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Originally published In Press as doi:10.1074/jbc.M706835200 on September 28, 2007

J. Biol. Chem., Vol. 282, Issue 49, 35703-35711, December 7, 2007
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Crystal Structure of Cel44A, a Glycoside Hydrolase Family 44 Endoglucanase from Clostridium thermocellum*

Yu Kitago{ddagger}, Shuichi Karita§, Nobuhisa Watanabe{ddagger}1, Masakatsu Kamiya, Tomoyasu Aizawa, Kazuo Sakka§, and Isao Tanaka{ddagger}

From the {ddagger}Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 0600810, Japan, §Major of Sustainable Resource Science, Graduate School of Bioresources, Mie University, Tsu 5148507, Japan, and Faculty of Advanced Life Sciences, Hokkaido University, Sapporo 0600810, Japan

Received for publication, August 16, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The crystal structure of Cel44A, which is one of the enzymatic components of the cellulosome of Clostridium thermocellum, was solved at a resolution of 0.96Å. This enzyme belongs to glycoside hydrolase family (GH family) 44. The structure reveals that Cel44A consists of a TIM-like barrel domain and a β-sandwich domain. The wild-type and the E186Q mutant structures complexed with substrates suggest that two glutamic acid residues, Glu186 and Glu359, are the active residues of the enzyme. Biochemical experiments were performed to confirm this idea. The structural features indicate that GH family 44 belongs to clan GH-A and that the reaction catalyzed by Cel44A is retaining type hydrolysis. The stereochemical course of hydrolysis was confirmed by a 1H NMR experiment using the reduced cellooligosaccharide as a substrate.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Cellulose is the major component of the earth's biomass. Some microorganisms, that can use cellulose as a carbon source produce various cellulases and can efficiently degrade cellulose. Most herbivorous animals and many insects have these types of microorganisms in their digestive systems. Some organisms that have cellulolytic ability were confirmed to produce a huge protein complex called cellulosome, which can efficiently degrade crystalline cellulose (1). In the cellulosome, a noncatalytic protein termed the scaffolding protein is associated with several enzymatic proteins by cohesin-dockerin interaction. The scaffolding protein contains modules called cohesin, and each enzymatic protein has a module named dockerin. In the scaffolding protein and the enzymatic proteins, modules are connected to each other by a flexible linker as observed by electron microscopy (2, 3) and small angle x-ray scattering (4, 5). The enzymatic proteins include cellulose binding modules (CBMs)2 and various cellulases, such as endoglucanases, xylanases, mannanases, cellobiohydrolases, glucosidases, etc. This variety makes it possible to degrade crystalline cellulose into oligo-, di-, and monosaccharides.

CelJ is one of the major enzymatic components of the cellulosome of Clostridium thermocellum strain F1, and its gene was cloned previously (6). It is a multidomain endoglucanase and consists of five modules and a signal peptide. The five components are a cellulose binding module that belongs to CBM family 30, a GH family 9 cellulase described as Cel9D, a GH family 44 cellulase described as Cel44A, a dockerin domain, and a CBM domain (CBM44) (7, 8). The Cel44A domain was cloned, and its cellulase activities were measured (9). The recombinant Cel44A consists of 519 amino acid residues with a molecular mass of 58 kDa, and it can degrade cellooligosaccharide, xylan, lichenan, and various celluloses, such as acid-swollen cellulose, ball-milled cellulose, and carboxymethylcellulose (CMC). In addition, Najmudin et al. reported that Cel44A has xyloglucanase activity (8). This broader substrate recognition indicates that Cel44A plays a central role in the early stages of cellulose degradation. The structural information of Cel44A was expected to explain this broad substrate recognition as well as the detailed reaction mechanisms of GH family 44 enzymes. Here we report the crystal structure of recombinant Cel44A and two mutant structures in complex with two kinds of substrates. Combined with biochemical experiments, these structures revealed the detailed reaction mechanisms of this enzyme.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Protein Expression and Purification—The Cel44A gene was amplified by PCR using the primers listed in Table 1 and inserted into the vector pQE-30 (Qiagen). The cloned Cel44A vector was transformed into Escherichia coli JM109 by electroporation. E. coli cells were grown in LB medium at 37 °C with 100 µg/ml ampicillin to midexponential phase (A600 = 0.6) and for an additional 3 h after adding 1 mM isopropyl 1-thio-β-D-galactopyranoside to induce expression. Expressed protein was purified with HiTrap Chelating HP 5 ml (Amersham Biosciences), treated with thrombin (Sigma) to remove the His tag, and passed again through HiTrap Chelating HP 5 ml, and the flow-through fractions were collected. Finally, it was purified with HiLoad 26/60 Superdex 200 pg (Amersham Biosciences) and concentrated to A280 = 16.6 in 20 mM Tris-HCl buffer (pH 8.0). Vectors carrying the E186Q and E359Q mutants were cloned and amplified by PCR using the primers listed in Table 1 designed against wild-type Cel44A vector. The PCR products were treated with DpnI (New England BioLabs) to degrade template plasmids. The expression and purification procedures of these mutants were the same as described for the wild type.


