Structural Basis for the Substrate Speciﬁcity of a ... · model, the tetrasaccharide moiety is...

doi:10.1016/j.jmb.2006.01.014 J. Mol. Biol. (2006) 357, 1211–1225

Structural Basis for the Substrate Specificityof a Bacillus 1,3-1,4-b-Glucanase

Olaf J. Gaiser1†, Kirill Piotukh2†, Mondikalipudur N. Ponnuswamy1,3†Antoni Planas4, Rainer Borriss2 and Udo Heinemann1,5*

1Forschungsgruppe Kristallo-graphie, Max-Delbruck-Centrum fur MolekulareMedizin, Robert-Rossle-Str. 1013125 Berlin, Germany

2Institut fur BiologieHumboldt-Universitat zuBerlin, Chausseestr. 117, 10115Berlin, Germany

3Department ofCrystallography and BiophysicsUniversity of Madras, GuindyCampus, Chennai 600 025India

4Laboratory of BiochemistryInstitut Quımic de SarriaUniversidad Ramon Llull08017 Barcelona, Spain

5Institut fur Chemie—Kristal-lographie, Freie UniversitatBerlin, Takustr. 6, 14195 BerlinGermany

0022-2836/$ - see front matter q 2006 E

†O.J.G., K.P. and M.N.P. contribuwork.Abbreviations used: E$I, enzyme

enzyme–substrate; RMSD, root-meaE$P, enzyme–product; CBH-I, cellobLBHBs, low-barrier hydrogen bondE-mail address of the correspond

[email protected]

Depolymerization of polysaccharides is catalyzed by highly specificenzymes that promote hydrolysis of the scissile glycosidic bond by anactivated water molecule. 1,3-1,4-b-Glucanases selectively cleave b-1,4glycosidic bonds in 3-O-substituted glucopyranosyl units within poly-saccharides with mixed linkage. The reaction follows a double-displacementmechanism by which the configuration of the anomeric C1-atom ofthe glucosyl unit in subsite KI is retained. Here we report the high-resolution crystal structure of the hybrid 1,3-1,4-b-glucanaseH(A16-M)E105Q/E109Q in complex with a b-glucan tetrasaccharide. Thestructure shows four b-D-glucosyl moieties bound to the substrate-bindingcleft covering subsites KIV to KI, thus corresponding to the reactionproduct. The ten active-site residues Asn26, Glu63, Arg65, Phe92, Tyr94,Glu105, Asp107, Glu109, Asn182 and Trp184 form a network of hydrogenbonds and hydrophobic stacking interactions with the substrate. Theseresidues were previously identified by mutational analysis as significantfor stabilization of the enzyme–carbohydrate complex, with Glu105 andGlu109 being the catalytic residues. Compared to the Michaelis complexmodel, the tetrasaccharide moiety is slightly shifted toward that part of thecleft binding the non-reducing end of the substrate, but shows previouslyunanticipated strong stacking interactions with Phe92 in subsite KI.A number of specific hydrogen-bond contacts between the enzyme and theequatorial O2, O3 and O6 hydroxyl groups of the glucosyl residues insubsitesKI,KII andKIII are the structural basis for the observed substratespecificity of 1,3-1,4-b-glucanases. Kinetic analysis of enzyme variants withthe all b-1,3 linked polysaccharide laminarin identified key residuesmediating substrate specificity in good agreement with the structural data.The comparison with structures of the apo-enzyme H(A16-M) and acovalent enzyme–inhibitor (E$I) complex, together with kinetic andmutagenesis data, yields new insights into the structural requirementsfor substrate binding and catalysis. A detailed view of enzyme–carbohydrate interactions is presented and mechanistic implications arediscussed.

q 2006 Elsevier Ltd. All rights reserved.

Keywords: protein structure; oligosaccharide complex; protein–carbohydrateinteractions; active-site mutant; 1,3-1,4-b-glucanase
*Corresponding author
lsevier Ltd. All rights reserve

ted equally to the

–inhibitor; E$S,n-square deviation;iohydrolase I;s.ing author:

Introduction

H(A16-M) is a representative of the retaining endo-1,3-1,4-b-D-glucan 4-glucanohydrolases (1,3-1,4-b-glucanases, lichenases, EC 3.2.1.73) which selectivelycleave b-1,4 glycosidic bonds in 3-O-substitutedglucopyranosyl units. The sequence of H(A16-M) isderived from the 1,3-1,4-b-glucanase from Bacillusmacerans in which the 16 N-terminal residues havebeen replaced by the respective residues of the

d.

Figure 1. Subsite arrangement in the endo-1,3-1,4-b-glucanase H(A16-M). Schematic representation of theglucosyl-binding subsites and the glycosidic linkagebetween the glucopyranosyl rings of the tetrasacharideGlcb4Glcb4Glcb3Glc spanning subsites KIV to KI in theE$P complex (a) and the covalent enzyme–inhibitorcomplex with a cellobiosyl moiety bound in subsitesKIII/KII (b). The 3-O substituted glucosyl residue(subsite KI) is marked in grey.

1212 1,3-1,4-b-Glucanase Substrate Specificity

enzyme from Bacillus amyloliquefaciens. Natural sub-strates of lichenases are 1,3-1,4-b-D-glucans occurringabundantly in the endospermcellwalls of cereals andgrain.1 Plant and microbial glucanases belong todistinct groups of glycosyl hydrolases withoutdiscernible sequence similarity and unrelated three-dimensional structures, representing examples ofconvergent protein evolution toward the samesubstrate specificity. The plant enzymes belong tothe glycosyl hydrolase family 17 and adopt a (b/a)8TIM-barrel fold, whereas the microbial enzymes areclassified asmembers of family 16with a b-sandwicharchitecture. b-Glucanases are retaining glycosi-dases2 acting by general acid/base catalysis in adouble-displacement reaction mechanism.3,4 Depo-lymerization of b-D-glucans involves two Sn2-typenucleophilic substitutions at the anomeric C1 atom,both catalyzed by the general acid/base residue. Thefirst reaction step (glycosylation) is rate-limiting5 andrequires the action of the nucleophile, resulting in acovalently linkedglycosyl-enzyme intermediatewitha-configuration. The second step (deglycosylation)involves hydrolysis by an activated water moleculeyielding the b-product, thus the overall reactionproceeds with net retention of the anomeric configu-ration. The main hydrolysis products are thetrisaccharide (3-O-b-cellobiosyl-b-D-glucopyranose)and the tetrasaccharide (3-O-b-cellotriosyl-b-D-gluco-pyranose).

The catalytic residues of Bacillus 1,3-1,4-b-gluca-nases have been identified by means of mutationalanalysis, affinity labeling, and X-ray crystallography.Mutational analyses on the Bacillus licheniformis6 andB. macerans7 enzymes have identified Glu105 andGlu109 as the catalytic nucleophile and the generalacid/base, respectively (H(A16-M) numbering).Their functional roleshavebeenprobedbya chemicalrescue methodology.8 Kinetic and mechanisticstudies of the B. licheniformis b-glucanase haveshown that the substrate-binding site is composedof six subsiteswith four subsites (KIV toKI) beingonthe non-reducing end from the scissile glycosidicbond and two subsites (CI andCII) on the reducingend (Figure 1). By definition, the scissile b-1,4glycosidic bond is positioned between subsites KIand CI, whereas the b-1,3 glycosidic bond localizesbetween subsitesKII andKI.9 The structures of twowild-type Bacillus 1,3-1,4-b-glucanases fromB. macerans7 andB. licheniformis10 have been analyzedby X-ray crystallography followed by structuralstudies on engineered hybrid and circularly per-muted variants (reviewed by Heinemann et al.11).More recently, the structure of the 1,3-1,4-b-glucanasefrom Fibrobacter succinogenes, harboring a naturalcircular permutation, has been determined.12

Family-16 1,3-1,4-b-glucanases are essentially pureb-proteinswith jellyroll architecture consistingof twomajor antiparallel b-sheets.

