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Published online before print July 5, 2006, 10.1110/ps.062220206
Protein Science (2006), 15:1951-1960. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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Crystal structures of native and xylosaccharide-bound alkali thermostable xylanase from an alkalophilic Bacillus sp. NG-27: Structural insights into alkalophilicity and implications for adaptation to polyextreme conditions

Karuppasamy Manikandan1, Amit Bhardwaj2, Naveen Gupta3, Neratur K. Lokanath4, Amit Ghosh3, Vanga Siva Reddy2 and Suryanarayanarao Ramakumar1,5

1 Department of Physics, Indian Institute of Science, Bangalore 560 012, India
2 International Centre for Genetic Engineering and Biotechnology, New Delhi 110 067, India
3 Institute of Microbial Technology, Sector 39-A, Chandigarh 160 036, India
4 Advanced Protein Crystallography Research Group, RIKEN Harima Institute, Hyogo 6795148, Japan
5 Bioinformatics Centre, Indian Institute of Science, Bangalore 560 012, India

(RECEIVED March 15, 2006; FINAL REVISION May 15, 2006; ACCEPTED May 15, 2006)


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Conclusions
 Materials and methods
 References
 
Crystal structures are known for several glycosyl hydrolase family 10 (GH10) xylanases. However, none of them is from an alkalophilic organism that can grow in alkaline conditions. We have determined the crystal structures at 2.2 Å of a GH10 extracellular endoxylanase (BSX) from an alkalophilic Bacillus sp. NG-27, for the native and the complex enzyme with xylosaccharides. The industrially important enzyme is optimally active and stable at 343 K and at a pH of 8.4. Comparison of the structure of BSX with those of other thermostable GH10 xylanases optimally active at acidic or close to neutral pH showed that the solvent-exposed acidic amino acids, Asp and Glu, are markedly enhanced in BSX, while solvent-exposed Asn was noticeably depleted. The BSX crystal structure when compared with putative three-dimensional homology models of other extracellular alkalophilic GH10 xylanases from alkalophilic organisms suggests that a protein surface rich in acidic residues may be an important feature common to these alkali thermostable enzymes. A comparison of the surface features of BSX and of halophilic proteins allowed us to predict the activity of BSX at high salt concentrations, which we verified through experiments. This offered us important lessons in the polyextremophilicity of proteins, where understanding the structural features of a protein stable in one set of extreme conditions provided clues about the activity of the protein in other extreme conditions. The work brings to the fore the role of the nature and composition of solvent-exposed residues in the adaptation of enzymes to polyextreme conditions, as in BSX.

Keywords: alkali thermostable; GH10 xylanase; solvent-exposed acidic residues; solvent-exposed basic residues; polyextremophilicity; alkalophilic organism


   Introduction
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 Abstract
 Introduction
Results
 Discussion
 Conclusions
 Materials and methods
 References
 
Xylanases (EC 3.2.1.8 [EC] ) are xylan-degrading enzymes belonging to glycosyl hydrolases that catalyze the hydrolysis of internal beta-1,4 bonds of xylan backbones. Xylan is the major hemicellulose component of the plant cell wall, and its hydrolysis by xylanases has potential economical and environmentally friendly applications (Shallom and Shoham 2003). Xylanases are mainly used in the paper pulp bleaching industry to replace the use of toxic chlorine-containing chemicals (Beg et al. 2001). Based on sequence and structure, most xylanases are classified into families 10 and 11 (Henrissat and Davies 1997), and a few belong to family 8, of the glycosyl hydrolases (De Vos et al. 2006; http://afmb.cnrs-mrs.fr/CAZy). To date, several GH10 xylanase structures have been solved (Derewenda et al. 1994; White et al. 1994; Dominguez et al. 1995; Schmidt et al. 1998; Fujimoto et al. 2000; Teplitsky et al. 2000, 2004; Canals et al. 2003; Natesh et al. 2003; Payan et al. 2004; Pell et al. 2004a,b; Ihsanawati et al. 2005). However, none of them is from an alkalophilic organism that can grow in alkaline conditions.