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  		TABLE 1
Primers used for cloning and mutagenesis of Cel44A

 
Enzyme Assay—Cellulase activity of Cel44A mutants was measured by incubation of 5.7, 5.8, and 5.7 µg of protein at 60 °C in 50 mM sodium phosphate buffer (pH 6.5) in the presence of CMC (10 mg/ml; Sigma) with 300 µl of the reaction mixture. Released reducing sugars were determined with 3,5-dinitrosalicylic acid reagent (10), using glucose as the standard. After incubation, 900 µl of 3,5-dinitrosalicylic acid reagent was added and heated in a boiling bath for 5 min. The absorbance was measured at 570 nm. One unit of enzyme activity was defined as the amount of enzyme required to release 1 µmol of reducing sugar from CMC in 1 min.

The enzymatic kinetics of Cel44A against cellohexaitol was determined by measuring the rate of release of reducing sugars also using 3,5-dinitrosalicylic acid reagent (10) with glucose as the standard. Cellohexaitol was prepared following the previous report (11). 25 mg of cellohexaose was reduced with 5 mg of sodium borohydride in 1 ml of water at room temperature. After 2 h, the solution was neutralized with Amberlite IR-120H cation exchange resin H+ form (Sigma), and its supernatant was collected. Then the solution was freeze-dried. Assays were carried out at 60 °C in 10 mM potassium sodium phosphate buffer (pH 7.0). The reaction system included 1–6 mM cellohexaitol and 3.26 µg of protein in a 30-µl solution. The reaction velocities were calculated by estimating the release of reducing sugars against six substrate concentrations.

Chronological Examinations of 1H NMR Measurements of the Reaction Product—Cel44A protein was purified without the thrombin treatment and was finally dissolved in 10 mM phosphate buffer (pH 7.4). Then the enzyme and substrate cellohexaitol were freeze-dried and were dissolved in D2O twice. Final concentration of the substrate cellohexaitol was 5 mg/ml in D2O with 10 mM phosphate buffer, pH 7.4. After adding ~1.0 mg of Cel44A enzyme, the 1H NMR spectra were recorded using a Bruker DRX500 spectrometer at 22.5 °C at 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 125, 185, 365, and 665 min after the reaction started. The reference data were measured without enzyme.

Crystallization and Data Collection—As a result of the screening using the Hampton Crystal screen, crystal screen 2, PEG/ion screen, grid screen PEG 6000, and grid screen PEG/LiCl, the initial Cel44A crystals were obtained under the conditions of the Hampton Crystal screen 2 kit 27: 0.1 M MES-NaOH, pH 6.5, 10 mM ZnSO4, 25% (w/v) PEG monomethyl ether 550. The screening experiments were performed by the sitting drop vapor diffusion method with a 100-µl reservoir and 1:1 µl protein/reservoir ratio at 293 K. The initial crystallization conditions were optimized to 0.1 M MES-NaOH, pH 5.8, 6 mM ZnSO4, 20% (w/v) PEG monomethyl ether 550 or 14% (w/v) PEG 3350 for wild-type Cel44A. In addition, the conditions for the E186Q mutant included 0.1 M MES-NaOH, pH 5.8, 10 mM ZnSO4, 16–20% (w/v) PEG monomethyl ether 550. In both cases, the hanging drop vapor diffusion method was used with a 1-ml reservoir and 2:2 µl protein/reservoir ratio at 293 K. Crystals grew to about 0.05 x 0.2 x 0.5 mm and were soaked in cryoprotectant containing 20% glycerol when the diffraction data were collected at 100 K. Some wild-type crystals grown in the presence of 14% (w/v) PEG 3350 grew to 0.1 x 0.4 x 0.7 mm. All crystals belonged to space group P212121, and unit-cell parameters are listed in Table 2. The asymmetric unit contained one Cel44A molecule with a Matthews' coefficient VM of 2.09 Å3 Da–1, which suggests that the estimated solvent content is ~41.0%.


