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ISSN: 2155-9821
Journal of Bioprocessing & Biotechniques

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Structure and Catalytic Mechanism of a Glycoside Hydrolase Family-127 β-L-Arabinofuranosidase (HypBA1)

Chun-Hsiang Huang1, Zhen Zhu1, Ya-Shan Cheng2,3, Hsiu-Chien Chan1, Tzu-Ping Ko4, Chun-Chi Chen1, Iren Wang4, Meng-RuHo4, Shang-Te Danny Hsu4,6, Yi-Fang Zeng7, Yu-Ning Huang7, Je-Ruei Liu5,7,8*and Rey-Ting Guo1*
1Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
2Genozyme Biotechnology Inc., Taipei 106, Taiwan
3AsiaPac Biotechnology Co., Ltd., Dongguan 523808, China
4Institute of Biological Chemistry, Taiwan
5Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan
6Institute of Biochemical Sciences, Taiwan
7Institute of Biotechnology, Taiwan
8Department of Animal Science and Technology, National Taiwan University, Taipei 106, Taiwan
Corresponding Authors : Rey-Ting Guo
Industrial Enzymes National Engineering Laboratory
Tianjin Institute of Industrial Biotechnology
Chinese Academy of Sciences, Tianjin 300308, China
Tel: 86-022-84861999
Fax :86 - 022-24828701
E-mail: [email protected]
  Je-Ruei Liu
Department of Animal Science and Technology
National Taiwan University
Taipei 106, Taiwan
Tel: 886-2-3366-6011
Fax: 886-2-3366-6001
E-mail: [email protected]
Received June 28, 2014; Accepted July 23, 2014; Published August 03, 2014
Citation: Huang CH, Zhu Z, Cheng YS, Chan HC, Ko TP, et al. (2014) Structure and Catalytic Mechanism of a Glycoside Hydrolase Family-127 ß-LArabinofuranosidase (HypBA1). J Bioprocess Biotech 4:171 doi: 10.4172/2155-9821.1000171
Copyright: ©2014 Huang CH, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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The β-L-arabinofuranosidase from Bifidobacterium longum JCM 1217 (HypBA1), a DUF1680 family member, was recently characterized and classified to the glycoside hydrolase family 127 (GH127) by CAZy. The HypBA1 exerts exo-glycosidase activity to hydrolyze β-1,2-linked arabinofuranose disaccharides from non-reducing end into individual L-arabinoses. In this study, the crystal structures of HypBA1 and its complex with L-arabinose and Zn2+ ion were determined at 2.23-2.78 Å resolution. HypBA1 consists of three domains, denoted N-, S- and C-domain. The N-domain (residues 1-5 and 434-538) and C-domain (residues 539-658) adopt β-jellyroll architectures, and the S-domain (residues 6-433) adopts an (α/α)6-barrel fold. HypBA1 utilizes the S- and C-domain to form a functional dimer. The complex structure suggests that the catalytic core lies in the S-domain where Cys417 and Glu322 serve as nucleophile and general acid/base, respectively, to cleave the glycosidic bonds via a retaining mechanism. The enzyme contains a restricted carbohydrate-binding cleft, which accommodates shorter arabino oligosaccharides exclusively. In addition to the complex crystal structures, we have one more interesting crystal which contains the apo HypBA1 structure without Zn2+ ion. In this structure, the Cys417-containing loop is shifted away due to the disappearance of all coordinate bonds in the absence of Zn2+ ion. Cys417 is thus diverted from the attack position, and probably is also protonated, disabling its role as the nucleophile. Therefore, Zn2+ ion is indeed involved in the catalytic reaction through maintaining the proper configuration of active site. Thus the unique catalytic mechanism of GH127 enzymes is now well elucidated.

