeled cellopentaose (C5) (A) and

cellohexaose (C6) (B) for 12 h. The wider arrows indicate the preferred cleavage site.



enzymes for specific applications. Deciphering the moleculardeterminants of catalysis is of high relevance to biotechnolo-gy with possible applications for the bioproduction of added-value chemicals.

This work was financially supported by grants from CNPq (474022/2011-4 and 310177/2011-1) and FAPESP (2008/58037-9). ARLD(2011/02169-4) and LCO (2011/13242-7) are FAPESP postdoctoralfellows. MVR is a FAPESP IC fellow (2012/12859-3). Authors’Contributions: A.R.L.D. designed the study, designed the site-directedmutagenesis, performed heterologous expression of the parentalenzymes in A. nidulans, analyzed the results and wrote themanuscript. M.V.R. performed the mutant cloning and expression,purification, biochemical characterizations, and capillaryelectrophoresis. L.C.O. conducted the normal-mode analysis, mole-cular docking and computational data evaluation and participated inmanuscript preparation. A.P.C. participated in circular-dichroismspectra measurement. B.A.D. and F.S. performed the biochemicalcharacterizations of the parental enzymes and capillary electrophor-esis and participated in manuscript preparation. D.A.P. built thephylogenetic tree and analyzed the data. F.M.S. revised the manuscriptand coordinated the study. All authors have read and approved thefinal manuscript.

References

Basma M, Sundara S, Calgan D, Vernali T, Woods RJ. 2001. Solvatedensemble averaging in the calculation of partial atomic charges. JComput Chem 22(11):1125–1137.

Bradford MM. 1976. Rapid and sensitive method for quantitation ofmicrogram quantities of protein utilizing principle of protein-dyebinding. Anal Biochem 72(1–2):248–254.

Buckeridge MS. 2010. Seed cell wall storage polysaccharides: Models tounderstand cell wall biosynthesis and degradation. Plant Physiol154(3):1017–1023.

Buckeridge MS, Rocha DC, Reid JSG, Dietrich SMC. 1992. Xyloglucanstructure and post-germinative metabolism in seeds of Copaiferalangsdorfii from savanna and forest populations. Physiol Plant 86(1):145–151.

Buckeridge MS, Santos HP, Tin�e MAS. 2000. Mobilisation of storage cell wallpolysaccharides in seeds. Plant Physiol Biochem 38:141–156.

Carpita NC, McCann MC. 2000. The cell wall. In: Buchanan BB, GruissemW, Jones R, editors. Biochemistry and molecular biology of plants.Rockville: American Society of Plant Physiologists. p 52–109.

Cota J, Alvarez TM, Citadini AP, Santos CR, de Oliveira NetoM, Oliveira RR,Pastore GM, Ruller R, Prade RA,MurakamiMT, Squina FM. 2011.Modeof operation and low-resolution structure of a multi-domain andhyperthermophilic endo-beta-1,3-glucanase from Thermotoga petro-phila. Biochem Biophys Res Commun 406(4):590–594.

Damasio AR, Ribeiro LF, Furtado GP, Segato F, Almeida FB, Crivellari AC,Buckeridge MS, Souza TA, Murakami MT, Ward RJ, Prade RA, PolizeliMLTM. 2012. Functional characterization and oligomerization of arecombinant xyloglucan-specific endo-beta-1,4-glucanase (GH12) fromAspergillus niveus. Biochim Biophys Acta 1824(3):461–467.

Dunbrack RL, Jr. 2002. Rotamer libraries in the 21st century. Curr OpinStruct Biol 12(4):431–440.

Geiser DM, Gueidan C, Miadlikowska J, Lutzoni F, Kauff F, Hofstetter V,Fraker E, Schoch CL, Tibell L, Untereiner WA, Aptroot A. 2006.Eurotiomycetes: Eurotiomycetidae and Chaetothyriomycetidae. Myco-logia 98(6):1053–1064.

Gilbert HJ, Stalbrand H, Brumer H. 2008. How the walls come crumblingdown: Recent structural biochemistry of plant polysaccharide degrada-tion. Curr Opin Plant Biol 11(3):338–348.

Gloster TM, Ibatullin FM, Macauley K, Eklof JM, Roberts S, Turkenburg JP,Bjornvad ME, Jorgensen PL, Danielsen S, Johansen KS, Borchet TV,Wilson KS, Brumer H, Davies GJ. 2007. Characterization and three-

dimensional structures of two distinct bacterial xyloglucanases fromfamilies GH5 and GH12. J Biol Chem 282(26):19177–19189.

Grigoriev IV, Nordberg H, Shabalov I, Aerts A, Cantor M, Goodstein D, KuoA, Minovitsky S, Nikitin R, Ohm RA, Otillar R, Poliakov A, Ratnere I,Riley R, Smirnova T, Rokhsar T, Dubchak I. 2012. The genome portal ofthe Department of Energy Joint Genome Institute. Nucleic Acids Res40(Database issue):D26–D32.

Heckman KL, Pease LR. 2007. Gene splicing andmutagenesis by PCR-drivenoverlap extension. Nat Protoc 2(4):924–932.

Hollup SM, Salensminde G, Reuter N. 2005. WEBnm@: A web applicationfor normal mode analyses of proteins. BMC Bioinform 6:52.

Iglesias N, Abelenda JA, Rodino M, Sampedro J, Revilla G, Zarra I. 2006.Apoplastic glycosidases active against xyloglucan oligosaccharides ofArabidopsis thaliana. Plant Cell Physiol 47(1):55–63.