The crystal structure of an enzyme–inhibitor (E$I)complex with an epoxybutyl cellobioside inhibitorcovalently bound in subsites KIII/KII13 is the onlyavailable structure of a bacterial 1,3-1,4-b-glucanasein liganded form. However, containing a cellobiosyl

unit bound in subsites KIII/KII, this structure doesnot reveal protein–carbohydrate interactions in sub-site KIV and the catalytic site (subsite KI), andthereforeprovides only limited informationabout thesubstrate specificity of 1,3-1,4-b-glucanases and themolecular mechanism of catalysis. This covalent E$Icomplex structure was used to model protein–carbohydrate interactions within the enzyme–sub-strate (E$S) complex of the B. macerans 1,3-1,4-b-glucanase.14 Residues presumed to contribute tosubstrate bindingwere selectedon thebasisof theE$Smodel and subsequently subjected to extensivemutational analysis15,16 allowing the assignment ofindividual enzyme–substrate interactions withinparticular subsites.

We report here the structure of an enzyme–ligandcomplex solved by X-ray crystallography to inves-tigate the contribution of key residues to ligandbinding in different glucosyl-binding subsites,aiming at illuminating the molecular basis forsubstrate specificity and the high selectivity ofenzymatic depolymerization. Here, the catalyticallyimpaired 1,3-1,4-b-glucanase H(A16-M)E105Q/E109Q

in which both the nucleophile (Glu105) and thegeneral acid/base (Glu109) have been mutated tothe isosteric glutamine residues was used forco-crystallization experiments with the substrateb-glucan hexasaccharide. The latter was designed tobe bound in subsites KIV to CII with the scissileb-1,4 glycosidic bond between subsites KI and CI,and the b-1,3 bond between subsites KII and KI(Figure 1). Structure analysis of the protein–carbohydrate complex, however, reveals a tetra-saccharide moiety bound in subsites KIV to KIwith a b-1,3 bond located between subsites KII andKI as expected for natural ligands of 1,3-1,4-b-glucanases. We therefore conclude that the crystalstructure represents the non-covalent complex

1,3-1,4-b-Glucanase Substrate Specificity 1213

between H(A16-M)E105Q/E109Q and its naturalreaction product, a b-glucan tetrasaccharide. Inaddition we used mutant enzyme kinetics to assessthe binding properties of all b-1,3 laminarin whichprovides, together with kinetic data from naturallichenan substrates, a deeper understanding ofspecific enzyme–carbohydrate interactions.

Results

Crystal structure of H(A16-M)E105Q/E109Q incomplex with b-glucan tetrasaccharide

Model refinement converged to a final R-value of0.163 (RfreeZ0.197) considering diffraction data inthe resolution range from 34.5 A to 1.64 A (Table 1).More than 90% of all protein residues are in themost favorable regions of the Ramachandrandiagram as defined by PROCHECK17 with root-mean-square deviations (RMSD) from target values

Table 1. Data collection and refinement statistics

Crystallographic parametersSource Synchrotron EMBL-DESY

BW7BSpace group P212121Cell parameters (A) aZ75.77, bZ88.76, cZ154.79Wavelength (A) 0.8337

Data collection statisticsa

Resolution range (A) 34.46–1.64No. of reflectionsObserved 538,165Unique 118,568Used for Rfree calculation(7%)

8310

Completeness (%) 92.5 (83.1)Redundancy 4.5Rsym

b 0.057overall I/s(I) 11.7Rcryst

c 16.36Rfree

d 19.70

Model (4 protein–ligand complexes in asymmetric unit)Amino acid residues 856Protein atoms 6760Carbohydrate ligand atoms 180Water molecules 1596Ions (Ca2C/Zn2C) 4/3

Ramachandran plotMost favored (%) 90.2Additionally allowed (%) 9.8Generously allowed (%) 0.0Disallowed (%) 0.0

RMSD from ideal geometryBond lengths (A) 0.012Bond angles (deg.) 1.66Torsion angles (deg.) 5.11Protein Data Bank code 1U0A

a Values in parentheses are for the highest resolution shell(1.67–1.64 A).

b RsymZP

hkl jIKhIij=P

hklhIi, where I is the integrated

observed intensity, hIi is the average integrated intensity withthe summation over all observed reflections.

c RcrystZjFobsKFcalcj/Fobs, where Fobs and Fcalc are theobserved and calculated structure factors, respectively.

d Rfree is calculated as Rcryst using 7% of all reflectionsrandomly chosen and excluded from structure calculation andrefinement.

for bond lengths and angles of 0.012 A and 1.6598,respectively. The overall mean B value is 15.9 A2.The asymmetric unit contains four crystallo-

graphically independent protein molecules, eachshowing clear electron density of four b-D-glucosylresidues in the substrate binding cleft, coveringsubsites KIV to KI. Well-ordered, discrete watermolecules are observed in subsites CI and CII,ruling out the presence of disordered glucosemoieties from the substrate hexasaccharide. Thus,only a tetrasaccharide and not the hexasaccharideadded to the crystallization buffer is found in thecrystal. Traces of wild-type enzyme resulting frompartial deamidation of Gln105 and Gln109 in thecatalytic center of H(A16-M)E105Q/E109Q duringprotein purification and/or crystallization veryprobably resulted in substrate cleavage. Thisinterpretation is supported by the observation ofweak residual or contaminant enzyme activity in alichenin-plate activity test with Congo-Red stainingwhen H(A16-M)E105Q/E109Q was incubated for twomonths at room temperature, pH 6. The hexasac-charide is a very good substrate for the wild-typeenzyme.16 The tetrasaccharide moiety identified inthe active site therefore represents the naturalreaction product. The high-resolution structure ofH(A16-M)E05Q/E109Q in complex with the producttetrasaccharide (Figure 2) is well defined by theelectron density and allows precise determinationof side-chain conformations and protein–carbohydrate interactions.The four protein molecules in the asymmetric

unit are very similar in conformation (RMSD overall Ca atoms: 0.142 A) and show identical secondarystructure. H(A16-M) is essentially an all b-proteinconsisting of two major all-antiparallel b-sheetsarranged atop each other. The seven-strandedb-sheets are bent and adopt a jellyroll architecturewith a convex and a concave face. The latter forms acleft in which the substrate is bound and hydro-lyzed. The substrate binding cleft comprises sixglucosyl-binding subsites and a major surface loop(residues 22–35) partially covering the cleft at thenon-reducing end of a bound substrate. This loop islinked to the inner b-sheet through a disulfide bondinvolving Cys32 and Cys61. Secondary structureanalysis reveals four small segments with a-helicalgeometry. On the convex side of the molecule,across from the active site, a calcium ion is boundwith nearly perfect pentagonal-bipyramidal geo-metry, coordinated to the protein backbone carbo-nyl oxygen atoms of Pro9, Gly45 and Asp207, thecarboxylate oxygen Od1 from Asp207 and threewater molecules. The seven ligands are between2.2 A and 2.5 A apart from the central calcium ion.Each two of the four protein–carbohydratecomplexes of the asymmetric unit are related by2-fold non-crystallographic symmetry with a zincion occupying a position on the symmetry axis.These zinc ions have almost perfect tetrahedralgeometry and coordinate to His145 Nd1 and Asp161Od2 from one proteinmolecule and to the equivalentresidues from the symmetry-related molecule.

Figure 2. Structure of H(A16-M)E105Q/E109Q in complex with b-glucan tetrasaccharide bound in the carbohydratebinding cleft. (a) Side-view of the protein–carbohydrate complex with the non-reducing end of the tetrasaccharidemoiety pointing to the left. (b) View along the substrate binding cleft from the reducing end. The protein is depicted asribbon diagram, b-strands and a-helices are colored light yellow and red, respectively. The carbohydrate moiety isshown as space-filling model with carbon and oxygen atoms colored grey and red, respectively.


A third zinc site was identified near His166 ofprotein chain 1, but not at the equivalent positionsof the three remaining protein molecules present inthe asymmetric unit. A total of 1596 watermolecules were located in the asymmetric unit.