Glycoside hydrolase; Arabinofuranose; β-LArabinofuranosidase; Crystal structure; Synchrotron radiation
Araf : Arabinofuranose; BSA: Bovine Serum Albumin; DUF: Domain of Unknown Function; EDTA: Ethylenediaminetetraacetic Acid; GH: Glycoside Hydrolase; HEPES: (4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid); HRGPs: Hydroxyproline-Rich Glycoproteins; Hyp: Hydroxyproline; MIRAS: Multiple Isomorphous Replacement with Anomalous Scattering; MR: Molecular Replacement; RMSD: Root-Mean-Square Deviations; SEC/MALS: Size Exclusion Chromatography-Multi-Angle Light Scattering; SLS: Static Light Scattering
Arabinofuranose (Araf) is broadly distributed in nature as an important component of glycoconjugates, most existing as α-L-forms. However, certain β-L-arabinofuranosyl linked residues are also found in bacteria and plants, including mycobacterial cell wall arabinans, the hydroxyproline (Hyp)-rich glycoproteins (HRGPs), glycopeptide hormones and biopolymers [1-9]. In spite of the abundance of β-LAraf- containing sugars (L-Araf) in bacterial and plant cells, degradation and metabolism of these polysaccharides remain largely undiscovered due to the lack of knowledge of corresponding degradative enzymes.
In the previous studies, the β-L-arabinofuranosidase (HypBA1, belonging to GH127 family) and β-L-arabinobiosidase (HypBA2, belonging to GH121 family) from Bifidobacterium longum JCM 1217 that participate in the β-L-arabinooligosaccharides metabolisms have been characterized [10,11]. In the hydrolysis of Hyp-linked β-L-arabinooligosaccharides, HypBA2 releases the β-1,2-linked Araf disaccharide (β-Ara2) from Araf-β-1,2-Araf-β1,2-Arafβ-Hyp (Ara3- Hyp). HypBA1 subsequently liberates L-arabinoses by hydrolyzing β-Ara2 [10,11]. In addition, Ara-, Ara2- and Ara3-Hyp as well as Ara2- and Ara-Me (methanol) are also preferred substrates for HypBA1, but Ara4-Hyp, extensin, potato lectin, pNP-arabino-, galacto-, glucoand xylo-pyranosides are not. Based on the substrate specificity and catalytic products, HypBA1 was proposed to hydrolyze β-Larabinooligosaccharides in an exo-acting manner [11]. Moreover, Fujita mutated three glutamate residues which are strictly conserved among the HypBA1 homologous (Glutamates are commonly observed catalytic important residues among all GH families) and proposed that Glu322, Glu338 and Glu366 might be potential catalytic residues [11].
Very recently, the crystal structures of the ligand-free and L-Araf complex forms of HypBA1 (PDB ID: 3WKW and 3WKX) were determined [12]. Based on the structural analyses, biochemical experiments and quantum mechanical calculations, Ito showed that the nucleophile function is likely served by Cys417 rather than a glutamate [12]. In the meantime, we have determined the crystal structures of HypBA1 in native form, in apo form and in complex with its product L-Araf. Here, by analyzing these structures, the relationship between the glutamate residues, the catalytic cysteine, the Zn2+ ion, and the substrate in HypBA1 is elucidated, and implications on the retaining mechanism of GH127-family enzymes are discussed.
Materials and Methods
Protein expression, purification, crystallization and data collection
The expression and purification methods employed for the protein have been described before [13]. To obtain phase information by multiple isomorphous replacement (MIR), the apo HypBA1 crystals grown in 0.4 M ammonium acetate and 18% w/v polyethylene glycol 3350 [13] were used for preparing heavy atom derived crystals by using the Heavy Atom Screen Hg kit (Hampton Research). The apo crystals (isomorphous to native crystal) were soaked with various mercurycontaining reagents (final concentration 2 mM) in cryoprotectant solution (0.5 M ammonium acetate, 25% w/v polyethylene glycol 3350 and 5% w/v glycerol) for at least 1 hr.
To remove any metal ion from protein, the purified enzyme (both Se-Met protein and native protein are used here) was dialyzed against 100 mM EDTA for two times (at least 12 hrs for each time) before crystallization. The crystals of Se-Met protein without Zn2+ ion (apo crystal) diffracted X-rays better than the native crystals. The HypBA1- Araf complex crystal was obtained by soaking the native crystal switch a cryoprotectant solution containing 50 mM Araf.