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  		TABLE 2
Data collection and refinement statistics

Values in parentheses are for the highest resolution shell.

 
The diffraction data sets were collected at Photon Factory (Tsukuba, Japan) BL-5A for wild-type Cel44A and E186Q mutants complexed with cellohexaose, SPring-8 (Hyogo, Japan) BL38B2 for wild-type complexed with cellopentaose, and SPring-8 BL41XU for E186Q mutant complexed with cellohexaose. The crystals were soaked in cryoprotectant containing 1 mM cellohexaose and cellopentaose (Seikagaku Kogyo, Tokyo) for 2 h to introduce substrates. In the case of a high concentration of cellohexaose, the crystals were soaked in cryoprotectant containing cellohexaose at 20% saturation. All data sets were processed using the HKL2000 software package (12), and the diffraction data statistics are shown in Table 2.

Phasing, Model Building, and Refinement—The structure of Cel44A was solved by the multiwavelength anomalous dispersion method using the zinc ion as an anomalous scatterer for phase calculation. Zinc ions were included in the crystallization conditions and seemed to bind accidentally to the Cel44A molecule as described. The zinc ion was located using SHELXD (13) after analyzing the substructure structure factors using SHELXC (14), and the initial phase calculation and phase improvement by density modification were performed by SHELXE (15). Initial model building was performed with ARP/wARP (16) using the phase calculated by SHELXE. As a result, 499 of the 519 cloned residues were traced automatically with their side chains. The initial model was extended manually with O (17), and restrained refinement was performed using REF-MAC5 (18, 19) for highest resolution wild type and CNS (20) for the others. The water picking procedures were performed by the combined use of CNS and LAFIRE (21, 22). The refinement statistics are given in Table 2.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Overall Structure—The structure of the wild-type Cel44A was refined to a resolution of 0.96 Å, and the final model is composed of 510 amino acid residues. Three residues at the N terminus and six residues at the C terminus were disordered and no clear electron densities were observed. Three glycerol molecules and two ions, described later, were also identified. The structure shows that Cel44A is composed of two large domains, a β-sandwich domain and a TIM-like barrel domain. The β-sandwich domain consists of residues 7–23 in N-terminal and 418–516 in C-terminal segments. The TIM-like barrel domain is formed by residues 24–417 in the middle part of the enzyme (Fig. 1). The β-sandwich domain consists of 10 strands (β1–β2 and β19–β26) in two twisted β-sheets. The topology of this domain could be easily classified into "the composite domain of GH family 5, 30, 39, 51" in the Structural Classification of Proteins data base (23). The TIM-like barrel domain has 19 {alpha}-helices and 16 β-strands ({alpha}1–{alpha}19 and β3–β18). The core part of the TIM-like barrel domain is the TIM barrel fold ({alpha}/β)8, except that the first {alpha}-helix of a typical ({alpha}/β)8 becomes a short loop in the Cel44A structure. There are two additional inserted regions in the TIM-like barrel domain. The two regions are present between the second β-strand (β4) and second {alpha}-helix ({alpha}5) of ({alpha}/β)8 (Ala45–Pro86) and between the third β-strand (β7) and third {alpha}-helix ({alpha}7) of ({alpha}/β)8 (Gln110–Tyr157), respectively (Fig. 1b). The inserted regions make a small domain that includes an imperfect antiparallel β-sheet of six strands and four small {alpha}-helices. In the small domain, there is one ion located in the loop on the edge of the imperfect β-sheet and tightly ligated by the side chains of Glu54 and Glu153, the main chain carbonyl oxygen of Asp50 and Tyr155, and two water molecules. From its ligand coordination, this ion seems to be a calcium ion. The zinc ion, which was used as the anomalous scatterer at the phasing step, is bound on the surface of the protein as a regular tetragonal coordination with the side chains of Asp35, Glu401, the main chain carbonyl oxygen atom of Ala395, and the side chain of Glu126 of the adjacent molecule. Three glycerol molecules used as a cryoprotectant were also found at the surface of the protein molecule, one of which was located at a pocket-like hollow as described below. There is a planar and relatively strong unassigned electron density at the center of the TIM-like barrel domain. The shape of the electron density looks like a pyranose ring, but it is not clear.