Jovanovic I, Magnuson J, Collart F, Robbertse B, AdneyW, HimmelM, BakerS. 2009. Fungal glycoside hydrolases for saccharification of lignocellu-lose: Outlook for new discoveries fueled by genomics and functionalstudies. Cellulose 16(4):687–697.

Khademi S, Zhang D, Swanson SM, Wartenberg A, Witte K, Meyer EF. 2002.Determination of the structure of an endoglucanase from Aspergillus nigerand itsmode of inhibition by palladium chloride. Acta Crystallogr D BiolCrystallogr 58(Pt 4):660–667.

Kirschner KN, Woods RJ. 2001a. Quantum mechanical study of thenonbonded forces in water-methanol complexes. J Phys Chem A105(16):4150–4155.

Kirschner KN, Woods RJ. 2001b. Solvent interactions determine carbohy-drate conformation. Proc Natl Acad Sci USA 98(19):10541–10545.

Lang PT, Brozell SR, Mukherjee S, Pettersen EF, Meng EC, Thomas V, RizzoRC, Case DA, James TL, Kuntz ID. 2009. DOCK 6: Combiningtechniques to model RNA-small molecule complexes. RNA 15(6):1219–1230.

Master ER, Zheng Y, Storms R, Tsang A, Powlowski J. 2008. A xyloglucan-specific family 12 glycosyl hydrolase from Aspergillus niger: Recombinantexpression, purification and characterization. Biochem J 411:161–170.

Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination ofreducing sugar. Anal Chem 31(3):426–428.

Moustakas DT, Lang PT, Pegg S, Pettersen E, Kuntz ID, Brooijmans N, RizzoRC. 2006. Development and validation of a modular, extensible dockingprogram: DOCK 5. J Comput Aided Mol Des 20(10–11):601–619.

Naran R, Pierce ML, Mort AJ. 2007. Detection and identification ofrhamnogalacturonan lyase activity in intercellular spaces of expandingcotton cotyledons. Plant J 50(1):95–107.

Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM,Meng EC,Ferrin TE. 2004. UCSF Chimera—Avisualization system for exploratoryresearch and analysis. J Comput Chem 25(13):1605–1612.

Powlowski J, Mahajan S, Schapira M, Master ER. 2009. Substrate recognitionand hydrolysis by a fungal xyloglucan-specific family 12 hydrolase.Carbohydr Res 344(10):1175–1179.

Prates ET, Stankovic I, Silveira RL, Liberato MV, Henrique-Silva F, Pereira N,Jr., Polikarpov I, SkafMS. 2013. X-ray structure andmolecular dynamicssimulations of endoglucanase 3 from Trichoderma harzianum:Structural organization and substrate recognition by endoglucanasesthat lack cellulose binding module. PLoS ONE 8(3):e59069.

Sagermann M, Matthews BW. 2002. Crystal structures of a T4-lysozymeduplication-extension mutant demonstrate that the highly conservedbeta-sheet region has low intrinsic folding propensity. J Mol Biol 316(4):931–940.

Sali A, Blundell TL. 1993. Comparative protein modelling by satisfaction ofspatial restraints. J Mol Biol 234(3):779–815.

Sambrook J, Fritsch EF, Maniatis T. 1988. Molecular cloning: A laboratorymanual. New York: Cold Spring Harbor Laboratory. 1659p.

Sandgren M, Shaw A, Ropp TH, Wu S, Bott R, Cameron AD, Stahlberg J,Mitchinson C, Jones TA. 2001. The X-ray crystal structure of theTrichoderma reesei family 12 endoglucanase 3, Cel12A, at 1.9 A resolution.J Mol Biol 308(2):295–310.

Sandgren M, Stahlberg J, Mitchinson C. 2005. Structural and biochemicalstudies of GH family 12 cellulases: Improved thermal stability, and ligandcomplexes. Prog Biophys Mol Biol 89(3):246–291.


Segato F, Damasio AR, Goncalves TA, de Lucas RC, Squina FM, Decker SR,Prade RA. 2012. High-yield secretion of multiple client proteins inAspergillus. Enzyme Microb Technol 51(2):100–106.

Shapiro AL, Vinuela E, Maizel JV, Jr. 1967. Molecular weight estimation ofpolypeptide chains by electrophoresis in SDS–polyacrylamide gels.Biochem Biophys Res Commun 28(5):815–820.

Soding J, Biegert A, Lupas AN. 2005. The HHpred interactive server forprotein homology detection and structure prediction. Nucleic Acids Res33(Web Server issue):W244–W248.

Song S, Tang Y, Yang S, Yan Q, Zhou P, Jiang Z. 2013. Characterization oftwo novel family 12 xyloglucanases from the thermophilic Rhizomucormiehei. Appl Microbiol Biotechnol 97(23):10013–10024.

Vincken JP, Beldman G, Voragen AG. 1997. Substrate specificity ofendoglucanases: What determines xyloglucanase activity? CarbohydrRes 298(4):299–310.

Yoshizawa T, Shimizu T, Hirano H, Sato M, Hashimoto H. 2012.Structural basis for inhibition of xyloglucan-specific endo-beta-1,4-glucanase (XEG) by XEG-protein inhibitor. J Biol Chem 287(22):18710–18716.

Znameroski E, Glass NL. 2013. Using a model filamentous fungus to unravelmechanisms of lignocellulose deconstruction. Biotechnol Biofuels6(1):6.

Supporting Information

Additional supporting information may be found in theonline version of this article at the publisher’s web-site.



understanding the function of conserved variations in the catalytic loops of fungal glycoside...

Documents