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Structure, Vol. 12, 623–632, April, 2004, 2004 Elsevier Science Ltd. All rights reserved. DOI 10.1016/j.str.2004.02.020 Parallel Substrate Binding Sites in a ␤-Agarase Suggest a Novel Mode of Action on Double-Helical Agarose

Julie Allouch,1 William Helbert,2 ␤(1,4) linkages as well as the occurrence of 3,6-anhydro- Bernard Henrissat,1 and Mirjam Czjzek1,* L-galactose bridges in the polysaccharide backbone fa- 1 4 Architecture et Fonction des Macromole´ cules vor the formation of twisted ribbons, with alternating C1 1 Biologiques and C4 chair conformations for the D-galactopyranose UMR 6098 and 3,6-anhydro-L-galactose rings, respectively (Arnott Centre de la Recherche Scientifique and et al., 1974; Rochas et al., 1986). The earliest model of Universite´ s Aix-Marseille I and II the agarose gel network involves the aggregation of 31 chemin Joseph Aiguier parallel double-helical structures stabilized by interchain F-13402 Marseille Cedex 20 hydrogen bonds (Arnott et al., 1974). The parallel double- 2 Ve´ ge´ taux Marins et Biomole´ cules helix model is reported to have a 9.5 A˚ axial periodicity, UMR 7139 (CNRS/UPMC/Laboratoires Goe¨ mar) each single chain being a left-handed 3-fold helix with Station Biologique de Roscoff a pitch of 19.0 A˚ . Each strand is translated axially relative Place Georges Teissier, BP 74 to the first one by half this distance (Arnott et al., 1974). 29682 Roscoff Cedex, Bretagne In agarose gels, helix propagation is interrupted by the France occurrence of natural structural discontinuities or de- fects, resulting from the occasional and random replace- ment of 3,6-anhydro-L-galactose by L-galactose-6-sul- Summary fate. Self-association of these polysaccharides into large crystalline clusters is thus prevented, and, instead, Agarose is a gel-forming polysaccharide with an they most probably form three-dimensional networks in ␣-L(1,4)-3,6-anhydro-galactose, ␤-D(1,3)-galactose re- aqueous solutions, consisting of crystalline zones of peat unit, from the cell walls of marine red algae. double helix aggregates separated by amorphous zones ␤-agarase A, from the Gram-negative bacterium containing single-stranded helices and random coil Zobellia galactanivorans, is secreted to the external (Bhattacharjee et al., 1978). medium and degrades agarose with an endo-mecha- Certain marine can utilize agarose as a car- nism. The structure of the inactive mutant ␤-agarase bon source. The enzymatic breakdown of agarose can A-E147S in complex with agaro-octaose has been be performed by two types of agarases, namely those ␤ ␤ solved at 1.7 A˚ resolution. Two oligosaccharide chains able to cleave the -1,4-galactosidic bonds ( -agarases) ␣ are bound to the protein. The first one resides in the and those able to cleave the -1,3-anhydro-L-galactosi- ␣ channel, spanning subsites Ϫ4toϪ1. A dic bonds ( -agarases). While the former are well docu- ␣ ␤ second oligosaccharide , on the opposite mented, -agarases are poorly known. -agarases are side of the protein, was filled with eight sugar units, found in three different families of the glycoside hy- parallel to the active site. The crystal structure of the drolase (GH) classification (http://afmb.cnrs-mrs.fr/CAZY), ␤-agarase A with agaro-octaose provides detailed in- namely in families GH-50, GH-86, and GH-16. Family GH16 formation on agarose recognition in the catalytic site. is one of the largest families and groups together The presence of the second, parallel, binding site sug- of varying substrate specificity such as keratan-sulfate ␤ gests that the might be able to unwind the endogalactosidases, -1,3-glucanases, mixed linkage ␤ double-helical structure of agarose prior to the cata- -1,3(4)-glucanases, xyloglucan endotransferases, and ␬ ␤ lytic cleavage. -carrageenases together with -agarases. Amino acid sequence alignment of the members of this heteroge- neous family reveals the existence of several subfamilies Introduction that coincide with the experimentally determined sub- strate specificity of the enzymes (Allouch et al., 2003). Agarose, an essentially neutral polysaccharide, is the Three-dimensional structures have been solved for major gelling component of agar. It is widely used as several of these subfamilies, namely ␤-1,3-1,4-gluca- a texturing and gelling agent in the food and medical nase (Hahn et al., 1995a, 1995b; Juncosa et al., 1994; industries as well as for numerous chromatographic and Keitel et al., 1993), ␬-carrageenase (Michel et al., 2001), electrophoretic techniques (De Ruiter and Rudolph, and, more recently, ␤-agarase (Allouch et al., 2003). 1997). This hydrocolloid is found in the cell walls of Structural data for the xyloglucan endotransferase sub- various marine red algae (Rhodophyta), where it is laid family is expected to be soon available, since the crystal- out as highly ordered molecules, either associated to lization of this enzyme from Populus tremula x tremu- the cellulose microfibrils or in the microfibril-less inter- loides has been reported recently (Johansson et al., cellular matrix (Kloareg and Quatrano, 1988). Agarose 2003). Despite this apparent wealth of structural data, ␤ is an alternating copolymer of 3-linked -D-galactopyra- no structure of any family GH-16 enzyme in complex ␣ nose and 4-linked 3,6-anhydro- -L-galactopyranose with its native substrate has been reported to date. This (Figure 1). Conformational analyses and X-ray diffraction information is particularly important in understanding the ␣ studies have shown that the succession of (1,3) and diversity of substrate specificity within family GH-16. Two ␤-agarases, ␤-agarase A and ␤-agarase B (here- *Correspondence: [email protected] after referred to as ␤-agaA and ␤-agaB), have been iso- Structure 624