Figure 1
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  		FIGURE 1.
a, two side views of the Cel44A molecule. It consists of a TIM-like barrel domain and a β-sandwich domain. The crystal structure includes a zinc cation, calcium cation, and three glycerol molecules. The glycerol molecules are shown as stick models. b, topology diagram of Cel44A. {alpha}-Helices are shown as arrows, and β-strands are shown as cylinders.

 
Crystal Structures Complexed with Substrates Cellopentaose and Cellohexaose—Cellohexaose and cellopentaose, which were identified as substrates of Cel44A, were introduced into the wild-type crystals using the crystal soaking technique, and these complex structures were solved at 1.75 and 1.6 Å resolution, respectively. In both cases, the electron density maps showed cellotetraose binding (Fig. 2a). Cellotetraose seems to be one of the reaction products. The locations of four pyranose rings of the molecule clearly show the presence of the four subsites –1 to –4 for recognition of pyranose rings at the nonreducing end. These subsites exist at one of the clefts on the surface of the Cel44A molecule, and some hydrogen bonds between Cel44A protein and substrate are expected, as shown in Fig. 2b. The pyranoses located at –1, –3, and –4 subsites interact with Trp392, Tyr71, and Trp64, respectively. Trp64 and Tyr71 face each other diagonally over the substrate at subsite –3, and Asn46 at subsite –1 is located at the opposite side of the substrate with Trp392. Two residues, Glu186 and Glu359, are located at the reducing end of cellotetraose, and these positions strongly suggest that these two are the catalytic residues. The orientation of 1'-OH of the pyranose at subsite –1 is unusual. It is almost perpendicular to the sugar plane, and this pyranose ring seems to be distorted from the relaxed chair form to the boat form (Fig. 2c).

Enzyme Assay of E186Q and E359Q Mutants—In order to confirm the catalytic residues suggested by the structures, E186Q and E359Q mutants were cloned and purified, and enzyme assays for these mutants were performed with the wild type as a reference. The specific activity of purified wild-type Cel44A enzyme was 40 units/mg, and no cellulase activity was detected with both mutants (Fig. 3); these two residues were therefore confirmed to be catalytic residues.

E186Q Mutant Structure Complexed with Cellopentaose and Cellohexaose—The crystal structures of E186Q mutant complexed with cellopentaose and cellohexaose were solved at 2.0 and 1.8 Å resolution, respectively. In the case of cellopentaose complex, four pyranose rings of the substrate were clearly observed at subsites –1 to –4 in the electron density map (Fig. 4a). Although the electron density of the nonreducing end pyranose ring is poorer than those of the other four, its existence could be clearly recognized. This nonreducing end pyranose ring sticks out into the solvent region from the surface of the Cel44A molecule, and there were no specific residues for substrate recognition. In this structure, the pyranose ring located at the –1 subsite is in the relaxed chair form, and one water molecule sits at the β-side of the ring. Unlike the wild type, the 1'-OH of the reducing terminal was clearly observed as a normal β-side conformation. In the case of cellohexaose, cellohexaose itself was also observed in the electron density map as with cellopentaose. Four pyranose rings from the reducing end were located at subsites –1 to –4, and other two pyranose rings from the nonreducing end are also observed in the solvent region (Fig. 4b).