The desirable properties of xylanases in the paper industry are stability and activity at high temperature and alkaline pH (Collins et al. 2005). Only one report is available on an alkali-active (pH 9.0) thermostable (at 338 K) GH10 xylanase (GSX) structure (PDB code 1r85) from a thermophilic organism, Geobacillus stearothermophilus T-6 (Teplitsky et al. 2004). The study enzyme, the extracellular endoxylanase BSX (~41 kDa), belongs to the GH10 family and is from an alkalophilic Bacillus sp. NG-27 (Gupta et al. 2000; Leelavathi et al. 2003). The enzyme is optimally active at 343 K (thermostable) and at a pH of 8.4 (alkali-stable). It does not contain any cysteine residues, precluding any thermostability due to disulfide bridge(s). While the factors responsible for the thermal stability of GH10 xylanases have been analyzed (Ihsanawati et al. 2005), not much has been discussed regarding the alkaline stability of GH10 xylanases. The crystal structure of BSX fills this knowledge gap on a molecular basis for the thermo-alkalophilic stability of the study enzyme through a cross-comparative study, which could form the basis for improving the thermo-alkaline stability. This is the first report that describes the crystal structure of an alkali thermostable GH10 xylanase from an alkalophilic organism.

The crystal structures of BSX alone and in complex with xylosaccharides were solved at 2.2 Å. For a comparative study, the two alkalophilic extracellular xylanase homologs, BHX and BFX, from alkalophilic organisms Bacillus halodurans (GenBank accession no. AAN03480 [GenBank] ) and Bacillus firmus (Chang et al. 2004), respectively, were identified through sequence database searches (BLAST [Altschul et al. 1990]) and structurally modeled. We have attempted to decipher the causative factors for the alkaline stability of BSX and delineate the alkali stability of GH10 xylanases in general, and have therefore compared it with other thermostable GH10 xylanases whose pH optimum was known from the literature. The structural features that are likely to be responsible for the alkaline stability of the enzyme are identified and discussed. Furthermore, based on protein surface similarity between the alkalophilic BSX and halophilic proteins, we predicted that BSX could be active at high salt concentration, and this was verified through biochemical experiments. The present study has enabled us to address the question of polyextremophilicity, as to whether deciphering structural features of a protein stable in one set of extreme conditions could provide clues about the stability of the protein in other extreme conditions.


   Results
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 Abstract
 Introduction
 Results
Discussion
 Conclusions
 Materials and methods
 References
 
The overall structure
The crystal structures of native and xylosaccharides-bound BSX were determined using the molecular replacement method and refined at 2.2 Å (Table 1). BSX folds as the ubiquitous (beta/{alpha})8-barrel, a structural fold common to many glycosyl hydrolases. In the BSX structure, Glu 149 at the C terminus of beta4 and Glu 259 in the middle of beta7 are identified as the catalytic acid/base and the nucleophile residues, respectively, from the multiple sequence alignment of BSX with other homologous xylanases (Fig. 1). The side chains of catalytic glutamate residues are at a distance of 5.5 Å, suggesting that the enzymatic reaction takes place by the retaining mechanism. Conserved aromatic residues line the long active-site groove.


Figure 1
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  		Figure 1. Multiple sequence alignment of GH10 xylanases analyzed. Boxed regions 100–115, 265–285, and 321–334 indicate the three inserted (>10) amino acid stretches that are unique to alkalophilic xylanases (see text for details). The secondary structures of BSX are shown on top of the sequence alignment, where coils and arrows represent helices and strands, respectively.

 


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  		Table 1. Crystal parameters and refinement statistics of the protein and the complex

 
In the substrate-bound crystal structure, experimental electron density is seen for a xylotriose and a xylobiose in molecule A and for a xylotriose and a xylose in molecule B. The interaction pattern of bound xylosaccharides at the active site region is different from that found in other homologous xylanases.