The enzyme–product (E$P) complex revealssmall conformational changes of the hybrid proteincompared to free H(A16-M), reflected in an averageRMSD of 0.66 A for all 214 Ca atoms. The p-helicalsegment (residues Gly96 to Thr101) flanking thenon-reducing end of the carbohydrate bindingcrevice undergoes rearrangement with shifts ofapproximately 0.9 A for the main-chain atoms anda 238 turn of the His99 side-chain, which effectivelynarrows the rim at the entrance of the substratebinding cleft, whereas no appreciable main-chainstructural changes could be detected for theresidues at the bottom of the substrate bindingcleft. The conformational changes in the loopcomprising residues Asn11 to Thr17 with maximalatom shifts of 3.2 A for the backbone atoms and4.7 A for the side-chain atoms can be attributed tolocal differences in the crystallographic environ-ment.

Carbohydrate conformation

The four crystallographically independentprotein–carbohydrate complexes of the asymmetricunit are characterized by well defined glucosylresidues in subsites KIII to KI, whereas theglucosyl unit in subsite KIV shows consistentlyweaker electron density and slightly higher Bvalues, which can be attributed to fewer proteincontacts and increased conformational flexibility.Average B values of the glucosyl atoms in subsitesKI, KII, KIII, and KIV are 16.7, 18.6, 23.4 and34.6 A2, respectively. The high resolution of thecrystal structure allows unambiguous determination

of sugar configuration, conformation, linkage-typeand protein–carbohydrate interactions.

The tetrasaccharide ligand in the E$P complexdisplays an extended conformation (Figure 3). Allfour glucosyl units are in 4C1 chair conformation,and torsion angle analysis shows that the glycosylbonds are all near the global energy minimum(Table 2).18 The E$P complex displays no gluco-pyranosyl ring distortion in subsite KI as found inthe E$S complex of cellobiohydrolase I (CBH-I),19

implying that strain is induced primarily byadditional E$S interactions in subsite CI. However,the glucosyl unit in subsite KI (Glc-I) directlyinteracts with Phe92, which causes Glc-I to deviateslightly from the ideal extended conformation.

Protein–carbohydrate interactions in substratebinding sites KIV, KIII and KII

The deep extended binding cleft can accommo-date six b-D-glucosyl units covering subsitesKIV toKI at the non-reducing end and subsites CI andCII at the reducing end, with the scissile b-1,4glycosidic bond positioned between subsites KIand CI. The substrate-binding cleft is decoratedwith residues acting as hydrogen donors oracceptors to form hydrogen bonds to the glycosylunits in distinct subsites. Amino acids witharomatic side-chains additionally promote binding,positioning and processive turnover of the poly-saccharide substrate by means of hydrophobicstacking interactions with the glucopyranose rings.

Enzyme–ligand interactions are summarizedin Table 3 and Figure 4. The glucosyl unit in subsiteKIV (Glc-IV) has only few contacts with theenzyme and shows elevated conformational flexi-bility as indicated by weaker electron density andincreased average B value (34.6 A2). Glc-IV formsa weak water-mediated hydrogen bond between

Figure 3.Carbohydrate conformationandaromatic side-chain interactions. Stereodrawingof the ligand-binding sitewiththe seven-stranded inner b-sheet and themajor loop covering the distant subsites of the substrate binding cleft. The producttetrasaccharide Glcb4Glcb4Glcb3Glc from one protein–carbohydrate complex of the asymmetric unit is shown in ball-and-stick representation with the jFojKjFcj difference electron density phased with the refined protein model, omitting thecarbohydrate. Aromatic side-chains lining the binding cleft are colored blue, other residues important for catalysis are inyellow, and the isosteric equivalents of the nucleophile and the general catalyst, Gln105 and Gln109, are shown inmagenta.


the O6 hydroxyl and the side-chain Nh1 of Arg65,and another interaction between the O2 hydroxyland the aromatic side-chain of Tyr24, termed X-H/p(Phe) hydrogen bond.20 In the free enzyme, theguanidinium group of Arg65 forms a stronghydrogen bond to the Tyr24 phenolic hydroxylgroup, whereas, upon ligand binding, Arg65 losescontact to Tyr24, approaching the carbohydrateligand (maximal atom shift of 3.2 A) and interactingnow with the O2 and O3 hydroxyls of the glucosylunit in subsite KIII (Glc-III) and the carboxylateside-chain of Glu63 which attributes a central role tothis residue in binding and positioning the distalglucosyl units in subsites KIII/KIV. The O2

hydroxyl of Glc-III forms an additional hydrogenbond with Ne2 of His99. The glucosyl unit bound insubsite KII (Glc-II) interacts with the side-chains ofboth Asn26 and Glu63 through hydrogen bondsbetween the O6 hydroxyl and Nd2, and thecarboxylate oxygen atoms, respectively, whereasthe O2 hydroxyl of Glc-II forms a water-mediatedhydrogen bond with Oh of Tyr94. The aromaticphenolic ring of Tyr94 additionally promotessubstrate binding and positioning by means ofhydrophobic stacking interactions with Glc-II.In contrast to the E$S complex model, whichsuggested hydrogen bonding interactions of

Table 2. Conformation of the tetrasaccharide ligand in the E$

Complex Glycosidic bond

E$P-1 (1–214) Glc2-(b1,3)-Glc1Glc3-(b1,4)-Glc2Glc4-(b1,4)-Glc3




a Torsion angles are defined as: f, O5i –C

1i –O

4iK1–C

4iK1 and j, C1

i –O4iK

Asn182 and Ser90 with Glc-II and the glucosylunit in subsite KI (Glc-I), respectively, Asn182bonds to the O4 hydroxyl of Glc-I, whereas Ser90makes no direct contact to any of the glucosylresidues but forms a strong hydrogen bond withAsn182 Od1. Moreover, Asn26 and Glu63 interactexclusively with Glc-II, whereas kinetic analysisassigned to them a major role in binding Glc-III.16

Protein–carbohydrate interactions in substratebinding site KI (catalytic site)

The active center involves the isosteric equiva-lents of the nucleophile and the acid/base catalyst,Gln105 and Gln109, respectively, which areinvolved in a precisely adjusted hydrogen bondnetwork with the auxiliary residues Trp103 andAsp107. Gln105 forms strong hydrogen bonds toTrp103 Ne1 and the carboxylate oxygen Od1 ofAsp107 as observed for Glu105 in the wild-typestructure (Glu105wt Oe1–(2.9 A)–Trp103wt Ne1;Glu105wt Oe2–(2.3 A)–Asp107wt Od1),14 whereasthe side-chain functional group of Gln109 interactswith Asp107 Od2 and Gln119 Ne2 by direct andwater-mediated hydrogen bonds, respectively.Asp107 Od2 additionally contacts a firmly posi-tioned water molecule which itself coordinates to

P complex

fa (deg.) ja (deg.)

K73.6 118.7K56.8 240.8K33.6 233.9

K78.6 113.7K88.4 258.9K33.0 229.8

K73.9 111.1K51.6 240.9K28.0 225.6

K76.8 117.4K54.7 243.4K43.3 232.3

1–C4iK1–C

5iK1 (IUPAC-IUB, 1971).

Table 3. Enzyme-b-glucan tetrasaccharide hydrogen bonding and stacking interactions

Substrate Protein

Residue Atom Residue Atom Distancea (A)

Glc-IV O6 Arg65 Nh1 2.4 3.5O2 (X–H/p) Tyr24 (Ring) 3.4 u(x)Z12.18b

Glc-III O2 His99 N32 2.8O2 Arg65 Nh2 2.9O3 Arg65 Nh1 3.2

Glc-II O2 Tyr94 Oh 3.1 2.6O3 Asn26 O 3.0 3.0O6 Asn26 Nd2 2.8O6 Glu63 O31 3.2 2.6O6 Glu63 O32 2.6

Stackingc Tyr94 (Ring) 4.3

Glc-I O1 Gln105 N32 3.1O1 Asp107 Od2 2.9O1 Asp107 Od2 3.2 2.7O1 Gln119 N32 3.2 3.3O1 Asn121 Nd2 3.2 2.9O1 Tyr123 Oh 3.4 3.2O1 Gln109 O31 3.2 2.8O2 Gln105 O31 2.7O2 Tyr123 Oh 3.2 3.0O4 Asn182 Nd2 3.1O6 Asn182 Od1 3.4O6 Gln109 O31 2.7O6 Trp184 N31 2.9

Stacking Phe92 (Ring) 4.7

a Average distances of four complexes in the asymmetric unit. Where two distances are given, they characterize contacts from thesubstrate atom to a bridging water oxygen to the protein atom.

b For the X–H/p hydrogen bond, the distance is measured between donor atom X and the aromatic midpoint; angle u(x) iscalculated between the vector from atom X to the center of the ring and the ring normal axis.

c Distance from center to center of interacting aromatic ring systems.