The X-ray diffraction datasets were collected at the beam line BL13B1 and BL13C1 of the National Synchrotron Radiation Research Center (NSRRC, Taiwan). The data was processed using the program of HKL2000 [14]. Prior to structural refinements, 5% randomly selected reflections were set aside for calculating Rfree as a monitor [15].
Size exclusion chromatography-multi-angle light scattering (SEC/MALS)
Absolute molecular weight of the purified protein was determined by static light scattering (SLS) using a Wyatt Dawn Heleos II multiangle light scattering detector (Wyatt Technology) coupled to an AKTA Purifier UPC10 FPLC protein purification system using a Superdex 200 10/300 GL size-exclusion column (GE Healthcare). HypBA1 protein with 0.75, 1.4, 1.6, 10, and 20 mg/ml concentration were applied to the size-exclusion column with a buffer containing 20 mM HEPES (pH 7.0) and 0.02% NaN3 by a flow rate of 0.5 ml/min. A 1.8 mg/ml concentration of BSA was used for the system calibration as a control. The absolute molecular weights of individual peaks in the size-exclusion chromatogram were determined by the SLS data in conjunction with the refractive index measurements (Wyatt Optilab rEX, connected downstream of the LS detector). A standard value of refractive index, dn/dc=0.185 ml/g, was used for proteins and the buffer viscosity h=1.0164 cP at 25°C was calculated using SEDNTERP. The value of reference refractive index, 1.3441 RIU, was taken directly from the measurement of the Wyatt Optilab rEX when buffer only passing through the reference cell.
Sedimentation velocity
Sedimentation velocity was performed using an XL-A analytical ultracentrifuge (Beckman Coulter, Fullerton, CA) using absorption optics. HypBA1 and control reference buffer (approximately 400 μL) were added to 12 mm thick epon double-sector centerpieces in an AN60-Ti rotor and spun at 20°C and 45,000 rpm after an initial 90- min temperature equilibration period. Detection of concentrations as a function of radial position and time was performed by optical density measurements at wavelength of 280 nm and absorbance profiles were recorded every 3 min. The protein samples were in the buffer containing 20 mM HEPES, pH 7 with a concentration of ca. 6 μM. The buffer density and viscosity were calculated by SEDNTERP [16] and the sorted data were analyzed with the standard c(s) model [17] in SEDFIT version 14.3. The plot of analytical ultracentrifugation concentration profiles and their fits were made with the software GUSSI, provided by Dr. Chad Brautigam in Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, USA).
Structural determination and refinement
The HypBA1 structure was solved by MIR with anomalous scattering (MIRAS) using one native and two mercury datasets [(C2H5HgO)HPO2 and C(HgOOCCH3)4]. In our previous studies, mercury can bind to free Cys residues very easily and the phase problem can be easily solved by using at least two mercury datasets by MIR [18-21]. The mercury atom binding sites were located using AutoSol wizard of PHENIX using 2.4 Å resolution cutoff [22]. Two mercury atom binding sites were identified and following density modification for phasing improvement was performed (figure of merit = 0.33). Automatic initial model building was carried out by AutoBuild of PHENIX. A 94% completeness model (620 residues with side chains) was built. Subsequent model building and structural refinement were carried out by using the programs COOT [23] and CNS [24].
The apo (treated with EDTA; no Zn2+ ion observed) and productcomplex (HypBA1-L-Araf) structures were determined by using the molecular replacement (MR) method with PHASER [25] using refined native structure as a search model. The 2Fo-Fc difference Fourier map showed clear electron densities for most amino acid residues. Subsequent refinements by incorporating ligands and water molecules were according to 1.0 σ map level. The refined structures were validated by using RAMPAGE [26]. Data collection and refinement statistics of these crystals are summarized in Table 1 and Supplementary Table S1. All Figures were prepared by using PyMOL [27].
X-ray fluorescence scan analysis
To investigate the metal ion presented in the HypBA1 crystals, fluorescence scan analysis of the protein crystal was performed at beam line BL13B1 of the National Synchrotron Radiation Research Center (NSRRC, Taiwan). The wavelength from 12 eV to 25588 eV was used to scan the crystal to see if there is any metal ion existed.