Table 1. Data Reduction and Refinement Statistics ␤-AgaA-E147S/ ␤-AgaA-E147S/ Native Data Statistics Octa-Agarose Dodeca-Agarose

Space group P3221 P21 Beamline ID14-eh1 ID14-eh4 Wavelength (A˚ ) 0.934 0.9793 Resolution (A˚ ) 37.2–1.7 41.5–1.7 Figure 1. Schematic Drawing of the Repeating Disaccharide Unit Total data 148,960 255,194 of Agarose Unique data 34,045 60,409 Redundancya 4.4 (4.5) 4.2 (4.2) The ␤(1,4) bond cleaved during hydrolysis by ␤-agarases is labeled. Completeness (%)a 95.3 (98.4) 98.8 (97.8) I/␴Ia 12.1 (6.8) 7.2 (2.2) R (%)a,b 4.2 (11.0) 7.3 (32.4) lated from the marine bacterium Zobellia galactanivor- sym ans Dsij and have been characterized (Jam et al., Refinement submitted). The catalytic domains of these enzymes Resolution range (A˚ ) 37.2–1.7 41.5–1.7 share 45% sequence identity, and their experimentally N of unique reflections 32,767 60,150 c determined 3D structures are highly similar (Allouch et Rwork (Rfree) 15.5 (18.2) 17.2 (21.3) al., 2003). At the biological level, a notable difference Rmsd bondsd (A˚ ) 0.015 0.025 Њ between the two enzymes is that ␤-agaA is secreted in Rmsd angles ( ) 1.762 2.2 the extracellular medium, while ␤-agaB is most probably Quality of Ramachandran plot attached to the outer cell membrane (Jam et al., submit- % of residues in most 85.5 87.1 ted). In addition, ␤-agarase A contains a 130 residue favored regions supplementary module of unknown function at its C % of residues in additional 14.5 12.9 terminus, separated from the catalytic domain by a 13 allowed regions residue linker peptide (Jam et al., submitted). The bio- % of residues in generously 0.0 0.0 chemical analysis of the reaction products of the two allowed regions catalytic domains has established that both enzymes No. of atoms act by an endo-mechanism. Only subtle differences Protein 2171 4340 have been found between the two enzymes: the catalytic Water 355 726 module of ␤-AgaA preferably degrades longer oligosac- Ion (Ca) 1 2 charides and has an overall slower reaction rate than Agarose1 43 86 ␤-AgaB (Allouch et al., 2003). Family GH-16 ␤-agarases Agarose2 74 180 perform catalysis with mechanism leading to net reten- a Values in parenthesis correspond to the highest resolution shell. b ϭ⌺| Ϫ | ⌺ tion of the anomeric configuration (Potin et al., 1993) via Rsym I Iav / I, where the summation is over all symmetry- a double displacement mechanism as first suggested by equivalent reflections. c Koshland (Koshland, 1953). The two catalytic residues R calculated on 5% of data excluded from refinement. d Rmsd, root-mean-square deviation. involved in this mechanism (acid/base and nucleophile) have been identified in a related family GH-16 member (Juncosa et al., 1994) and correspond to glutamates 147 ride, spanning subsites Ϫ4toϪ1 in the active site chan- and 152, respectively, in ␤-AgaA. nel. A second binding site on the surface of the enzyme, Like all members of family GH-16 with a known 3D parallel to the active site cleft and occupied by an intact structure, the crystal structures of the catalytic domains agaro-octaose, is identified and described. of native ␤-AgaA and ␤-AgaB reveal a jelly-roll fold, formed of two seven-stranded ␤ sheets interconnected Results and Discussion by extended loops. The concave ␤ sheet forms a long active site cleft, surrounded by the extended loops and Overall Structure runs a full 36 A˚ across the surface of the enzymes. Crystals of the Zobellia galactanivorans Dsij ␤-AgaA- This structural feature, together with the biochemical E147S in complex with the natural substrate agaro- analysis of the reaction products indicate that these two octaose, obtained by cocrystallization trials, belong to ␤ -agarases are capable of cleaving randomly a single space group P3221, with cell dimensions a ϭ b ϭ 51.38 A˚ agarose chain. The detailed analysis of the residues in and c ϭ 205.36 A˚ . The structure of the complex was the active site channel, as well as the structural compari- determined at 1.7 A˚ resolution by molecular replacement son with the related enzymes within family GH-16 helped with AMoRe using the native ␤-AgaA structure as the to pinpoint a number of residues most probably forming search model. The coordinates describing one enzyme the substrate binding subsites. However, a description molecule with two oligosaccharide chains per asymmet- of the details of agarose recognition is not possible ric unit were refined to a final R factor and Rfree of 15.5% in absence of an enzyme/substrate complex. Here we and 18.2%. A second crystal form has been obtained report the crystal structure of an inactive ␤-agaA by soaking experiments of mutant ␤-AgaA-E147S in obtained by site-directed replacement of the catalytic presence of agaro-dodecaose. In this case, the asym- nucleophile by a serine (referred to below as ␤-AgaA- metric unit contained two enzyme molecules each in E147S) in complex with long agaro-oligosaccharides. complex with two oligosaccharide chains, leading to