Figure 4. Enzyme–carbohydrate interactions in subsites KI to KIV. Schematic depiction of hydrogen bond andhydrophobic stacking interactions in the enzyme–ligand complex. Glc-I to Glc-IV mark glucosyl residues bound insubsites KI to KIV. Amino acid side-chains are shown with their functional groups. Hydrogen bonds with donor-acceptor distance below 3.5 A are shown as broken lines (numbers are donor-acceptor distances in A). Water moleculesmediating hydrogen bonds are depicted as black balls. Hydrophobic interactions are also indicated . Note that theb-1,3 linkage between Glc-II and Glc-I positions both 6CH2-OH glucosyl side-chains on the same side of thetetrasaccharide.



Asn121 Nd2 from subsite CI. The newly formedanomeric carbon (C1) of the tetrasaccharide reactionproduct resides 3.2 A away from Gln105. BothGln105 and Gln109 support carbohydrate bindingand positioning by hydrogen bond interactionswith Glc-I in the catalytic subsite. Gln105 interactswith both the O1 and O2 hydroxyls, whereas Gln109is in direct contact to the O6 hydroxyl and alsowater-mediated hydrogen bonds to the O1 hydroxylby means of its bidentate functional side-chaingroup. Additional protein–carbohydrate inter-actions in subsite KI include hydrogen bondsbetween Asn182 Nd2 and Glc-I O4, Trp184 Ne1 andGlc-I O6, Asp107 Od2 and Glc-I O1 and, in addition,hydrophobic stacking interactions between Glc-Iand the phenyl ring of Phe92. In cellulases, thetryptophan residue in subsite KII promotes sub-strate binding by stacking interactions with thebound glucopyranosyl ring, whereas in lichenasesthe aromatic ring system of Trp184 is positionedalmost perpendicular to the glucosyl ring in subsiteKI, forming the aforementioned hydrogen bond toGlc-I O6. The conformation of Trp184 is additionallystabilized by strong hydrophobic stacking inter-actions with Phe30 (4.1 A center-to-center distance).The pair Phe30/Trp184 was earlier proposed to beresponsible for the structural integrity of thecatalytic cleft through attachment of the majorloop to the inner b-sheet.16 The b-configured O1

hydroxyl of Glc-I is involved in an extendedhydrogen bond network including the active-siteresidues Gln105, Asp107 and Gln109.

Substrate binding sites CI and CII

In subsitesCI/CII no electron density indicatingthe binding of glycosyl residues is observed.Instead, a well-defined hydrogen bond network is

Table 4. Catalytic activity of B. macerans 1,3-1,4-b-glucanase v

Substra

Lichenan16

Enzymea kcat (sK1) Km (mg mlK1)

DDGslich

(kcal molK1)

WT 1191G53 0.45G0.08 (2.6Y24W 787G22 0.14G0.03 K0.47 (5.0Y24F 481G31 2.19G0.36 1.59 (1.7Y24A 325G32 3.85G0.77 2.21 (7.0N26A 262G28 4.7G1.0 2.47 (4.5E63D 112.6G8.6 4.85G0.80 3.03 (9.7E63Q 8.08G0.32 1.47G0.18 3.95 (3.9R65A 811G33 1.95G0.21 1.19 (9.0A98W 908G23 0.61G0.05 0.37 (3.7H99D 276G13 0.26G0.07 0.59 (1.9H99R 147.1G4.5 0.89G0.08 1.78 (1.7S90A 324G15 0.33G0.05 0.65 (5.6Y123F 627G20 0.16G0.04 K0.24 (6.5Y123A 40.4G1.8 0.13G0.04 1.36 (2.1W184Y 163.8G7.6 0.68G0.12 1.54 (9.5E131Q 302.4G9.2 2.37G0.20 1.94 (2.1W192A 40.5G1.9 3.13G0.33 3.41 (2.1

a Residue numbering according to the sequence of H(A16-M).b Subsites that are only partially occupied with laminarin are indi

established around the b-configured O1 hydroxyl inthe active site and in subsiteCI, suggesting that thehexasaccharide substrate used for co-crystallizationhas been catalytically processed. Most likely,H(A16-M)E105Q/E109Q has been re-activated bypartial spontaneous deamidation of Gln105 and/or Gln109 to yield the catalytically competentnucleophile and general acid/base Glu105 andGlu109, respectively. Yet, subsites CI/CII are notcompletely empty but are occupied with a numberof water molecules forming an extended hydrogenbond network which involves the O1 hydroxyl ofGlc-I and additional contacts to residues coveringsubsites CI/CII. These include Oh of Tyr123,which also stacks with the active-site residueTrp103, Gln119 Ne2, Asn121 Nd2, and Glu132 Oe1/e2.The water coordinated to Asn121 Nd2 has a fullyoccupied tetrahedral coordination sphere beingfurther bound to Asp107 Od2 from the active siteand to two additional water molecules which bothinteractwith thegeneral acid/base equivalentGln109Ne2, and also with Glc-I O1 and Gln119 Ne2,respectively.Thesefirmlypositionedwatermoleculesappear to be central for the intricate water-mediatedhydrogen bond network in subsitesCI/CII.

Catalytic activity of wild-type and mutant 1,3-1,4-b-glucanases against laminarin

The catalytic activity of the B. macerans 1,3-1,4-b-glucanase and a set of active-site variants againstlaminarin was determined and compared with thehydrolysis of lichenan16 by these enzymes (Table 4).All active-site residues of the B. macerans 1,3-1,4-b-glucanase are identical with those in the hybridenzyme H(A16-M) which is the subject of thisstructural study. As expected, the efficiency indegrading the all b-1,3-linked laminarin is greatly

ariants with lichenan and laminarin substrates

te

Laminarin

kcat (sK1)

Km

(mg mlK1)DDGs

lam

(kcal molK1) Subsiteb

3G0.06)!10K1 6.73G0.300G0.13)!10K1 4.05G0.24 K0.74 (KV), KIV1G0.10)!10K1 9.46G0.84 0.49 (KIV)G0.4)!10K2 5.88G0.56 0.76 (KIV)G0.5)!10K2 14.7G2.6 1.64 (KIII), KIIG0.9)!10K3 9.1G1.5 2.31 (KIII), KIIG0.1)!10K3 4.42G0.20 2.43 (KIII), KIIG0.1)!10K2 6.18G0.10 0.63 (KIII)5G0.23)!10K1 5.76G0.66 K0.33 KIII8G0.11)!10K1 5.41G0.59 0.04 KIII8G0.18)!10K1 11.1G1.8 0.57 (KIII)G0.4)!10K2 5.61G0.69 0.87 KI0G0.12)!10K1 2.29G0.12 K1.27 KI, CIG0.3)!10K2 3.9G1.3 1.27 KI, CIG0.6)!10K2 7.19G0.73 0.69 (KI)7G0.11)!10K1 3.82G0.49 K0.24 (CI)G0.2)!10K2 6.1G1.1 1.56 (CI)

cated in parentheses.


reduced in comparison with the mixed-linkedlaminarin with a drop in kcat/KM by a factor of7!104. Below, the contributions of individualactive-site residues to lichenan and laminarinhydrolysis are discussed in detail with respect tothe structure of the enzyme–product complex.