Structural features
The "WP" sequence-structure-interaction motif: Positioning of tryptophans for xylosaccharide binding
The two Trp–Pro peptides (Trp 235–Pro 236 and Trp 267–Pro 268) in the (+) side of the active site region may be referred to as a "WP" sequence-structure-interaction motif. The peptide bond between Trp and Pro is cis, and Trp side chains are in the tg+ or tg– conformation. In both cases, as seen in the xylosaccharides-bound BSX structure, the Trp is positioned in such a way that the aromatic ring of Trp is sandwiched between the proline ring and the sugar ring (Fig. 2). The centroids of the indole rings of the Trps are at an average distance of 4.0 Å from the proline rings. The centroid-to-centroid distances of the sugar ring at the +2 subsite and the six- and five-membered rings of Trp 267 are 4.7 Å and 4.6 Å, respectively. The corresponding distances for the ring of the xylose moiety at the +3 subsite and Trp 235 are 4.1 Å and 5.3 Å, respectively. This indicates favorable van der Waals interactions of the Trps with the sugar moiety and the prolines. Also, reduction in the accessible surface area of Trps (for the Trp 235 side chain, percent relative accessibility changes from 40 to 24, and for Trp 267, it changes from 48 to 21) is noticed upon xylobiose complexation, implying interactions between the xylosaccharide moiety and the Trp rings. It has been shown in a different context that the large aromatic group, namely Trp, provides a higher association constant and binding enthalpy in the course of enzymatic reactions (Chavez et al. 2005). The occurrence of WP in two independent positions with similar conformational and interaction features in BSX (Fig. 2) has encouraged us to term this as a WP sequence-structure-interaction motif. The two WP motifs together may help in the efficient binding of the carbohydrate moiety of the xylan polymer in the active site cleft of BSX. The motif is also present in the related xylanase structure (GSX) (Zolotnitsky et al. 2004) with similar conformational features. The WP motif is present in the sequences of BHX and BFX.


Figure 2
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  		Figure 2. Stereo view of the interaction of xylobiose with "WP" motifs. The 2Fo-Fc electron density of xylobiose was contoured at 1.0{sigma}. Broken lines indicate hydrogen-bonding interactions between the enzyme and xylose moieties. Active site subsites are numbered.

 
Metal binding site: Importance of Mg2+ for the enzymatic activity
The crystal structure revealed a metal binding site, found at the C-terminal end of the catalytic domain. The presence of a metal binding site was not anticipated from an earlier modeled structure (Mande et al. 2000) and biochemical studies. As MgCl2 was present in the crystallization soup used for crystallizing BSX, a bound Mg2+ ion is seen at the C-terminal region of the protein. The Mg2+ is coordinated by two side-chain oxygen atoms from Asn 292 and Asp 354 and three waters that are roughly in a plane; a main-chain carbonyl oxygen atom of Arg 351; and a water molecule on either side of the plane (Fig. 3). A Zn2+-binding site is present at an equivalent position in GSX (PDB code 1r85) with similar coordination.


Figure 3
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  		Figure 3. The coordination of Mg2+ seen in the native BSX structure with a 2Fo-Fc electron density map contoured at 2.0{sigma}. Waters are shown as small gray spheres.

 
Subsequently, to explore the biological role of bound Mg2+ found in the crystal structure of BSX, we investigated experimentally the effect of Mg2+ on the activity of xylanase. Interestingly, it was found that the activity of BSX increased in the presence of Mg2+ in a concentration-dependent manner (Fig. 4), and maximum activity was observed at 6 mM MgCl2. Therefore, these results clearly provide evidence, for the first time, of the requirement of Mg2+ for the activity of the alkali thermostable xylanase for its biological function. The bound metal ion presumably provides additional structural stability to the C-terminal region of the enzyme, particularly to the last secondary structural element {alpha}8, as two of the metal-coordinating residues, Arg 351 and Asp 354 (the C-terminal residue), belong to {alpha}8.


Figure 4
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  		Figure 4. Change in the activity of BSX when assayed with different concentrations of MgCl2. The activity of BSX after the metal ions were chelated out was taken as baseline activity.