The unbiased averaged electron density map clearly final R and Rfree factors of 17.2% and 21.3%. Further reveals density for four units of an agaro-oligosaccha- crystallographic statistics are reported in Table 1. As Parallel Substrate Binding Sites in ␤-Agarase A 625

with the native structure, the enzyme unit in ␤-AgaA- countered in glycosidases (Davies et al., 1998, 1995). E147S encompasses amino acids 21–288. No major This is attributed to the necessary deformation of the conformational or structural changes are observed upon pyranose ring at the site of cleavage during catalysis. binding of the oligosaccharide. Briefly, the enzyme folds As a consequence, the enzymes have evolved not to as a jelly-roll, which consists almost exclusively of ␤ optimally bind the ground state substrate conformation, sheets and surface loops. Each sheet is formed by seven but to more favorably interact with the transition state. ␤ strands. The molecule has a slightly elongated shape Indeed, distorted sugar rings in the Ϫ1 subsite have with a prominent cleft crossing the concave ␤ sheet on been reported in hen egg white (Hadfield et one side, forming the catalytic channel, typical of endo- al., 1994), a (Sulzenbacher et al., 1996), and a acting glycosidases. This cleft, at least 36 A˚ long, origi- chitobiase (Tews et al., 1996). They have subsequently nates from the twist and bending of the two ␤ sheets, been observed in several other glycoside surrounded by the loop regions as already described (Czjzek et al., 2000; Davies et al., 1998; van Aalten et earlier (Allouch et al., 2003; Hahn et al., 1995b). The al., 2001; Varrot et al., 2002). Furthermore, it is possible overall root-mean-square deviation of the main chain that the relatively low affinity of the ϩ1 binding site, atoms is 0.413 A˚ for 268 residues between the native often observed in glycoside hydrolases, also contributes structure and the structure of the complex. Besides the to the lack of binding of oligosaccharides accross the replacement of E-147 by a serine introduced to obtain point of cleavage. Here, the mutation of the catalytic a catalytically inactive protein, only two residues of the nucleophile E-147 into a serine creates an empty space binding groove undergo conformational changes. First, which enables the preferential binding of the reducing the side chain of R-176 is reorientated to form a hydro- end of the oligosaccharide exclusively in the ␣ configu- gen bond with the hydroxyl group O3 of 3,6-anhydroga- ration. The resulting sugar mimicks the covalent glyco- lactose of the sugar unit in subsite Ϫ2 and O5 and O4 syl-enzyme intermediate found in retaining glycosi- of galactose in subsite Ϫ3. Second, N-177, which is in dases. Structures of trapped covalent glycosyl-enzyme a ␤ strand conformation in the native structure, adopts intermediates have been described for several retaining a310 helix conformation in the complex. In addition, two glycoside hydrolases (Burmeister et al., 1997; Davies et loops differ slightly in their spatial localization (29–40 al., 1998; Ducros et al., 2002; MacKenzie et al., 1998; and 173–182). Due to the relative high resolution of the Notenboom et al., 1998; Numao et al., 2003; Varrot and data on the complex, five residues displaying double Davies, 2003; Vocadlo et al., 2001; White et al., 1996). conformation have been identified: M-36, M-185, S-200, In our case, because of spontaneous mutarotation, the E-54, and R-57. The first three residues are also in dou- reducing end of the agaro-oligosaccharide present in ble conformations in the native structure, unlike the lat- the crystallization medium is a mixture of ␣- and ter two. None of these residues is involved in substrate ␤-anomers. During cocrystallization, the enzyme has binding. On the contrary, seven residues in double con- probably selected the oligosaccharide and the binding formation in the native structure are stabilized in a single mode that fitted best the active site channel, viz., placing conformation in the present, complexed structure. the terminal ␣-anomer of agaro-octaose in subsite Ϫ1. These are Q-41, K-104, K-127, R-204, E-216, E-231, and The trapping of ␣-anomer products in the Ϫ1 subsite of D-258. However, only K-127 is involved in substrate retaining glycosidases mutated at the nucleophile has binding, and this residue forms a hydrogen bond (3.38A˚ ) been reported previously for a mannanase (Jahn et al., to the hydroxyl-O5 of one of the anhydro-3,6-galactose 2003) and a cellulase (Ducros et al., 2003). As a conse- units (unit R; for numbering see Figure 3B). quence, only the subsites on the “glycon” side of the cleavage point are occupied by agaro-octaose. Only the first four sugar rings are visible, the others being Binding of Agaro-Oligosaccharides in the Active disordered outside of the active site. The presence of Site Channel four subsites on the glycon side is in agreement with The Fo-Fc difference density for the two complex crystal the experimental subsite determination reported earlier forms that we have obtained unambiguously showed a (Allouch et al., 2003). Figures 3A and 4A illustrate the tetrasaccharide spanning the active site cleft subsites overall hydrogen bonding network formed between the from Ϫ4toϪ1 (subsite numbering according Davies et active site residues and the substrate molecule, also al. [1997]), and the oligosaccharide could readily be built summarized in Table 2. The D-galactopyranose unit in into the density (Figure 2A). The average B values range the Ϫ1 subsite is stabilized by numerous hydrogen from 12 to 30 A˚ 2 for the different pyranose units. No bonds, involving residues D-149, E-152, D-181, E-254, deformation of the pyranose ring at the Ϫ1 subsite is Q256, and W-138. Of these, E-254 and Q-256 are cer- observed; in fact, the proximal sugar adopts a relaxed tainly involved in the specific recognition of the galac- 4 C1 conformation and adopts the ␣-anomeric configura- tose unit, since they form tight hydrogen bonds to both tion. Most probably, the bound oligosaccharide extends O4 and O6, which are determinant for this type of sugar beyond the binding cleft of the enzyme on the nonreduc- unit (Table 2). The catalytic acid/base residue is at 4.18 A˚ ing end, since cleavage of the octasaccharide by the from the C1 atom of the galactose unit in the Ϫ1 subsite. inactivated enzyme is highly improbable. However, the A well-defined water molecule, positioned between this supplementary sugar units are probably disordered and C1 atom and E-152, at a distance of 3.44 A˚ to C1 and therefore not defined, since they are not stabilized by hydrogen bonded to the acid/base (2.67 A˚ ), is at an ideal any hydrogen bonds and/or contacts with the enzyme. position for the catalytic reaction. Therefore we consider Difficulties in obtaining enzyme/substrate complexes that the galactose in subsite Ϫ1 is correctly positioned spanning the cleavage point have frequently been en- with respect to the catalytic acid/base residue. Structure 626