Discussion

Carbohydrate binding modules and processingenzymes have been widely studied with the aim ofilluminating their structure and function in mole-cular detail. Despite substantial contribution frombiochemistry and structural biology providinginsight into the global architecture of 1,3-1,4-b-glucanases, the molecular details of carbohydratebinding at the site of hydrolysis (catalytic subsiteKI)remained elusive. The lack of structural informationof 1,3-1,4-b-glucanases in complex with naturalligands therefore prevented the detailed analysis ofsubstrate specificity, selectivity and processivity ofenzymatic carbohydrate depolymerization. Here wedescribe the high-resolution crystal structure ofH(A16-M)E105Q/E109Q in complex with the naturalreaction product, b-D-glucan tetrasaccharide, allow-ing the identification of structural features respon-sible for substrate specificity in subsitesKIV toKI.

Enzyme conformation and carbohydrate binding

Only very small main-chain movements in theactive site of H(A16-M) are observed in the E$Pcomplex compared to the free enzyme. The pairGln105/Asp107 shows limited side-chainrearrangements upon the isosteric replacement ofGlu105 (E105Q), as well as Arg65 and His99. Thelack of high-resolution structural data illuminatingnative protein–carbohydrate interactions promptedthe modelling of an E$S complex between 1,3-1,4-b-glucanase and a b-glucan hexasaccharide cover-ing subsites KIV to CII.14 However, the E$S modeldid not take into account distortion of the glucopyr-anosyl ring in subsite KI. In addition, the energeticcontributions of hydrophobic stacking interactions,hydrogen bonds and electrostatic interactionsdepend on the local environment of functionalgroups, which are difficult to assess properly. TheE$S model, therefore, may suggest a binding modedifferent from that in the E$S complex in solution orthat observed in the E$I and the E$P complex. Thelatter shows the tetrasaccharide moiety to beslightly shifted toward that part of the cleft bindingthe non-reducing end of the substrate, therebyaccommodating a pattern of hydrogen bondspartially different from that in the E$S model. Thisis in agreement with previous results from kineticanalysis16 and resembles the ligand shift observedin the case of cellobiohydrolase I (CBH-I) com-plexed with cellobiose and other cellooligo-saccharides.19,21 In the E$P complex, the isostericequivalent of the nucleophile, Gln105, is at adistance of 3.2 A from the anomeric carbon C1 of

Glc-I, whereas the acid/base equivalent Gln109resides 3.8 A away from the O1 hydroxyl of Glc-Iwhich in both cases would exclude direct inter-action and efficient catalysis in the E$S complex.The observed carbohydrate shift suggests that, inthe E$S complex, the glucosyl residues of lichenansubstrates do not perfectly match with their cognatesubsites due to the enzyme’s active site architecture,which strongly promotes productive substratebinding and hydrolysis if the glucosyl residues tobe bound at the site of catalysis (subsites KI/CI)are in strained or distorted conformation. Uponcleavage of the scissile bond, however, the oligo-saccharide product is free to adopt a ground-stateconformation in which the glucosyl residues matchwell with subsites KIV to KI. The associatedchange in free energy is assumed to provide muchof the driving force for hydrolysis. This implies anE$P affinity, which may give rise to productinhibition at high product concentrations. In E$Pcomplexes of some glycoside hydrolases, glucose/xylose units in subsites KI and KII form slightlyshorter hydrogen bonds and seem to be strongerbound than in the E$S (Michaelis) complex or in acovalent intermediate. Sometimes, additionalhydrogen bonds are observed as in the case ofCel5A from B. agaradhaerens22 or the b-1,4-xylanasefrom B. circulans.23 The binding pattern in distantsubsites of E$S and E$P complexes is nearlyidentical22 as well as in an E$P complex and thecorrespondent covalent intermediate.24

Molecular basis of substrate specificity

The substrate binding site of polysaccharidedegrading enzymes includes a number of subsitesthat accommodate preferentially saccharyl residueswith a sugar-type specific OH-substitution pattern.The architecture of these subsites further allowsdiscrimination of substrates with different types ofglycosidic linkages that determine the orientation ofneighboring monosaccharyl residues within apolymeric substrate. The active site of glycosylhydrolases has evolved to efficiently performhydrolysis of the scissile glycosidic bond which, inthe case of retaining endo-glucanases, involves thenucleophile, the general acid/base catalyst and anumber of auxiliary residues providing structuraland functional support for productive substratebinding and transition-state stabilization.

Tyr24 and Asn26 from the major loop coveringsubsites KIV to KII participate in ligand binding,as well as His99 from subsite KIII. Substratebinding is further entropically favored by therelease of nine coordinated water molecules andsubsequent binding of the glucosyl residues insubsites KII and KI, supported by hydrophobicstacking interactions with Phe92 and Tyr94 (sub-sites KI and KII, respectively). In addition, Trp192from the a-helical segment in subsite CI maypromote productive binding and hydrolysis bymeans of hydrophobic stacking interactions.Tryptophan residues in subsiteCI are characteristic


for cellulases of families 5, 7 and 12 and assumed toplay an important role in catalysis by imposingconformational strain (ring distortion) in theglucosyl residue in subsite KI22,25,26 which mayprovide a substantial amount of activation energyfor hydrolysis. The hydrogen bond network invol-ving Tyr94, Glu63, Asn182, Glu105, Glu109, Asn121and Glu132 in subsites KII, KI and CI is furtherconsidered to contribute to productive bindingand transition-state stabilization. In subsites KIIand KI, the number of hydrogen bonds reaches amaximum (Table 3), consistent with the need forhighly specific interactions close to the site ofcatalysis.

In b-glucans two neighboring glucopyranosylunits linked by a b-1,3 bond have the orientationwith the 6-CH2-OH side-chains on the same side ofthe glucopyranosyl rings, whereas a b-1,4 linkagecauses the monosaccharide units to alternate withrespect to the positioning of the 6-CH2-OH side-chains.18 In the complex, binding of a laminari-biosyl unit in subsites KII/KI is accomplished bystrong hydrogen bond interactions of Asn26 andGlu63 with the O6 hydroxyl of Glc-II, as well asTrp184 Ne1 and Gln109 with the O6 hydroxyl of Glc-I in the catalytic subsite, which strongly favorsbinding of b-1,3 linked glucopyranose units over acellobiosyl residue in subsites KII/KI. Theneighboring subsites KIII and CI accommodateglucopyranose rings with b-1,4 linkage to theglycosyl residues in subsites KII and KI, respect-ively, thereby promoting substrate binding withalternating orientation of the 6-CH2-OH side-chains. In subsite KIII, strong hydrogen bondsbetween the side-chain guanidinium group ofArg65 and both O2 and O3 hydroxyls of Glc-IIIcontribute to substrate specificity, whereas theactive site architecture and the geometry of subsitesKI and CI strongly favors selective cleavage of thescissile b-1,4 glycosidic bond. Based on the previousenzyme–substrate model,14 Asn121 and Glu131were proposed as potential interaction partnersfor the O6 hydroxyl group of the glucosyl residuebound in subsite CI. Interestingly, both candidateresidues coordinate to a firmly bound watermolecule in the E$P complex, which additionallycontacts Tyr123 Oh. This water molecule maytherefore mimic the position of the O6 hydroxylgroup of the substrate glucopyranosyl residue insubsite CI. Subsites KIV and CII are less dis-criminative with respect to sugar conformation andappear to accommodate both linkage-types.

The reported structure of the E$P complex andmutational analysis show that the glucopyranoserings in subsitesKIII toKI form a number of directhydrogen bonds to the protein with their O2, O3 andO6 hydroxyl groups, whereas the single X–H/pinteraction between Tyr24 and the O2 hydroxyl ofGlc-IV may allow binding in different confor-mations and a more variable positioning of theglucosyl unit. The all b-1,3 polysaccharide lami-narin with all sugar rings in the same orientationadopts a bent conformation in contrast to the

extended form of b-1,4 linked glucans. As a result,laminarin would miss interactions in subsites KIVandCII, and partiallyKIII andCI, and is thereforeprocessed only slowly as shown by kinetic analysisresulting in a 7!104-fold reduction of kcat/Km

(Table 4). Cellooligosaccharides are not cleaved bylichenases, because productive binding in subsitesKII to KIV is strongly disfavored by the b-1,4linkage, which orients the glucopyranosyl residuesin regular alternation with the 6-CH2-OH side-chains at approximately 1808.