Results and Discussion
The overall structure
In this study, the crystal structures of native (with a Zn2+ ion) and apo (no Zn2+ ion observed) and in complex with L-arabinose (HypBA1-L-Araf) were determined. HypBA1 folds into three domains, designated N-domain (N-terminal domain), S-domain (all α-domain; substrate-binding domain) and C-domain (C-terminal domain) (Figure 1A). Both adopting β-jellyroll folds, the N- (residues 1-5 and 434-538) and C-domain (residues 539-658) consist of 10 β-strands, and 7 β-strands and 3 small α-helices, respectively. The S-domain (residues 6-433) comprises an (α/α)6-barrel and accommodates an L-Arafbinding cavity, so it is considered as the catalytic domain (Figure 1A).
In order to assess the unique structural feature of the N- and C-domains, a structural homology searches with DALI [28] were carried out (Figure S1). It reveals that N-domain (colored in green, broken square) of HypBA1 displays structural similarity to C-terminal domains of xyloglucanase (GH44, colored in cyan), human α-galactosidase (GH27, colored in magenta), β-xylosidase (GH39, colored in yellow) and α-L-rhamnosidase (GH78, colored in orange) with RMSD of 3.0 Å (512 Cα atoms with 14% sequence identity), 3.5 Å (390 Cα atoms with 10% sequence identity), 2.6 Å (501 Cα atoms with 10% sequence identity) and 2.8 Å (1029 Cα atoms with 9% sequence identity), respectively [29-32]. On the other hand, C-domain (colored in green, broken square) of HypBA1 shows structural similarity to N-terminal domain of ErbB4 kinase (colored in cyan) and LKB1 (colored in salmon) with RMSD of 2.6 Å (274 Cα atoms with 4% sequence identity) and 3.5 Å (311 Cα atoms with 15% sequence identity), respectively [33,34] (SI Figure S1). However, their functional roles remain to be investigated. A possibility is that these domains might associate with substrate to facilitate catalytic reaction, as a carbohydrate-binding motif does.
In order to investigate whether the L-Araf (product) could cause conformational change, the native, apo and complex structures were superimposed and no obvious conformational change was observed (data not shown). However, a few regions which are close to the L-Araf binding cavity were missing in both the native and apo structures (residues 31-50 and 247-250 for native, residues 35-50 and 413-415 for apo), but all these loop regions can be clearly seen in the complex structure. Interestingly, the longest loop consisting of residue 31-50 was found stretching across the surface of the S-domain to cover, or cap, the ligand-binding cavity (Figure 1B). From the complex structure, the capping loop undergoes induced fit and is then stabilized through interacting with the L-Araf. Accordingly, the capping loop might play an important role in the catalytic reaction by regulating the substrate binding because one of the ligand binding residues, Gln45, is located on the capping loop and a similar case has been reported [35].
On the other hand, although not supplemented in the crystallization solution, a Zn2+ ion was observed adjacent to the active site as a strong electron density. It was further validated by using X-ray fluorescence scan analysis and anomalous difference Fourier map (Figure 2A). Based on the |Fo| - |Fc| omit map, it is clear that the Zn2+ ion forms a typical tetrahedral complex with four Zn2+ ion-chelating residues, including Glu338, Cys340, Cys417 and Cys418 and all the metal-to-ligand distances are about 2.3 Å (Figure 2A). Interestingly, even though no significant conformational change was caused by Zn2+ binding, the two Zn2+ ion-chelating residues Cys417 and Cys418 in the apo structure were shifted away from the original positions (Figure 2B). Therefore, when the Zn2+ ion is absent only a broken loop can be observed because of the absence of stabilizing interactions (Figure 2C).