Figure 2. Experimental Maps Defining the Ligand Molecules Electronic density around the galactopyranose units of the oligosaccharide in the active site channel (A) and on the surface binding site (B). The (Fo-Fc) Fourier-difference maps before refinement are shown in the top, while omit-maps at the end of refinement, calculated using only the enzyme model phases without substrate, contoured at 2.5␴ above the mean density are shown at the bottom.

The 3,6-anhydro-galactopyranose unit in subsite Ϫ2 polar residues will form hydrogen bonds to two adjacent is surrounded by W-73 on one side and F-174 and R-176 pyranose units and therefore form a discriminating pat- on the other side. In particular, R-176 forms a hydrogen tern for the binding of the characteristic-diose subunit bond to the O3 of the anhydro bridge and therefore of oligo-agarose chains (see Table 2). The alignment of is crucial for binding of 3,6-anhydro-galactose in this the sequence of ␤-AgaA with the other family GH-16 subsite. The D-galactopyranose unit in the Ϫ3 subsite ␤-agarases indicates that the residues involved in the is stabilized by stacking interactions with W-73 on one surface binding site observed here are not conserved side and F-179 on the other side. Two further hydrogen (data not shown) in the few other agarases whose se- bonds, involving R-176 and E-144, stabilize the sugar quences are available. This site is probably a feature ring in this subsite. Only two hydrogen bonds are formed specific of ␤-agarase A from Zobellia galactanivorans Dsij. (E-144 and N-71) with the 3,6-anhydro-L-galactopyra- In the vast majority of the documented cases, poly- nose unit in subsite Ϫ4, explaining the higher mobility saccharidases have evolved supplementary substrate of this sugar ring reflected by the higher B value of this binding sites by recruitment of independent carbohy- sugar (30 A˚ 2) with respect to the sugar rings in the other drate binding modules (CBM) (Bourne and Henrissat, subsites (12–16 A˚ 2). 2001; Gill et al., 1999; Henrissat and Davies, 2000). These CBMs are believed to enhance the activity of the en- The Surface Binding Site zymes on insoluble substrates by increasing the effec- The two crystal forms that we have obtained displayed tive enzyme concentration on the polysaccharide sur- well-defined and continuous electron density for oligo- face (Gill et al., 1999). Their removal by proteolysis or saccharides bound to a long surface binding site (Figure genetic manipulation reduces the activity of the enzyme 2B). Seven sugar rings were visible for the crystals in on insoluble polysaccharides but not on soluble sub- space group P3221 and eight in the P21 crystal form. strates (Tomme et al., 1995). By contrast, surface bind- Both complexes permit the unambiguous determination ing sites have been observed so far only in ␣- of the orientation of the bound oligosaccharide. The mean and related enzymes (Kadziola et al., 1994; Robert et B factor values vary from 15 to 33 A˚ 2 for the individual al., 2003; Skov et al., 2002). It has been proposed that sugar units bound to the surface site. The hydrogen these surface sites could help the adsorption of the bonding network and the residues forming this binding enzyme onto granular starch (Kadziola et al., 1998) or the site are depicted in Figures 3B and 4B and listed in guidance of substrate molecules for transglycosylation Table 2. The base of the binding site is formed by two events (Albenne et al., 2004). In the case of the surface tryptophan residues (W-87 and W-277), which are side binding site observed here in ␤-agarase A, the high num- by side, in an almost perpendicular orientation, enabling ber of enzyme/substrate interactions of the bound oligo- stacking interactions with two successive galactose saccharide is unlikely to be an artifact of crystallization units. In addition, a high number of hydrogen bonds but certainly points to a biological role. We hypothesize are formed with numerous asparagine and glutamine that this surface binding site is involved in the adhesion residues that border the oligosaccharide chain on both of the enzyme to its natural substrate, which is insoluble sides (Figures 3B and 4B). Remarkably, each of these in its naturally occurring form of a gel in algal cell walls. Parallel Substrate Binding Sites in ␤-Agarase A 627