Hydrophobic interactions

The glycosyl residues in subsites KI to KIV arenot only involved in numerous hydrogen bondinginteractions but also in additionally importanthydrophobic stacking interactions (Figures 2 and3). However, the aromatic side-chains of Tyr24(subsite KIV), Tyr94 (KII) and Phe92 (KI) areslightly off-positioned relative to the interactingsugar rings. The functional implications of thisbinding feature may reside in the precisely tunedaffinity modulation of a polymeric substrate cove-ring multiple subsites. This is of particular import-ance for enzymes following a multiple attackmechanism, by which sequential cleavage ofglycosyl bonds within a polysaccharide chain isachieved by sliding the reaction product to presentthe next scissile glycosidic bond without productdissociation from the enzyme. As outlined forCBH-I, this can be explained by a model in whichthe residues mediating hydrophobic stacking inter-actions are slightly off-positioned relative to theenergy minimum of the hydrogen bonding pat-tern,19 implying that aromatic residues smoothenthe energy profile of individual subsites and favorefficient substrate movement in the binding cleft.27

Although there is no clear experimental evidencefor a multiple attack mechanism in family-161,3-1,4-b-glucanases, the offset observed betweenhydrophobic and hydrogen-bonding interactions inthe E$P complex may be an important featureinvolved in product release: Once the glycosidicbond has been hydrolyzed, the resulting oligosac-charide is able to slide inside the binding cleft,resulting in a slightly different positioning relativeto the initial substrate with concomitant modifi-cation of enzyme–product affinity.The conserved aromatic residue at the entrance of

the substrate binding cleft appears to be importantfor substrate binding.28 In the case of F. succinogenes1,3-1,4,-b-glucanase, substitution of Trp203 byphenylalanine leads to an increase of Km (reducedsubstrate affinity) and a larger increase of kcat, thusresulting in an overall improved catalytic efficiency(kcat/Km). The more bulky tryptophan side-chainproviding a larger hydrophobic surface area favorshydrophobic stacking interactions and initial sub-strate binding compared to phenylalanine. How-ever, effective sliding of the substrate upon cleavageis impaired, resulting finally in reduced catalyticefficiency. In H(A16-M), Tyr24 is the only protein


residue in direct contact to Glc-IV (Table 2). Tyr24represents the aromatic residue at that end of thecleft binding the non-reducing end of the substratewhich is functionally conserved in 1,3-1,4,-b-gluca-nases, underlining its importance for initial sub-strate binding. Interestingly, Y24W is one of the rareamino acid substitutions that positively influencesubstrate binding and transition state stabilizationas shown by a threefold reduction in Km anda negative change in activation energy(DDGswK0.47 kcal molK1).16 Comparative anal-ysis assessing the contribution of individual sub-sites to the observed Km effect allows the conclusionthat, upon substitution of Tyr24 by tryptophan, thelatter creates effectively a new subsite termed KVby interacting with a glucosyl unit beyond subsiteKIV at the non-reducing end of the substrate.16

The catalytic machinery

The catalytic machinery of bacterial endo-1,3-1,4-b-glucanases is highly conserved among lichenasesof family 16 and involves two acidic catalyticresidues and various auxiliary residues assistingin productive substrate binding and catalysis. Thecatalytic residues of H(A16-M) are anchored in oneb-strand with the nucleophile Glu105 being firmlypositioned between Trp103 and Asp107 by stronghydrogen bond interactions. In the case of the freeenzyme, the acid/base catalyst Glu109 does notinteract with Asp107 but forms a hydrogen bond toGln119. In the E$P complex, the active-site residuesslightly reorient their side-chains to form a con-tinuous hydrogen bond pattern involving inter-actions between Trp103 Ne1 and Gln105 Oe1, Gln105Ne2 and Asp107 Od1, Asp107 Od2 and Gln109 Ne2,and water-mediated hydrogen bonds betweenAsp107, Gln109 and Glu119 (Figure 5). Thisarrangement positions the side-chain functionalgroup of Gln109 nearly parallel with the glucopy-ranosyl ring in subsite KI and allows interactionswith both the glycosidic oxygen of the scissile bondand the O6 hydroxyl. The direct interaction of theacid/base catalyst with the O6 hydroxyl group ofGlc-I appears to be an important feature of catalysis.This hydrogen bond pattern in the catalytic subsiteKI is common to retaining glycosidases of families7, 12 and 16 (clans B/C) with the general acid/basepositioned to protonate the glycosidic oxygen syn tothe endocyclic C1–O5 bond.19,24,25 Of particularimportance for catalysis is the hydrogen bondinteraction between the O2 hydroxyl group of theglucosyl residue in subsite KI (Glc-I) and thenucleophile. This structural feature has beenvisualized in complexes of cellulases from families5, 7 and 10.25,29,30 The energetic contribution of thehydrogen bond with the O2 hydroxyl makes up to10 kcal molK1 and appears to be a commonstructural feature of b-glycosidases.31,32

Assuming the protonation states of the nucleo-phile (Glu105) and the acid/base catalyst (Glu109)in both the free enzyme and the E$P complex to besimilar, the small conformational differences to

the isosteric equivalents Gln105, Gln109 and theintermediate Asp107 (maximal atom shifts of 2.0 A,1.0 A, and 2.7 A, respectively) can be attributed tothe presence of the reaction product and the alteredhydrogen bond network of the side-chain amidegroups with the auxiliary residue Asp107. In fact,significant pKa differences between the amidegroup of Gln105 and the carboxylate of Asp107could be the reason for the longer and thereforeweaker hydrogen bond between Gln105 Ne2 andAsp107 Od1 (2.9 A) compared to the pair Glu105wt

Oe2/Asp107wt Od1 (2.3 A), in which the hydrogenbond is supposed to have low-barrier character.Asp107 not only stabilizes the nucleophile but alsocontributes to carbohydrate binding by forming ahydrogen bond to the b-configured O1 hydroxyl inthe E$P complex (2.9 A). Auxiliary aspartateresidues of both B. macerans and B. licheniformislichenases (Asp105 and Asp136, respectively) makesimilar charge contributions of 3.3–3.7 kcal molK1

to the rate-limiting glycosylation step throughstabilization of the nucleophile, as shown by isostericreplacement of these aspartate residues foruncharged asparagine residues (200–300-folddecrease of kcat).

6,7 This moderate effect does,however, not exclude a much more significant roleof the auxiliary aspartate in the deglycosylation step.Reaction step-dependent conformation changes ofthe auxiliary Asp107 in H(A16-M) are shown inFigure 5.

Trp103 is conserved among lichenases but has noanalogs in other glycosidases of clan B. The lengthof the hydrogen bond to the nucleophile Glu105(Trp103 Ne1-Gln105 Oe1) is very similar in theavailable structures of apo-E, E$I and E$P withdonor–acceptor distances of 2.9 A each (Figure 5).The role of Trp103 may therefore be mainlyrestricted to structural support for the nucleophile.Substitution of Trp103 results in 600-fold reductionof kcat (4.0 kcal molK1) without affecting the pHdependence of the enzymatic reaction.33 Ser174 incellobiohydrolase I (T. reesei) and Asn158 in CelB2(Streptomyces lividans) from clan C may fulfillsimilar functions.