Interestingly, although there is only one HypBA1 monomer in an asymmetric unit, HypBA1 forms a dimer with a crystallographic symmetry-related molecule (Figure 3A and 3B). An analysis with PDBePISA shows that the contact interface encompasses 77 residues that bury a total surface area of about 2781 Å2 on the S- and C-domain [36]. The intermolecular forces include hydrogen bonds (not shown) and salt bridges (Figure 3C). To confirm that HypBA1 also forms a dimer in solution, size exclusion chromatography coupled with multiangle light scattering (SEC/MALS) was conducted. SEC/MALS offers an estimate of the absolute molecular weights in solution based on the angular dependence of scattered light intensity, which is less dependent on the molecular shapes. At protein concentrations of 0.75-1.60 mg/ ml, the SEC/MALS analysis using a Superdex 200 10/300 GL column indicates that its molecular mass is 134.9-138.8 kDa, corresponding to a dimeric form of HypBA1 (Figure 4A). As the elution peak of the SEC/MALS is relatively symmetric, the calculated molecular weight distribution indicates that the sample is monodispersed. Accordingly, our SEC/MALS data suggests that the HypBA1 protein exists as a very stable dimer in solution under low protein concentrations or at increased protein concentrations of 10 mg/ml or 20 mg/ml (data not shown).
Furthermore, attempting to determine the molecular weight of HypBA1 at an even lower concentration in solution, we applied sedimentation velocity experiment by using analytical ultracentrifugation (AUC-SV). At a concentration of 6 μM, the polypeptide was detected as a single species with a sedimentation coefficient of 7.77 S, which corresponds to a molecular mass of 143.0 kDa, the mass of a HypBA1 dimer (Figure 4B and 4C). Taken together, the results by employing two independent biophysical methods elucidate the oligomeric state of the recombinant HypBA1 in solution. At a much higher protein concentration (ca. 10 mg/ml), a symmetric elution peak was still observed and the corresponding molecular weight also coincided with the dimeric forms of HypBA1. The results are consistent with the AUC-SV data, which showed that HypBA1 was dimeric at a low protein concentration and only a small population of higher oligomers emerged when the protein concentration was increased (Figure 4). Both the results of the SEC/MALS and AUC-SV analyses are in a good agreement with the crystallographic findings that HypBA1 exhibits as a very stable dimerization propensity.
Ligand binding and substrate modeling
The |Fo| - |Fc| omit map and anomalous difference Fourier map of the bound L-Araf and Zn2+ ion are both very clear, respectively (Figure 2A). Based on the binding mode of L-Araf, there are ten hydrogen bonds between the sugar unit and eight amino acid residues including Gln45, His142, His194, His270, Glu322, Glu338, Tyr386 and Cys415 (Figure 5A), but not Glu366. According to the relative spatial positions of L-Araf and Zn2+ ion in the complex structure, Zn2+ ion might not directly involve in the catalytic reaction because the Zn2+ ion is distantly located to the C1 of product (5.0 Å) (data not shown). Moreover, the configuration of Zn2+ ion also makes it unlikely to activate a water molecule for catalysis (which would turn out an inverted α-sugar). This is because the Zn2+ ion has formed an almost perfect tetrahedral coordination with Glu338, Cys340, Cys417 and Cys418 (Figure 2A).
By analyzing the HypBA1-L-Araf complex structure, we believe that the bound L-Araf (product) corresponds to the -1 subsite. The space adjacent to the O2 atom is too small to accommodate a sugar residue. To further elucidate possible substrate binding mode, a twosugar- units substrate (Araf-β1,2-Araf; β-Ara2) was manually modeled into the potential subsites from -1 to +1 in the substrate-binding cavity (Figure 5B). The model was subsequently subjected to several cycles of energy minimization with CNS [24]. Interestingly, the location of the simulated model seems almost fit the size and shape of calculated cavity map (Figure 5B), which is generated by using the web server POCASA (POcket-CAvity Search Application) [37]. Therefore, the accuracy of the simulated model is justified. In this model, the side chain of Glu322 also binds to the O1 of the +1 sugar, which is in turn bound to Tyr386, and two other residues Gln44 and Tyr250 can interact with the O5 of the same +1 sugar. Beyond the +1 sugar, there is no room to accommodate additional L-Araf and Hyp units unless the capping loop is opened. How the enzyme binds to Ara2- and Ara3-Hyp remains to be elucidated.