Figure 2
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  		FIGURE 2.
a, the reaction product recognition of the minus subsite observed with Fo – Fc electron density map contoured 2.0{sigma} in the wild-type crystal structure complexed with cellohexaose. b, the substrate-recognizing residues and four hydrogen bonds between the reaction product and the wild-type Cel44A molecule in the crystal structure. c, close up stereoview of the reducing end pyranose ring. The Fo – Fc electron density map is shown as a gray surface contoured 2.0{sigma}. The map is shown in the region of 3.0 Å distance around the substrate in a and c.

 


Figure 3
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  		FIGURE 3.
Enzyme activities of wild type and E186Q and E359Q mutants. The release of reducing sugar from CMC by wild type and mutants was measured and plotted.

 
Interestingly, soaking with a higher concentration of cellohexaose gave different results. In this case, the structure was solved at 2.8 Å resolution, and eight pyranose rings were identified in the obtained electron density map (Fig. 5). The sugar chain stretches over the putative catalytic point, and there were five pyranose rings at the plus side and three at the minus subsites. The appearance of cellooctaose seems to be caused by the superposition of the several cellohexaose binding states. Previously, Sakon et al. (24) identified cellotetraose in the crystal structure despite using cellobiose, and they concluded that it was the result of reverse reaction. In our case, however, the reverse reaction is impossible, because one of the catalytic residues was mutated, and the inactivity of the mutant was confirmed by biochemical assay as described above. The pyranose rings located at the +3, +4, and +5 subsites interact with Trp327 and Trp331. These two residues are located at one side of the substrate, and Trp327 is located between subsites +3 and +4. The direction of the pyranose ring located at the +1 subsite is almost perpendicular to the pyranose ring plane located at the –1 subsite. In order to connect the pyranose rings at –1 and +1 subsites, the pyranose ring located at the –1 subsite cannot be in the relaxed chair form. The cellooctaose model, in which the pyranose ring of the –1 subsite is the boat form, is fit well into the observed electron density map, as shown in Fig. 5.

Determination of the Stereochemical Course of Hydrolysis—To confirm the retention mechanism of hydrolysis, the chronological examinations of 1H NMR measurements of the reaction product was performed for Cel44A. To avoid the overlap of peaks for the reactant and the products, the reduced cellohexaose, cellohexaitol, was used as a substrate. The enzymatic kinetics against cellohexaitol was calculated as km = 4.43 s–1, Kcat = 2362.9 µM from the reaction velocity measurements. Changes of the spectra for the anomeric region during the reaction at 22.5 °C are shown in Fig. 6. The spectra show that the superiority of the amount of β-anomer clearly exists at the early stage of the reaction before the two anomers come to equilibrium.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
Structure Comparison—Glycoside hydrolase simply hydrolyzes the glycosidic bond, and the GH family includes many enzymes because of the diversity of carbohydrates. Sequence-based classification can be correlated with the structural information, and such analysis has provided useful information for the substrate recognition and the enzymatic reaction mechanisms (25). The combination of structural and amino acid sequence-based information regarding the glycoside hydrolases and other carbohydrate-related proteins, such as glycosyl transferases, polysaccharide lyases, carbohydrate esterases, and CBMs is summarized in the carbohydrate-active enzymes (CAZy) data base (26), in which glycoside hydrolases fall into 110 families (GH family) based on their amino acid sequences. Half of the GH families fall into 14 superfamilies termed clans based on their structural similarities, and several features such as reaction mechanisms can be estimated by the classifications.


Figure 4
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  		FIGURE 4.
a, the Fo – Fc electron density map contoured 2.5{sigma} around substrate in the E186Q mutant complexed with cellopentaose. b, the Fo – Fc electron density map contoured at 2.5{sigma} around substrate in the E186Q mutant complexed with cellohexaose. In both panels, the map is shown in the region of 3.0 Å distance around the substrates, and the water molecule that is expected to attack in the second step of the reaction is indicated by a sphere.

 


Figure 5
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  		FIGURE 5.
The Fo – Fc electron density map contoured at 2.2{sigma} around subsite –1 in the E186Q mutant complexed with a high concentration of cellohexaose. The map is shown in the region of 3.0 Å distance around the substrate. Shown is the model of the E186Q mutant structure complexed with a high concentration of cellohexaose.