Substrate-assisted pKa modulation andmechanistic implications

In the free enzyme, Asp107 is in tight contact tothe nucleophile Glu105 through a strong hydrogenbond with presumably low-barrier character. Thedistance between Glu105 Oe2 and Asp107 Od1 is2.3 A, which is below the estimated limit of 2.55 Afor low-barrier hydrogen bonds (LBHBs).34 Studieson enzymes with a catalytic residue sharing theproton with a neighboring aspartate in LBHBmanner demonstrate a significant decrease of pKa

with a concomitant acidic shift of the catalyticpH optimum of such a system, in contrast to asimple electrostatic repulsion of two carboxylategroups.35

The enzymatic reaction of retaining b-glucanasescalls for a mechanism by which the pKa of the


acid/base catalyst cycles along the reaction coordi-nate: The same residue acts as a general acid in thefirst step (glycosylation) whereas it is the generalbase in the second (deglycosylation) step. Its pKa

drops from the enzyme–substrate complex to thecovalent glycosyl-enzyme intermediate due toremoval of the negative charge of the nucleophilewhen it becomes covalently bound in the inter-mediate.36 Additional pKa shift assistance is pro-posed for family-16 glucanohydrolases as studiedhere. Upon formation of the glycosyl-enzymeintermediate, the side-chain of Asp107 rotatesabout 808 to form a new hydrogen bond with thedeprotonated acid/base catalyst Glu109, assistingits pKa drop which results from elimination of thenegative charge of the nucleophile. This situationcorresponds to that observed in the X-ray structureof the covalent E$I complex with an epoxyalkylglycoside inhibitor.13 After deglycosylation, Asp107returns to its initial position, leaving Gln109 tointeract with Gln119 and a network of four watermolecules in the area of subsite CI. As to whetherAsp107 and Trp103 play a similar role in deglyco-sylation by means of stabilizing the leavingcarboxylate is unknown, since glycosylation is therate-limiting step in kinetics of lichenases even foractivated substrates.8 Similarly, in the case ofcellobiohydrolase I (CBH-I), Asp214 interacts withboth catalytic carboxylates simultaneously, givingpriority to the nucleophile Glu212 in the unli-ganded form of the enzyme (Glu212–(2.6 A)–Asp214–(3.3 A)–Glu217), but moving closer to thecatalyst Glu217 in the E$S (Michaelis) complex

Figure 5. Mechanistic role of catalytic and auxiliary aminHydrogen bond network between Trp103, Glx105, Asp107, Gapo-E, E$I and E$P denote free enzyme, enzyme–inhibitor comand enzyme–product complex, respectively. Hydrogen bondsgiven in A.

(Gln212–(3.5 A)–Asp214–(2.7 A)–Glu217).19 Thisaltered hydrogen bond pattern may be either aconsequence of the E212Q substitution or anindication of the involvement of Asp214 in pKa

cycling of the catalytic carboxylates. If the latter istrue, the observed Asp214 proton transfer could bethe missing link for lichenases indicating thatbreaking the low-barrier hydrogen bond (LBHB)between the nucleophile Glu105 and Asp107 maystart already in the Michaelis complex. In bothCelB2 (S. lividans) and EGI (Fusarium oxysporum),the auxiliary aspartate residues stabilize the depro-tonated form of the nucleophile. However, in CelB2Asp104 has no contact to the general acid/basecatalyst even after leaving the nucleophile in theglycosyl-enzyme intermediate.24 The EGI analogAsp199 forms hydrogen bonds with both thenucleophile and the acid/base catalyst in the freeenzyme.25 In the covalent intermediate, interactionwith the nucleophile still has LBHB character, whilethe catalyst switches to interaction with thesubstrate already in the Michaelis complex.The role of a tryptophan residue in subsite KI of

b-retaining glycosidases sharing the b-sandwichfold requires special attention. In the complex ofendoglucanase Cel2B from S. lividans with theproduct 2-F-cellotriose, O6 of the fluoro-substituted2-F-glucosyl unit in subsite KI forms hydrogenbonds to both Trp24 Ne1 and Glu203 Oe2, the generalacid/base.24 It was suggested that, in the case ofCel2B, O6 of Glc-I is involved in proton transferalong the hydrogen bond network comprisingTrp24 Ne1, Glc-I O6, Glu203 Oe2 and the glycosidic

o acids in bacterial lichenases along the reaction cycle.lx109 and Gln119 in the different structures of H(A16-M);plex with the covalently bound epoxybutyl cellobioside,are shown as dotted lines with donor–acceptor distances


oxygen, with the O6 hydroxyl group acting ashydrogen acceptor for Trp24 and as hydrogendonor for the acid/base catalyst Glu203, whichfinally protonates the oxygen of the scissile bond.24

A very similar hydrogen bond pattern is observedin the complex of EGI from F. oxysporum with anuncleavable thiooligosaccharide25 as well as in thecomplex of cellobiohydrolase I (CBH-I) fromT. reesei with cellotetraose.19 In the E$P structurepresented here, both Trp184 and Gln109 formhydrogen bonds to the O6 hydroxyl of Glc-Iwhereas the bidentate side-chain functional groupof Gln109 forms weak water-mediated hydrogenbonds to both the endocyclic O5 and the b-con-figured O1 hydroxyl, the latter being equivalent tothe glycosidic oxygen in the substrate. Thishydrogen bond network observed in the E$Pcomplex, therefore, appears to be equally wellsuited for proton transfer to the glycosidic oxygenand stabilization of the transition state, therebyallowing carbohydrate assisted pKa modulation ofthe general acid/base, which implicates afunctional induced fit mechanism for catalysis.

Structural interpretation of mutant enzymekinetics

Kinetic analysis identified residues important forcatalysis and allowed a classification as beingimportant for (i) ground-state substrate bindingand/or (ii) transition-state stabilization. A completemutational analysis of residues that may interactwith the oligosaccharide substrate was performedon B. macerans 1,3-1,4-b-glucanase.16

According to kinetic analysis of B. macerans 1,3-1,4-b-glucanase, residues in subsite KIII mediatethe strongest affinity and contribute most to theground-state substrate binding with the alaninevariants Y24A, N26A and E63A showing a five to20-fold effect in Km. Tyr24 and Asn26 are situated onthe major loop covering subsites KIV to KII and,though flexible, tightly attached to the inner b-sheetthrough the disulfide bond Cys32-Cys61 and thestacking interaction between Phe30 and Trp184.Similar loop motifs can be found in glycosidasesfrom families 11 and 12. The E$S model of H(A16-M) suggested Tyr24 to form a hydrogen bond withthe O3 hydroxyl of Glc-III and to stack with Glc-IV.However, in the E$P complex Tyr24 makes only asingle p-H-bond contact to the O2 hydroxyl of Glc-IV, whereas the conformation of Glc-III positions theO3 hydroxyl at 4.6 A distance from Tyr24 Oh,excluding a direct hydrogen bond interaction. Incontrast to the E$S model, Asn26 and Glu63 have nocarbohydrate interactions in subsite KIII but formhydrogen bonds to the O6 hydroxyl of Glc-II. Thekey residues for transition-state stabilization areGlu63 and Asn182 with DDGs values of 6.5 and5.5 kcal molK1, respectively.16 The function ofAsn182 (subsite KI) is more than just positioningof Glu63 and/or Trp184, since it contributes more totransition state stabilization than Trp184 itself (5.5versus 4.0 kcal molK1),16 demonstrating a key role of

this residue in catalysis. The E$S model predictedAsn182 to form a hydrogen bond with the O6

hydroxyl of the glucosyl unit in subsite KII,whereas in the E$P complex Asn182 contributes tocarbohydrate binding by interacting with the O4

hydroxyl of Glc-I. In addition, Asn182 is involved inan extended hydrogen bond network along Arg65(subsite KIII), Glu63 (subsite KII), Asn182, Trp184(subsite KI), Glc-I O6 and the general acid/baseequivalent Gln109. Interestingly, the destabilizationintroduced by the double mutation E63D/N182Q(7.4 kcal molK1) is larger than the sum of theindividual contributions (3.0 kcal molK1 and3.3 kcal molK1 for E63D and N182Q, respectively),indicating a cooperative effect of both Glu63 andAsn184 in transition-state stabilization through thehydrogen bond interactions in subsitesKII andKI.On the contrary, the effects caused by N182Q andS90A mutations are additive in spite of the fact thatstrong hydrogen bonds between Ser90 Og andAsn182 Od1 are present in all available structuresof apo-E, E$I and E$P (2.6, 2.8 and 2.6 A, respec-tively). Phe92, previously supposed to be too faraway from the substrate to make any productivecontacts, turns out to stack with Glc-I, supportingthe results from kinetic analysis, which assigned anenergetic contribution to transition-state stabili-zation of 4.8 kcal molK1 to this residue.