 
A comparison of the structure of BSX with those of other GH10 xylanases
In order to understand the molecular basis for the alkaline stability of the study enzyme, the primary and the tertiary structure of BSX and its close homolog GSX have been compared with those of other thermostable xylanases with known 3-D structures and characterized to be active at a pH close to neutral (Table 2). The comparison is, therefore, expected to elucidate the features of relevance to alkali thermostable xylanases.


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  		Table 2. Details of solvent-exposed residues for the extracellular GH10 xylanase structures

 
Primary structure comparison
The alkaline xylanases (BSX, BHX, BFX, and GSX) have higher numbers of acidic residues than others and have three major inserted stretches of >10 amino acids (100–115, 265–285, and 321–334; numbering based on BSX) (Fig. 1). Notably, Asp and Glu conserved among the alkalophilic xylanases are less conserved in nonalkalophilic xylanases. The extra domain present in GSX is absent in BSX and also in BHX and BFX (Fig. 1). It may be noted that in BSX, the percentage of acidic amino acids (Asp/Glu) in the three inserted stretches (which may be called "acidic-rich additions") common to alkalophilic GH10 xylanases is 25% as opposed to 18% Asp/Glu when the overall protein length is taken into account. Moreover, they occur in the catalytic/activity face of the enzyme (Sterner and Hocker 2005). It has been suggested that adaptation to extreme conditions can take place through insertions or exchanges of short sequences without the requirement of gradual changes over the entire protein chain (Besir et al. 2005).

A comparison of solvent-exposed charged residues
Protein surfaces are known to be responsible for protein stability under diverse environmental conditions and are especially important for extracellular enzymes. It has been shown from a comparative structural genomics study on mesophilic and thermophilic proteins that a significant amino acid substitution, which differentiates between the two, occurs at the solvent-exposed sites (Chakravarty and Varadarajan 2002). Hence, it is pertinent to compare the surfaces of alkaline xylanase with those of neutrophilic xylanases, and it is possible due to the availability of the BSX crystal structure. The percentage of Asp and Glu in 10% accessibility bins (percent side-chain accessibility of a residue in protein) for the analyzed xylanase structures is depicted in Figure 5A. The percentage of Asp and Glu in the last bin, which corresponds to solvent-exposed residues, is higher for alkaline-stable xylanases than for neutrophilic xylanases. Furthermore, the percentage of solvent-exposed acidic residues is higher than the solvent-exposed basic residues for BSX and other alkaline xylanases (BHX and BFX) from alkalophilic organisms (Table 2). Importantly, the alkali thermostable GH10 xylanases from alkalophilic organisms are clearly separated from their neutrophilic counterparts when the percentage of solvent-exposed basic residues is plotted against the percentage of solvent-exposed acidic residues (Fig. 5B). At the same time, the percentage of exposed polar residues (Asn, Gln, Ser, and Thr) (Suhre and Claverie 2003) is reduced in alkaline xylanases (Table 2). Similar calculations carried out by us for the alkaline cellulase protein (PDB code 1g01; Shirai et al. 2001) from alkalophilic Bacillus sp. KSM-635 showed consistent results in that the percentage of solvent-exposed acidic residues (12.6%) is higher than the percentage of solvent-exposed basic residues (2.2%).


Figure 5
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  		Figure 5. (A) Histogram plot of percentage of acidic residues in different relative side-chain accessibility bins. The percentage of acidic residues is considerably higher for GH10 alkaline xylanases in the last bin. (B) 2D plot of percentage of solvent-exposed basic residues vs. the percentage of solvent-exposed acidic residues. Thermo (1nq6, 1i1w, 1xyz, and 1bg4) and Alkali (BSX, BHX, and BFX) represent neutrophilic and alkalophilic GH10 xylanases, respectively. 1vbu and 1r85 are GH10 xylanases from the hyperthermophilic organism Thermotoga maritima and the thermophilic organism Geobacillus stearothermophilus T-6, respectively.