 
The clan GH-A is the biggest clan superfamily and includes 17 kinds of GH families. GH-A is defined by three features: (i) ({alpha}/β)8 TIM barrel fold; (ii) the positions of two catalytic residues, which are located at the ends of the fourth and seventh β strands of the TIM barrel (27), and (iii) the hydrolysis catalytic mechanism of retention. The clan GH-A is also termed as the 4/7 superfamily because of the positions of catalytic residues. The distance between these two residues is ~5Å.

Cel44A can be classified into clan GH-A because it has all three features described above. The distance between two catalytic residues (Glu186 and Glu359) is ~5.3 Å, and as described below, the retention reaction mechanism of Cel44A was confirmed by 1H NMR. Furthermore, the seven counterparts of eight key residues, which include the catalytic residues and have been identified to be functionally conserved in clan GH-A (24, 28, 29), are also observed in Cel44A: Arg42, Asn185, Glu186, His283, Tyr285, Glu359, and Trp392. A catalytic triad composed of serine-histidine-glutamate seems also to be common to clan GH-A. The catalytic triad plays a role in lowering the pKa of the acid/base residue and is critical for enzymatic activity (30). Cel44A has the counterpart triad, Thr358-His283-Glu186.

Cel44A has a β-sandwich domain termed "the composite domain of GH family 5, 30, 39, 51." All of these four GH families belong to clan GH-A. In these four families, there are 62 crystal structures in CAZy, and six structures shown in Table 3 have both the TIM barrel and the composite domain of GH family 5, 30, 39, 51 (31–35). Despite the low level of amino acid identity of 11–16%, Cel44A has several features in common with these six structures. (i) The core topologies of both the TIM barrel and the β-sandwich correspond well, although there are some differences in the small insertion domains and the detailed structures. (ii) The β-sandwich domain is composed of nine strands from the C terminus and one strand from the N-terminal region. The only exception is GH 30 human acid-β-glucosidase, in which an additional two strands are from the N-terminal region. (iii) The spatial relationships between the TIM barrel domain and β-sandwich domain are quite similar. (iv) The spatial positions of the catalytic residues correspond well. These comparisons support the idea that Cel44A belongs to clan GH-A.


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  		TABLE 3
Six crystal structures compared with Cel44A

Statistic values are the results of the comparison with Cel44A. PDB, protein data bank.

 
Substrate Recognition—The crystal structures of the wild-type and the E186Q mutant complexed with substrates showed that Cel44A has nine subsites from +5 to –4. The subsites and substrate recognition pattern are summarized in Fig. 7. As described under "Results," substrate recognition is achieved by the interactions between aromatic residues and pyranose rings and several hydrogen bonds with hydroxyl groups of pyranose rings. Differences in the affinity of each subsite are expected by the environment in the catalytic cleft. Around the catalytic center, subsites –1, –2, and –3 form hydrogen bonds, and subsites –1, –3, and –4 form aromatic stacking with substrates. In contrast, subsites +1 and +2 have no potential for pyranose ring recognition, and subsites +3, +4, and +5 have only weak interactions by one-side aromatic stacking. These features indicate that the subsites on the minus side have higher affinity for pyranose ring recognition than those on the plus side. Therefore, it is suggested that subsites –1 to –4 seem to catch the chain first when the sugar chain approaches the catalytic cleft. This is consistent with the results of the enzymatic assay for various cellooligosaccharides in which Cel44A showed no enzymatic activity against cellobiose, cellotriose, or cellotetraose (9). This is because these three saccharides would be caught at subsites –1 to –4 with higher affinity, and the reducing end of each sugar chain would not project into subsite +1 over the catalytic center. On the other hand, cellopentaose is cleaved into glucose and cellotetraose with low activity, and cellohexaose is hydrolyzed into cellobiose and cellotetraose. Cellopentaose binds to subsites +1 to –4, and the pyranose ring projecting into subsite +1 is dissociated and released. In the case of cellohexaose, four pyranose rings at the nonreducing end are caught by subsites –1 to –4, and two pyranose rings at the reducing end project into subsites +1 and +2. The state in which one or two pyranose rings project into the plus subsites seems to be relatively unstable, because cellopentaose and cellohexaose seem to bind subsites –1 to –5 and –1 to –6, where –5 and –6 are the extra sites, as observed in the structure of the E186Q mutant complexed with these substrates. The prospective difference in substrate recognition between plus and minus subsites is also reasonable with regard to the reaction mechanism of Cel44A described below. In the reaction mechanism of retention, the reducing side of the reaction products must be released immediately after cleavage, because it should be replaced by a water molecule for nucleophilic attack in the second step of the reaction.