Figure 3. Substrate Binding Sites of ␤-Aga- rase A Detailed view of the substrate molecules, highlighting a selection of residues important for substrate binding (A) in the active site cleft of ␤-agarase A and (B) at the surface binding site of ␤-agarase A. Aromatic residues are colored in purple, polar residues in yellow, positivly charged residues in light blue, and the catalytic acid base as well as the mutated nucleophile are colored in red. (C) A global view of the two agaro-oligosaccharide chains bound to the active site channel and the sur- face site of the enzyme. These figures were prepared with Molscript (Kraulis, 1991).

Interestingly, the oligosaccharide chains in the two bind- unwinding of the double-helical structure of agarose by ing sites are parallel to each other and their nonreducing ␤-agaA is reminiscent, but different, from the mode of ends converge to only about 16 A˚ from each other. The action of helicases that use ATP to processively unwind curvature of the chains (Figure 3C) is such that their DNA (Delagoutte and von Hippel, 2002). continuation suggest that they could merge in the paral- lel double-helical structure of agarose (Arnott et al., Structure of Agarose 1974). In order to verify this (Figure 5), we have con- The structural model of double-helical agarose chains structed a model by extending the agarose chains be- has been obtained from low-resolution fiber diffraction yond the enzyme surface and ending up in the parallel data (PDB idcode: 1aga) (Arnott et al., 1974). While af- double-helical structure (PDB code: 1aga). The chains fording a model of ordered junction zones, this approach could be built readily with no steric clash, and with did not allow the description of the disordered regions glycosidic bond torsional angles in the allowed regions separating the ordered double-helical regions and that of the Ramachandran plot of the agarose glycosidic give rise to the three-dimensional network of agarose bonds dihedral angles (Haggett et al., 1997). This gels. Moreover, because of the low resolution of the strongly suggests that the enzyme may not only bind fiber diffraction data, the very existence of double- two parallel agarose chains, but that it may be able to helical regions has been challenged by some authors locally separate the two strands of the parallel double (Guenet et al., 1993; Jimenez-Barbero et al., 1989). The helix of agarose by acting like a wedge. This is in agree- parallel binding sites that we have observed in the agaro- ment with experimental data showing that ␤-agaA is oligosaccharide/agarase complexes strongly support the capable of degrading agarose gels (Jam et al., submit- parallel chain structure of Arnott (Arnott et al., 1974). The ted). A similar mode of action has already been proposed structure of the complexes not only depicts the detailed for a starch binding domain from glucoamylase (Sori- interactions of a ␤-agarase with an agaro-oligosaccha- machi et al., 1997; Southall et al., 1999). The possible ride, but also provides the first 1.7 A˚ resolution structure Structure 628

Figure 4. Schematic Description of the Ligand/Enzyme Interactions Highlighting the Residues Involved and Water Molecules (A) Tetra-agaose in the catalytic active channel spanning subsites Ϫ4toϪ1. (B) Octa-agarose at the surface binding site. The subsites are labeled L to R running from the reducing to the nonreducing end of the oligosaccharide. This figure was prepared with LIGPLOT (Wallace et al., 1995). of an agaro-octaose single chain. In the single chain chain at the cleavage site, it is generally observed that state, the agarose molecule is considerably more ex- polysaccharide binding proteins select a conformation tended and less twisted than in the double-helical struc- of their ligands from a preexisting conformation occurring ture. With the exception of the distortion of the substrate in solution (Imberty, 1997; Kogelberg et al., 2003). The

Table 2. List of Direct Hydrogen Bonds between the Agarose Units and Enzyme Residues Oligo-Agarose in the Catalytic Active Channel Oligo-Agarose at the Surface Binding Site Residue Name Residue Name Agarose Atom and Atom Distance in A˚ Agarose Atom and Atom Distance in A˚