Kinetic effects observed for a series of enzymevariants with either the natural substrate lichenanor the all b-1,3 linked laminarin provided additionalclues about the structural basis for the catalyticperformance of 1,3-1,4-b-glucanases. Complexdestabilization upon mutation of key residueswith both lichenan and laminarin as substrateswas most prominent in the case of the amino acidsubstitutions N26A, E63D, E63Q, Y123A, andW192A (Table 4), pointing at the importance ofsubsites KII/KI (and partially KIII/CI) for thebinding of a laminaribiosyl unit. In contrast, thesubstitutions Y24A, Y24F, R65A, and H99R hadlittle or no effect on catalysis with laminarin assubstrate but substantially impaired hydrolysis oflichenan, indicating that subsitesKIV/KIII did notcontribute to laminarin binding. This is wellconsistent with the mechanistic model of enzyme–ligand interaction, which implies that a b-1,3 linkedlaminaribiosyl unit is bound in subsites KII/KI,but the polysaccharide laminarin fails to interactwith the enzyme in distant subsites due to its bentconformation. Interestingly, introducing bulkyhydrophobic residues by the substitutions Y24W,A98W (subsite KIII), as well as Y123F led to anincreased catalytic efficiency of laminarin depoly-merization. The reason may reside in the improvedsubstrate binding through hydrophobic stackinginteractions with the tryptophan side-chains insubsites KIV/-III (Y24W, A98W), and removal ofunfavorable interactions involving the phenolichydroxyl group of Y123 in subsite CI.

Substitution of Phe59 in B. licheniformis lichenase,the analog of Phe30 in H(A16-M), by alanine (F59A)results in transition-state destabilization by


3.7 kcal molK1.15 The detrimental effect on catalysisis similar to that of the mutation W184Y inH(A16-M), which causes destabilization by4.0 kcal molK1. Since in this case the stackinginteraction can be maintained, it is a strongindication that the functional role of Trp184 is notonly in providing hydrophobic stacking inter-actions but also hydrogen bond interactions topromote substrate binding and catalysis. Thesignificant stabilizing effect of Trp184 is similar tothat of Glu63, Phe92 and Asn182 and supports theidea of its role as potential hydrogen bond donor. Itis especially noteworthy that any catalytic assist-ance system based on an O6 hydroxyl group wouldhave a pronounced effect in modulating thecatalytic efficiency.

In summary, the crystal structure of (A16-M)E105Q/

E109Q bound to a b-glucan tetrasaccharide, togetherwith kinetic and mutagenesis data, has furnishednew insight into the structural requirements forsubstrate binding and catalysis by providing adetailed view of protein–carbohydrate interactionsin an enzyme–product complex.

Materials and Methods

Recombinant gene construction and protein isolation

Recombinant genes of H(A16-M) containing the singlemutations E105Q and E109Q were constructed with themethod of splicing-by-overlap extension,37 using thehybrid gene H(A16-M)38 in the vector pTZ19R as atemplate and previously described primers.7 The EcoRV-HindIII fragment of the mutated gene H(A16-M)E109Q

was then cloned into the H(A16-M) gene carrying themutation E105Q to obtain the double mutant H(A16–M)E105Q/E109Q. The recombinant protein was expressed inEscherichia coli DH5a cells and purified from cell extractsapplying ion-exchange and size-exclusion chromatog-raphy using HiLoad MonoS and Superdex 75 columns(Amersham Biosciences), respectively. For the finalpurification step, 50 mM sodium phosphate buffer(pH 7.0) and 0.1 mM CaCl2 was used. Protein purityexceeded 98% as estimated from SDS-polyacrylamide gelelectrophoresis.

Synthesis of the b-glucan hexasaccharide

The hexasaccharide Glcb4Glcb4Glcb3Glcb4Glcb4Glc-OMe was prepared by enzymatic transglycosylationcatalyzed by the wild-type 1,3-1,4-b-glucanase fromB. licheniformis. The tetrasaccharide Glcb4Glcb4Glcb3Glcobtained by enzymatic depolymerization of barleyb-glucan2 was transformed into the correspondingb-fluoride and condensed with b-methyl cellobioside inhydroorganic media (acetonitrile/water) following theprocedure reported.39 Alternatively, the same compoundwas obtained in higher yield by using the glycosynthasemethodology: a-cellobiosyl fluoride, a-laminaribiosylfluoride, and b-methyl cellobioside were condensedsequentially by the action of two glycosynthases, E134A1,3-1,4-b-glucanase from B. licheniformis and E197A cellu-lase 7B from Humicola insolens, to yield the targethexasaccharide in 80% overall yield after purification.40

The hexasaccharide substrate used for co-crystallization

was pure as judged by NMR and mass spectrometryanalyses (not shown).

Crystallization and X-ray data collection

H(A16-M)E105Q/E109Q, concentrated to 10 mg mlK1

(0.42 mM), was pre-incubated with the substrate b-glucanhexasaccharide prior to crystallization in a molar ratio of1:10 and mixed with an equal volume of crystallizationbuffer containing 0.01 M zinc sulfate heptahydrate, 0.1 MMes (pH 6.5) and 25% (v/v) PEG monoethyl ether 550.Diffraction-quality crystals were readily obtained within2–3 days by the method of hanging-drop vapor-diffusionat room temperature. Crystals were mounted on a nylonloop and flash-frozen in liquid nitrogen. Data werecollected on a MAR Research Imaging Plate detector atbeamline BW7B of the European Molecular BiologyLaboratory outstation at DESY (Hamburg). Crystals hadP212121 symmetry and diffracted beyond 1.6 A maximalresolution. Data were processed with DENZO, scaled andmerged using the program SCALEPACK.41 The details ofdata collection and refinement statistics are given inTable 1.

Structure analysis and refinement

The structure was solved by molecular replacementwith AMoRe42 using the native-like hybrid enzymeH(A16-M) (PDB accession code: 2AYH) as a searchmodel. The structure was refined with a stepwise increaseof the resolution range and the addition of Ca2C andZn2C and water molecules to the model. The b-glucanhexasaccharide ligand was not considered in this stage ofthe refinement to minimize model bias. Several rounds ofrefinement and model building were done using CNS,43

REFMAC44 and the interactive graphic program O.45 Thedifference electron density calculated with diffractiondata of the enzyme–substrate complex and the refinedphases of the apo-enzyme (apo-E) unambiguouslyrevealed the presence of a tetrasaccharide moietyconsisting of four covalently linked glucosyl units in thesubstrate binding cleft spanning subsites KIV to KI. Theprotein–carbohydrate model was further improved bychecking the jFoKFcj difference density map for boundwater molecules and alternative side-chain confor-mations.

Enzyme kinetics with laminarin

Enzyme-catalyzed hydrolysis of laminarin was per-formed by incubating wild-type and mutant forms ofendo-1,3-1,4-b-D-glucanase from B. macerans (7 nM–70 mM)with laminarin (0.1–10 mg mlK1) in citrate-phosphatebuffer (6.5 mM citric acid, 87 mM NaH2PO4 (pH 7.2)),0.1 mM CaCl2 at 50 8C. Initial rates were obtained bydetermining the net release of reducing sugars in linear1 min intervals. Reactionswere stoppedbyadditionofDNSreagent.46 Data were analyzed in terms of activity asfunction of either time or enzyme concentration. Mutantvariants showing non-linearity were excluded from anal-ysis. Data v0 (sK1) versus [S]0 (mM) were fitted to theMichaelis–Menten model by non-linear regression.

Protein Data Bank accession code

The experimental data and atomic coordinates of therefined model were submitted to the RCSB Protein DataBank and are available under accession code: 1U0A.


Acknowledgements

O.J.G. was partly funded by the Bundesminis-terium fur Bildung und Forschung (BMBF) throughthe Leitprojektverbund Proteinstrukturfabrik, andM.N.P. was supported by a grant from the DeutscherAkademischer Austauschdienst (DAAD). Ongoingsupport of this work by the Fonds der ChemischenIndustrie is gratefully acknowledged.

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Edited by R. Huber

(Received 20 July 2005; received in revised form 30 December 2005; accepted 4 January 2006)Available online 25 January 2006

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