Figure 6
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  		FIGURE 6.
Time course of the 1H NMR spectra of Cel44A enzymatic reaction product using cellohexaitol as a substrate. The spectra were recorded at 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 125, 185, 365, and 665 min after the reaction started. a, typical 1H NMR spectra of the product. The reference spectrum was measured without enzyme. The doublet at {delta} 5.1 ppm corresponds to H1 of {alpha}-anomer, and the doublet at {delta} 4.6 ppm corresponds to H1 of β-anomer. b, the integrated peak intensities of both {alpha}- and β-anomer are plotted against the reaction time.

 


Figure 7
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  		FIGURE 7.
Schematic two-dimensional diagram of substrate recognition mechanisms based on four crystal structures complexed with substrates.

 
The distances between pyranose rings and aromatic rings of Tyr and Trp residues are relatively large (3.5–5.0 Å), and vacancy between substrates and the catalytic cleft can be observed in the crystal structures complexed with substrates. These relatively loose binding mechanisms are likely to make it possible for Cel44A to recognize various types of substrate, since Ahsan et al. (9) confirmed the degradation of each substrate in detail. For example, xylan is different from cellulose in the absence of a 6'-carbon and its hydroxyl group. Cel44A does not strictly recognize 6'-OH, and the degradation of xylan is possible. Also, CMC can be degraded by Cel44A at only the specific positions where the 2'- and 3'-OH are not modified into carboxymethyl groups, because 2'-OH and 3'-OH of the pyranose rings at subsites –1 and –3 are important for the substrate recognition of Cel44A. In the case of degradation of lichenan and glucomannan, only the β-1,4 binding can be cleaved. The inactivity against β-1,3-glucose and β-1,4-mannose main chain are confirmed (8). The inactivity against β-1,4-mannose main chain may be caused by the disagreement between subsites and the 2'-OH conformation of the mannopyranose main chain, since the 2'-OH conformation of pyranose ring at subsite –1 has an important role in forming the boat form conformation as described.

Interestingly, xyloglucanase activity of Cel44A was reported (8). The crystal structures complexed with substrates show that the catalytic cleft of Cel44A has some side clefts and pockets behind the 6'-OH positions (Fig. 8). These side clefts and pockets seem to fit the shapes of various sugar derivatives that have large branches at the 6'-OH of the glucan backbone. Actually, one glycerol molecule binds in one of these pockets, and the hydroxyl group of sugar branch seems to fit into this pocket.


Figure 8
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  		FIGURE 8.
The molecular surface of Cel44A around the substrates. The figure shows two types of substrate obtained from the E186Q mutants complexed with cellohexaose. In addition, three glycerol molecules are also shown as stick models.