 
Protein surface rich in acidic residues: Activity of BSX at high salt concentrations
The surfaces of halophilic proteins (those optimally active in high salt concentration) have a higher number of acidic residues than nonhalophilic proteins (Dym et al. 1995; Frolow et al. 1996; Bieger et al. 2003; Besir et al. 2005). Also, protein surfaces rich in acidic residues are implicated in the adaptation of enzymes to function under halophilic conditions (Dym et al. 1995; Frolow et al. 1996). The density of acidic residues on the surface of ferredoxin (PDB code 1doi) from Haloarcula marismortui is one per 173 Å2 (Frolow et al. 1996; Supplemental Fig. 1). On average, one surface-exposed acidic residue contributes 256 Å2 to the surface area of BSX. In contrast, the corresponding value for a neutrophilic thermostable xylanase whose structure has been solved earlier in our laboratory is 1024 Å2 (Natesh et al. 2003). The similar features in protein surface between the alkalophilic BSX and a halophile, i.e., a protein surface rich in acidic residues (Supplemental Fig. 1), prompted us to predict that BSX could be active at high salt concentration; hence, we have investigated the activity of the enzyme under various concentrations of NaCl (Fig. 6). The protein was soluble in the entire range of salt concentrations (0–4 M), and no significant loss of activity was detected up to 2 M NaCl. The enzyme started losing its activity from 2 M NaCl; however, it retained 33% activity even at 4 M NaCl. It implies that BSX displays relatively high activity at medium salt concentrations (2 M NaCl) and moderate activity even under high salt concentration (4 M NaCl), vindicating our prediction.


Figure 6
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  		Figure 6. Change in activity of BSX assayed with different concentrations of NaCl. The activity of BSX at 0 M of NaCl was taken as a baseline activity.

 

   Discussion
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 Abstract
 Introduction
 Results
 Discussion
Conclusions
 Materials and methods
 References
 
The stability of BSX at alkaline pH: Enhanced solvent-exposed Asp/Glu
The cell wall of alkalophilic Bacillus strains contains acidic polymers such as galacturonic acid, gluconic acid, glutamic acid, aspartic acid, and phosphoric acid in comparison with those of the neutrophilic Bacillus subtilis (Horikoshi 1999). From the same study (Horikoshi 1999), addressing the question of the mechanism of stabilization, it was suggested that negative charges on these acidic nonpeptidoglycan components may enable the cell surface of alkalophilic Bacillus to adsorb positively charged ions (Na+ or H3O+) and repulse hydroxide ions (present due to alkaline pH), and effectively may assist cells to grow in alkaline environments (Horikoshi 1999; Dubnovitsky et al. 2005). In BSX, firstly, the occurrence of acidic residues is higher (64 out of 97 charged residues, 66% of total); secondly, 70% of the negatively ionizable residues are found on the protein surface (Fig. 5A; Supplemental Fig. 1). In addition, the ratio of solvent-exposed acidic residues to solvent-exposed basic residues is found to be highest for BSX (Table 2; Fig. 5). While the intracellular enzymes being sequestered inside the cell surrounded by cell wall are not exposed to harsh conditions, extracellular enzymes (such as BSX), on the other hand, are exposed to harsh environments and have to evolve strategies for their stability and viability under adverse external conditions (Horikoshi 1999). Taken together, protein surfaces rich in acidic residues may help the protein carry out its enzymatic activity at the alkaline pH environment and protects the protein core from the OH– attack. From the present analysis, it appears that both cells and proteins follow a common strategy of having exteriors rich in acidic groups for the purpose of achieving stability under alkaline conditions. The negatively rich surrounding, which modulates the microenvironment and thus the pKa of the catalytic residues (Gutteridge and Thornton 2005; Li et al. 2005), may be important for the alkaline pH optimum of BSX.

Alkaline thermostability of BSX: Depletion of solvent-exposed Asn
A structural feature observed from the comparison of BSX with nonalkalophilic xylanase structures is that the surface-accessible Asn, which is an alkaline-susceptible as well as heat-labile residue, is less abundant in BSX (Table 2). Solvent-exposed Asn residues undergo deamidation and isomerization at increased temperature, in particular at alkaline pH (Gulich et al. 2002; Walden et al. 2004). The mutation of solvent-exposed Asn to Ala in Streptococcal protein G increased the alkaline stability of the protein (Gulich et al. 2002). The higher number of salt bridges and lower number of solvent-exposed Asn (Table 2) may contribute to alkaline thermostability of the BSX enzyme.