Proposed catalytic mechanism
As previously mentioned, three potential catalytic residues (Glu322, Glu338 and Glu366) have been proposed. Among them, Glu366 is too far away from the substrate-binding cavity and unlikely to participate directly in the catalytic reaction (Figure 1A). However, the mutant E366A had 16% activity left in a previous study [11]. Consequently, Glu366 might play a role in structural stability, although not participating directly in the catalytic reaction. By contrast, the residues Glu322 and Glu338 are more reasonable catalytic amino acids due to their proximal locations. In the crystal structure of HypBA1-L-Araf complex, Glu322 is hydrogen bonded to the O1 atom of the sugar and it is in a good position for the general acid/base role. In the absence of L-Araf, Glu322 is probably hydrogen bonded to His270, which is supposed to be protonated. When His270 turns to interact with the sugar, the proton may remain bound to Glu322, making it a good general acid catalyst. On the other side Glu322 is hydrogen bonded to Tyr336, which may also serve as a proton donor. Both His270 and Tyr336 are strictly conserved among the GH127 enzymes (Figure 3).
Regarding the nucleophile, Glu338 binds to the O2 atom but it is nearly 4 Å away from C1, too far and unlikely to undertake this role, although it can be fully ionized by binding to Zn2+. However, the finding that the mutant E338A only had 0.0013% activity left in a previous study [11] clearly shows that Glu338 plays an important role in ligand recognition and Zn2+ binding. Very recently, Ito solved the Zn2+ ion containing HypBA1 structure in ligand-free and complex forms [12]. The subsequent structure-based mutagenesis and biochemical analysis, in conjunction with quantum mechanical calculations, allowed Ito to make a clear proposal that the nucleophile should be Cys417 rather than Glu338 [12]. In our apo structure, the Cys417-containing loop is shifted away due to the disappearance of all coordinate bonds in the absence of Zn2+ ion (Figure 2C). Cys417 is thus diverted from the attack position, and probably is also protonated, disabling its role as the nucleophile. Therefore, Zn2+ ion is involved in the catalytic reaction through maintaining the proper configuration of active site.
As said by Ito, however, we cannot rule out other possibility of catalytic reaction mechanism, such as the utilization of two carboxylate residues (Glu322 and Glu338) separated by a suitable distance (5.4 Å) for retaining mechanism. In this case, the bound L-Araf should represent the +1 sugar and the -1 sugar would be severely skewed to fit into the limited space, which is not likely. On the other hand, a recent review suggests that some GH families employ novel mechanisms instead of typical carboxylate base/nucleophile, including substrateassisted mechanisms, proton transferring network, utilization of noncarboxylate residues and utilization of an exogenous base/nucleophile [38]. Interestingly, apart from Glu322, Glu338, Cys340, Cys417and Cys418, Tyr386 is also strictly conserved among several GH127 members (Figure 3). The side chain of Tyr386 is equally close to the C1 of L-Araf (3.2 Å) as is that of Cys417, and it may correspond to the non-carboxylate residue in an alternative mechanism. However, the lack of a base to subtract its proton renders Tyr386 a weak nucleophile. Consequently, the most reasonable catalytic mechanism may involve a Cys417-sugar intermediate, as shown in Scheme 1. Besides the use of a different nucleophile (Cys417 rather than an Asp or Glu) in the first step, the remaining steps are almost the same as those of classic retaining mechanism.
In summary, the native, apo and complex crystal structures of HypBA1 give us a first glimpse of the GH127 family with respect to protein folding and catalytic mechanism. The results presented here shall provide a critical starting point and a firm basis for further studies of the GH127 family. In addition to the catalytic S-domain, HypBA1 also contains N-domain and C-domain, the latter participating in dimer formation. To investigate the functions of this novel multidomain protein, further experiments with mutagenesis and truncation are required.
This work was supported by National High Technology Research and Development Program of China (2012AA022200), Academy of Sciences (2011B09030042), Tianjin Municipal Science and Technology Commission (12ZCZDSY12500) and National Science Council of Taiwan (NSC 102-2628-B-002- 007-MY2). The synchrotron data collection was conducted at beam line BL13B1 and BL13C1 of NSRRC (National Synchrotron Radiation Research Center, Taiwan).

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