 
Reaction Mechanisms—The result of the chronological 1H NMR experiment for the reaction products described above confirmed the reaction mechanism of retention for Cel44A. This result is consistent with several structural features, including the positions of the catalytic residues. Especially, the wild-type Cel44A structures complexed with two kinds of substrates are considered to be strong evidence of the catalytic mechanism of retention, in which pyranose ring distortion at subsite –1 was observed, and the 1'-OH group was clearly pointed to the β-side of the pyranose ring at the reducing end (Fig. 2c). And the water molecule, which is observed at the β-side of the reducing end pyranose ring in the E186Q mutant complexed with two kinds of substrates, overlaps with the position of the glycosidic oxygen between subsite +1 and –1 observed in the crystal structure of the E186Q mutant complexed with a higher concentration of cellohexaose. This water position is consistent with the nucleophilic attack at the second step of the reaction. Furthermore, there is no space for a water molecule at the {alpha}-side of the pyranose at subsite –1 in any of the crystal structures we have solved. The crystal structure of the E186Q mutant complexed with a high concentration of cellohexaose can be considered the snapshot of the instant just before the first step of the double displacement reaction. This structure shows that the sugar chain bends at a point between subsites –1 and +1 by pyranose ring distortion at subsite –1 from the relaxed chair form to the boat form and fits to the catalytic center, which is a shallow pocket-like hollow in the cleft. This distortion causes exposure of the C1 atom of the pyranose ring to the side of nucleophilic residue Glu359 and makes it possible to attack the C1 atom. Glu359 also plays a role in pulling the pyranose ring at subsite –1 by hydrogen bond with 2'-OH. On the other hand, the wild-type crystal structures complexed with substrates can be considered as the snapshot of the situation just after the final water attack. This is supported by the distorted shape and the unusual position of 1'-OH of the pyranose ring at subsite –1. On the basis of these results, the whole mechanism of the reaction can be described as follows. First, the cellulose chain, which sometimes has large sugar side chains, enters the recognition cleft and is held by some aromatic stacking and hydrogen bonds. Next, the bending of the cellulose chain by substrate recognition of the protein induces the distortion of the pyranose ring at subsite –1 with the help of the hydrogen bond between Glu359 and 2'-OH of the pyranose ring at subsite –1. This transformation makes it possible that the nucleophilic attack by Glu359 to the C1 atom of the pyranose ring at subsite –1. Then the cellulose chain is cleaved, and the intermediate is formed by covalent bonding between the C1 atom of the pyranose ring at subsite –1 and Glu359. Then the pyranose ring at subsite –1 returns to the relaxed chair form. After forming the intermediate, the fragment at plus subsites is released easily, because the fragment is not strongly recognized at the plus subsites. After release of the fragment at plus subsites, the water molecule can be located at a suitable position for nucleophilic attack to the C1 atom of the pyranose ring at subsite –1 from the β-side. Then the second step reaction occurs by the water molecule and the covalent bond between Glu359 and the C1 atom of the pyranose ring at subsite –1 is cleaved. Finally, the product at the minus subsite is released.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
We solved the crystal structure of Cel44A and explained its reaction mechanisms in detail. Cel44A has characteristics of GH 5, 30, 39, 51 families with regard to domain configuration, whole topology, and the position of catalytic residues. The catalytic mechanism of retention was confirmed for Cel44A by the 1H NMR experiments. The structural features and the revealed catalytic mechanism of retention suggest that Cel44A belongs to clan GH-A. The structural features correspond well to the broader substrate specificity revealed by the biochemical assay against various substrates (8, 24). Especially, the xyloglucanase activity reported previously (8) is consistent with the side clefts observed in the crystal structure. Both of the wild-type and the mutant structures also support the substrate recognition and the reaction mechanisms in which the distortion of the pyranose ring at subsite –1 plays a role as a trigger of the reaction. The distortion is caused by the hydrogen bond between 2'-OH of the pyranose ring at subsite –1 and nucleophile Glu359, and this is the main reason for β-1,4-glucanase activity.


   FOOTNOTES
 
* This work was supported by a research grant from the National Project on Protein Structural and Functional Analysis from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and structure factors (code 2e4t, 2eo7, 2e0p, 2ej1, 2eqd, and 2eex) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). Back

1 To whom correspondence should be addressed: Faculty of Advanced Life Sciences, Hokkaido University, N10W8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan. E-mail: nobuhisa{at}sci.hokudai.ac.jp.

2 The abbreviations used are: CBM, cellulose binding module; CMC, carboxymethylcellulose; GH, growth hormone; MES, 4-morpholineethanesulfonic acid; PEG, polyethylene glycol. Back


   ACKNOWLEDGMENTS
 
We thank Tomoko Yamasaki for technical support in the enzyme assays, Yoshitaka Umetsu (High Resolution NMR Laboratory, Graduate School of Science, Hokkaido University) for NMR measurements, Dr. Hirotaka Katsuzaki for technical support in the 1H NMR experiments, and Dr. Naoto Isono for technical support in the high pressure liquid chromatography experiments.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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