AGL Ϫ4 O2 E-144 O⑀2 2.77 AGL L O5 N-82 N␦2 3.04 O2 N-71 N␦2 3.54 GAL M O4 N-82 N␦2 3.20 O3 N-71 O␦1 3.21 O4 Q-85 O⑀1 3.09 O3 W-73 N 3.30 O4 Q-85 N⑀2 3.70 GAL Ϫ3 O2 E-144 O⑀1 2.65 O5 Q-85 O⑀1 3.11 O2 Y-69 O␩ 3.8 O6 Q-98 N⑀2 2.73 O3 E-144 O⑀1 3.57 AGL N O2 Q-98 N⑀2 3.65 O4 R-176 N␩2 3.07 O3 Q-96 N⑀2 3.59 O5 R-176 N␩2 2.94 O3 Q-85 O⑀1 2.94 AGL Ϫ2 O3 R-176 N␩2 3.54 GAL O O2 D-271 O␦2 2.75 O3 R-176 N␩1 2.98 O2 Q-96 O⑀1 3.12 O4 E-144 O⑀1 3.57 AGL P O3 D-271 O␦2 3.76 GAL Ϫ1 O1 D-149 O␦1 2.73 O4 D-271 O␦2 3.56 O1 D-149 O␦1 3.48 GAL Q O4 N-89 N␦2 3.71 O1 S-147 O␥ 3.58 O6 N-89 N␦2 2.95 O2 H-172 N␦1 3.63 O6 Q-92 N⑀2 3.2 O2 H-172 N⑀2 3.65 AGL R O2 K-127 N␨ 3.58 O4 E-254 O⑀1 2.65 O5 K-127 N␨ 3.40 O4 E-254 O⑀2 3.58 GAL S O3 K-127 N␨ 3.09 O4 Q-256 O⑀1 3.17 O4 K-127 N␨ 2.88 O6 R-199 O 3.39 Parallel Substrate Binding Sites in ␤-Agarase A 629

Table 3. Dihedral Angles around the Glycosidic Linkages of the Agarose Oligomers

Ring Linkage φ (Њ) ⌿ (Њ) G→A(␤-1,4 linkages)

Q-R (surface site) Ϫ47.6 Ϫ109.9 O-P (surface site) Ϫ83.1 Ϫ70.35 M-N (surface site) Ϫ55.2 Ϫ175.8 Ϫ3toϪ2 (catalytic site) Ϫ57.7 Ϫ159.5 In double helix Ϫ122.9 Ϫ113.0

A→G(␣-1,3 linkages) R-S surface site) Ϫ52.5 54.31 P-Q (surface site) Ϫ73.6 131.0 N-O (surface site Ϫ76.9 97.52 L-M (surface site) Ϫ86.5 44.4 Ϫ2toϪ1 (catalytic site) Ϫ73.4 132.6 Ϫ4toϪ3 (catalytic site) Ϫ126.25 140.5 In double helix Ϫ51.9 156.0

A, 3,6-anhydro-L-galactopyranose; G, D-galactopyranose. The atoms chosen for angle calculations were: ring 1 (ring O Ϫ C1 Ϫ

linking O) Ϫ ring 2 (Cn Ϫ Cnϩ1)

containing the mutation was transformed in XL1-Blue cells. Trans- formants were selected on LB ampicillin agar plates. Mutations were verified by automated DNA sequencing (Genome Express, Meylan, France), and the resulting construct was referred to as pET20b/ ␤-agaA-E147S.

Protein Expression and Purification Escherichia coli ORIGAMI (DE3) pLysS cells have been transformed with the resulting plasmid pET20b/␤-agaA-E147S. The mutant ␤-AgaA-E147S was expressed and purified in the same way as the native protein (Allouch et al., 2003; Jam et al., submitted). Cells were grown in M9 medium supplemented with 2% cas-aa with 100 ␮g/ml ampicillin, 15 ␮g/ml tetracyclin, 34 ␮g/ml chloram- Figure 5. Overall View of the Constructed Model of ␤-Agarase A in phenicol, and 15 ␮g/ml kanamycin. ␤-AgaA-E147S was expressed Interaction with an Unwinding Double Helix of Agarose using the pET20b vector (Novagen) as a His-tagged fusion protein in the periplasm of E. coli ORIGAMI (DE3) pLysS strain and purified first by metal affinity chromatography on a column of Chelating Fast structure of the agaro-oligosaccharide/agarase com- Flow Sepharose loaded with NiSO4 using a concentration gradient from 30 to 400 mM imidazole. A second purification step by exclu- plexes described here therefore provides a valid struc- sion chromatography on S100 Sephacryl (Amersham Pharmacia) tural model for isolated agarose chains. The respective using Tris 20 mM (pH 7.5), NaCl 200 mM yielded an electrophoreti- φ and ⌿ dihedral angles around the glycosidic bond cally pure protein. The yield was 1 mg/l of culture medium, and the (for a definition see Haggett et al. [1997]) are scattered purified protein was concentrated using a dialyzing concentrator around values of Ϫ76Њ/130Њ for the ␣-1,3 linkages and equipped with a polyethersulfone membrane (10 kDa cut-off) (Ami- values of Ϫ55Њ/Ϫ130Њ for the ␤-1,4 linkages (Table 3), con) to a final concentration of 3.5 mg/ml. compared to the values of Ϫ51.9Њ/156.0Њ for ␣-1,3 link- ages and Ϫ122.9Њ/Ϫ113 for ␤-1,4 linkages in the double- Agaro-Oligosaccharide Purification helical structure. The values observed in the agaro-oli- Agarose (1% w/v, GIBCO) was melted in 100 ml of boiling deionized Њ gosaccharide/agarase complexes are in complete water. The solution was cooled down to 40 C and maintained at this temperature to prevent gelation of the polysaccharide. Agarose agreement with those obtained by Haggett et al. in a was incubated with 500 ␮l overexpressed ␤-AgaB (1 nmol/ml) at molecular dynamics study of agaro-oligosaccharides 40ЊC for 3 hr. Then, the reaction was stopped by boiling the incuba- using explicit water molecules (Haggett et al., 1997). tion medium for 15 min. After cooling, the hydrolyzate was freeze- dried and stored at 4ЊC. Experimental Procedures The method applied to the purification of calibrated agaro-oligo- saccharides was adapted from Rochas et al. (1986). 1 ml of 1.5% Site-Directed Mutagenesis agaro-oligosaccharide mixture in deionized water, obtained as de- The QuikChange site-directed mutagenesis kit (Stratagene) was scribed above, was injected on a Biogel P2 (Biorad Laboratories) used to introduce a mutation in the agaa gene. Briefly, the pET20b/ column (100 ϫ 5 cm) and was eluted with deionized water at a flow agaa was entirely amplified by the Pfu Turbo DNA polymerase by rate of 35 ml/min. Fractionation was monitored with a Spectra- using two complementary mutated primers 5Ј ACT GAC GAC GAG System RI 150 refractometer. The most homogeneous fractions col- ACT CAG TCA ATT GAT ATT ATG GAA GGC 3Ј and 3Ј GG AAC TGA lected were directly subjected to crystallization assays without CTG CTG CTC TGA GTC AGT TAA CTA TAA TAC C 5Ј in which a further treatment. The degree of polymerization (DP) of oligosaccha- Glu codon (GAA) was replaced by a Ser codon (TCA) at position rides was determined using calibrated neooligoagarose provided 147. The parental plasmid was digested by DnpI, and the vector by Dextra Laboratories (Reading, UK). Structure 630