Multiple structural features are responsible for the extremophilicity of proteins
Each protein has its own adaptation and strategy for extremophilicity. It has been shown recently that electrostatic interactions and compactness are the most common features found in thermophilic proteins as compared with their mesophilic counterparts (Robinson-Rechavi et al. 2006). Similarly, a point may be raised that there may be more than one property responsible for the alkalophilicity of proteins. At the same time, a given structural feature such as higher number of salt bridges may contribute to more than one extremophilic property of a protein. In the present study, we have attempted to unveil the structural features of alkaline stability; they are the predominant solvent-exposed acidic residues and lower solvent-exposed Asn. These may be expected to be important among multiple factors responsible for alkaline stability. It appears that GH10 xylanases that have the conserved structural scaffold (beta/{alpha})8 TIM barrel have chameleon-like characteristics, with variable surface features facilitating their adaptations to different situations.

Activity of BSX at high salt concentrations: Implications for polyextremophilicity in proteins
We have predicted that BSX could be active at high salt concentrations, as it has surface features similar to those of halophilic proteins (Supplemental Fig. 1). The prediction was verified subsequently through biochemical experiments (Fig. 6). However, the activity gradually decreases with increasing salt concentration for BSX (Fig. 6), whereas for a halophilic protein (Madern et al. 2000) the activity is low at low salt concentrations and increases with increasing salt concentration. There must be subtle differences that eventually influence the nature of the changes in activity as a function of changes in salt concentration. Nevertheless, our work implies that certain structural features may contribute to more than one extremophilic property, i.e., the possibility of polyextremophilicity in proteins.

By comparing with other extremophiles, it may be seen that a particular strategy (for example, enhanced salt bridges or acidic protein surface) could be common for adaptation to more than one extreme condition. Understanding the relative contributions and the interplay of multiple features that enable adaptation to different extreme conditions is of great fundamental importance for studying the evolution of polyextremophilicity in proteins. This may also be of practical utility in designing enzymes for industrial applications.


   Conclusions
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 Abstract
 Introduction
 Results
 Discussion
 Conclusions
Materials and methods
 References
 
The work exemplifies the mechanism of adaptation of enzymes to function under alkaline conditions through changes in the nature and the composition of solvent-exposed residues. It may be surmised from the comparative study that alkalophilic GH10 xylanases from alkalophilic organisms are likely to have a surface rich in acidic residues, a relatively lower number of solvent-exposed basic residues (Fig. 5B), and a lower number of solvent-exposed Asn, as seen in BSX. The situation may be roughly described as "acidic residues outside and Asn inside" and may be expected to be important among the structural features for alkaline stability. Our analysis should aid the engineering of proteins with improved stability and activity in alkaline conditions, a requirement for biotechnological applications and in particular of xylanases in the paper pulp processing industry. Also, we have shown here through prediction and subsequent experimental verification that the study of structural features of a protein known to be stable in one set of extreme conditions may provide clues about the stability of the protein in other extreme conditions, i.e., the intriguing phenomenon of polyextremophilicity in proteins. The importance of polyextremophilicity in helping us to understand the evolutionary history of proteins and organisms merits extensive investigation.


   Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Conclusions
 Materials and methods
References
 
Crystallization, data collection, and structure solution
The purification, crystallization, and data collection of the native enzyme were carried out as described (Manikandan et al. 2005). The enzyme-complex crystals were grown in a reservoir solution containing 0.1 M NaCl, 0.2 M MgCl2, 0.1 M Tris-HCl (pH 8.5), and 15% PEG 8000. A synchrotron data set was collected for the xylosaccharides-bound BSX crystal at Spring-8 BL26B1 beamline ({lambda} = 1.0 Å) at 100 K (Table 1). Both data sets were processed using the programs DENZO and SCALEPACK from the HKL2000 package (Otwinowski and Minor 1997).

The native enzyme structure was solved by the molecular replacement method with the program AMoRe (Navaza 1994), using as a search model the crystal structure (PDB code 1hiz). There are two molecules in the asymmetric unit that corresponds to the Matthews coefficient (Matthews 1968) of 2.9 Å3 Da–1. Refinement using the parameters of Engh and Huber (1991) and model building were carried out using CNS1.1 (Brünger et al. 1998) and COOT0.31 (Emsley and Cowtan 2004), respectively. Five percent of randomly selected observed reflections were kept aside for the cross-validation (Brünger 1992). The refined native enzyme was used as a molecular replacement search model for the xylosaccharides-bound BSX. The bound xylooligosaccharides were located using difference Fourier maps and fitted. NCS restraint with weight of 100 was used in the final round of refinement of the xylosaccharides complex structure and applied only to protein atoms. The stereochemistry of the final models was analyzed with PROCHECK (Laskowski et al. 1993), and RMS deviations resulted in the proper values (Table 1). None of the models contains residues in the generously and disallowed region of the Ramachandran ({varphi}, {psi}) map (Ramachandran and Sasisekharan 1968; Table 1).

Metal binding and activity at high salt concentrations assay
Both the purified enzyme and the substrate Oat xylan (Sigma) were treated with 25 mM EDTA overnight at room temperature to remove any bound metal ions and then dialyzed extensively against 50 mM Tris-HCl (pH 8.4) buffer containing 0.9 M NaCl, followed by further dialyses against only 50 mM Tris-HCl (pH 8.4). To determine the Mg2+ ion requirement for xylanase activity, the reaction mix was supplemented separately with varying concentrations of MgCl2 (0–10 mM), and the enzyme activity was determined as described elsewhere (Gupta et al. 2000).

To study the activity at high salt concentrations, the purified BSX premixed with 50 mM Tris-HCl (pH 8.5) was incubated with various concentrations of NaCl for 24 h at room temperature, and then the activity was measured. One microgram (1 µg) of purified enzyme was used for each activity assay reaction volume of 100 µL. The data plotted in Figures 4 and 6 were the average values of three independent experiments.

Structure analysis
Secondary structure assignment was made using PROMOTIF (Hutchinson and Thornton 1996). The accessibilities of residues were calculated using the program NACCESS (S.J. Hubbard and J.M. Thornton, University College London). The solvent-exposed residues were identified using the cutoff value of ≥30% in the relative surface accessibility area of the side chain. Hydrogen bonds and the contacts between xylosaccharides and active site residues were found using HBPLUS (McDonald and Thornton 1994) and CONTACT of the CCP4 suite (CCP4 1994), respectively. Salt bridges were assigned when atoms of opposite charge were found within 4 Å of each other. Multiple sequence alignment was done with CLUSTALW (http://www.ebi.ac.uk/clustalw) and rendered using ESPRIPT (Gouet et al. 1999). Figures of molecules were drawn using PyMOL (DeLano Scientific).


   Footnotes
 
Supplemental material: see www.proteinscience.org

Reprint requests to: Suryanarayanarao Ramakumar, Department of Physics, Indian Institute of Science, Bangalore 560 012, India; e-mail: ramak{at}physics.iisc.ernet.in; fax: +91(080)2360-2602; or Vanga Siva Reddy, Plant Transformation Group, International Centre for Genetic Engineering and Biotechnology, New Delhi 110 067, India; e-mail: vsreddy{at}icgeb.res.in; fax: +91(011)2616-2316.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062220206.


   Acknowledgments
 
Facilities at the Supercomputer Education and Research Centre, the Interactive Graphics Based Molecular Modeling Facility, and the Distributed Information Centre (both funded by the Department of Biotechnology at the Indian Institute of Science, Bangalore) were used in this work. The intensity data for the native protein were collected at the X-Ray Facility for Structural Biology at the Indian Institute of Science, Bangalore, supported by the Department of Science and Technology (DST) and the DBT. We thank Dr. Debnath Pal for critical reading of the manuscript and helpful discussions. K.M. thanks CSIR (India) for a fellowship. We are extremely grateful to the anonymous referees for a very thorough reading of the manuscript and valuable suggestions.


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 Discussion
 Conclusions
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