Crystallization Coˆ te d’Azur (France), Goe¨ mar (St. Malo, France), and the Centre ␤-AgaA-E147S was concentrated to 3.5 mg/ml and stored in 50 mM National de la Recherche Scientifique (CNRS, France). Tris buffer, pH 7.5, with 25 mM NaCl. Crystallization conditions were first investigated using two sparse-matrix sampling kits (Molecular Received: November 11, 2003 Dimensions and Stura Footprint). Two crystal forms of the enzyme- Revised: January 6, 2004 substrate complex have been obtained. The first are issue of cocrys- Accepted: January 6, 2004 tallization trials of ␤-AgaA-E147S with agaro-octaose, purified as Published: April 6, 2004 described above. In this case, the crystallization solution contained 30% PEG 4000, 200 mM ammonium acetate, and 100 mM sodium References acetate, pH 4.6. Crystals were grown by mixing 2 vol of protein solution, equally containing about 4 mM agaro-octaose, with 1 vol Albenne, C., Skov, L.K., Mirza, O., Gajhede, M., Feller, G., D’Amico, of precipitant solution in a hanging-drop vapor diffusion setup at S., Andre, G., Potocki-Veronese, G., Van Der Veen, B.A., Monsan, P.,

20.5ЊC. Crystals grew within 1 week and are in space group P3221, et al . (2004). Molecular basis of the amylose-like polymer formation having unit cell parameters a ϭ b ϭ 51.38 A˚ and c ϭ 205.36 A˚ . catalyzed by Neisseria polysaccharea amylosucrase. J. Biol. Chem., Thereafter, a single crystal was soaked for 30 s in successive solu- 279, 726–734. tions with increasing glycerol concentrations up to a maximum of Allouch, J., Jam, M., Helbert, W., Barbeyron, T., Kloareg, B., Henris- 15%. sat, B., and Czjzek, M. (2003). The three-dimensional structures of The second crystal form was obtained by soaking experiments. two ␤-agarases. J. Biol. Chem. 278, 47171–47180. First, crystals of the mutant protein ␤-AgaA-E147S were grown in Arnott, S., Fulmer, A., Scott, W.E., Dea, I.C., Moorhouse, R., and conditions close to those of the native enzyme (Allouch et al., 2003). Rees, D.A. (1974). The agarose double helix and its function in aga- The crystallization solution contained 24% PEG 4000, 160 mM am- rose gel structure. J. Mol. Biol. 90, 269–284. monium acetate, and 80 mM sodium acetate (pH 4.6) and led to Bhattacharjee, S.S., Yaphe, W., and Hamer, G.K. (1978). 13C-n.m.r. crystals which belonged to space group P21 with unit cell parameters a ϭ 42.2 A˚ ,bϭ 135.0 A˚ ,cϭ 49.8 A˚ , and ␤ϭ101.8 A˚ . These spectroscopic analysis of agar, kappa-carrageenan and iota-carra- single crystals were then transferred to a mother liquor containing geenan. Carbohydr. Res. 60, C1–C3. approximately 10 mM agaro-dodecaose during one night and sub- Bourne, Y., and Henrissat, B. (2001). Glycoside hydrolases and gly- sequently soaked in 10% glycerol solution, prior to flash freezing. cosyltransferases: families and functional modules. Curr. Opin. Struct. Biol. 11, 593–600. Data Collection and Refinement Burmeister, W.P., Cottaz, S., Driguez, H., Iori, R., Palmieri, S., and Diffraction data at 1.7 A˚ resolution were collected on beamlines Henrissat, B. (1997). The crystal structures of Sinapis alba myrosi- ID14-EH1 and EH4 (ESRF, Grenoble, France) for ␤-AgaA-E147S/ nase and a covalent glycosyl-enzyme intermediate provide insights agaro-octaose and ␤-AgaA-E147S/agaro-dodecaose complexes, into the substrate recognition and active-site machinery of an respectively. S-glycosidase. Structure 5, 663–675.

The ␤-AgaA-E147S/agaro-octaose cocrystals (P3221) contained CCP4 (Collaborative Computational Project Number 4) (1994). The one molecule in the asymmetric unit, whereas for ␤-AgaA-E147S/ CCP4 suite: programs for protein crystallography. Acta Crystallogr. agaro-dodecaose (P21), two molecules were present in the asym- D 50, 760–763. metric unit. In both cases, the structure was solved by molecular Czjzek, M., Cicek, M., Zamboni, V., Bevan, D.R., Henrissat, B., and replacement, using AMoRe (CCP4, 1994; Navaza, 1994) and Esen, A. (2000). The mechanism of substrate (aglycone) specificity ␤-AgaA_CM as search models. The respective correlation and R in beta- is revealed by crystal structures of mutant factors of the molecular replacement are 64.9% and 34.2% for one maize beta-glucosidase-DIMBOA, -DIMBOAGlc, and -dhurrin com- solution for ␤-AgaA-E147S/agaro-octaose and 59.2% and 36.7% plexes. Proc. Natl. Acad. Sci. USA 97, 13555–13560. ␤ for two solutions for -AgaA-E147S/agaro-dodecaose. Davies, G.J., Tolley, S.P., Henrissat, B., Hjort, C., and Schulein, M. ␤ ␤ Both -AgaA-E147S/agaro-octaose and -AgaA-E147S/agaro- (1995). Structures of oligosaccharide-bound forms of the endoglu- dodecaose were built manually using TURBO (Roussel and Cambil- canase V from Humicola insolens at 1.9 A˚ resolution. Biochemistry lau, 1991). The refinement was performed with REFMAC 5 (CCP4, 34, 16210–16220. 1994). Water molecules were added using CCP4/wARP (Perrakis et Davies, G.J., Wilson, K.S., and Henrissat, B. (1997). Nomenclature al., 1997). The stereochemistry of the final structures was evaluated for sugar-binding subsites in glycosyl hydrolases. Biochem. J. 321, using PROCHECK (Laskowski et al., 1993). All refinement statistics 557–559. are summarized in Table 1. Davies, G.J., Mackenzie, L., Varrot, A., Dauter, M., Brzozowski, A.M., Schulein, M., and Withers, S.G. (1998). Snapshots along an enzy- ␤ Construction of the Model of the -AgaA in Complex matic reaction coordinate: analysis of a retaining beta-glycoside with Partially Unwound Helical Agarose . Biochemistry 37, 15280–15287. The model of ␤-agaA in complex with partially unwound helical De Ruiter, G.A., and Rudolph, B. (1997). Carrageenan biotechnology. agarose was obtained by simple duplication of the agaro-octaose Trends Food Sci. Technol. 8, 389–395. and agaro-tetraose units, as defined in the structural complex. These duplicated parts were then translated and rotated in a way Delagoutte, E., and von Hippel, P.H. (2002). Helicase mechanisms that they could be connected to the nonreducing ends of the two and the coupling of helicases within macromolecular machines. Part oligosaccharides in the complex structure respectively, with respec- I: structures and properties of isolated helicases. Q. Rev. Biophys. tive φ and ⌿ dihedral angles in the allowed region. These ends 35, 431–478. were then joined to the model of a double-helical agarose unit, the Ducros, V.M., Zechel, D.L., Murshudov, G.N., Gilbert, H.J., Szabo, coordinates of which were accessible through the Protein Data Bank L., Stoll, D., Withers, S.G., and Davies, G.J. (2002). Substrate distor- (accession number 1aga). The model construction was performed tion by a beta-mannanase: snapshots of the Michaelis and covalent- using the program TURBO (Roussel and Cambillau, 1991). intermediate complexes suggest a B(2,5) conformation for the tran- sition state. Angew. Chem. Int. Ed. Engl. 41, 2824–2827. Acknowledgments Ducros, V.M., Tarling, C.A., Zechel, D.L., Brzozowski, A.M., Frand- sen, T.P., von Ossowski, I., Schulein, M., Withers, S.G., and Davies, The authors wish to thank Murielle Jam, Tristan Barbeyron, Bernard G.J. (2003). Anatomy of glycosynthesis: structure and kinetics of the Kloareg, and Gurvan Michel (Station Biologique, Roscoff, France) Humicola insolens Cel7B E197A and E197S glycosynthase mutants. for useful discussions. We would also like to thank Pedro Coutinho Chem. Biol. 10, 619–628. for helpful advice and discussions. We are also indebted to the Gill, J., Rixon, J.E., Bolam, D.N., McQueen-Mason, S., Simpson, ESRF staff on beamlines ID14-EH1 and EH4 for help during data P.J., Williamson, M.P., Hazlewood, G.P., and Gilbert, H.J. (1999). The collections. This work was funded by the Re´ gion Provence-Alpes- type II and X cellulose-binding domains of Pseudomonas xylanase A Parallel Substrate Binding Sites in ␤-Agarase A 631

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Accession Numbers

Coordinates and observed structure factor amplitudes have been deposited in the Protein Data Bank (accession code 1URX).