Molecular Docking of Bacillus pumilus Xylanase and Xylan Substrate Using Computer Modeling

CHINESE JOURNAL OF BIOTECHNOLOGY Volume 23, Issue 4, July 2007 Online English edition of the Chinese language journal

Cite this article as: Chin J Biotech, 2007, 23(4), 715–718.

RESEARCH PAPER

Molecular Docking of Bacillus pumilus Xylanase and Xylan Substrate Using Computer Modeling LIN Jin-Xia1,2, ZHANG Liao-Yuan1,2, ZHANG Guang-Ya1, FANG Bai-Shan1* 1

Key Laboratory of Industrial Biotechnology, Hua Qiao University, Fujian Quanzhou 362021, China

2

Biological Engineering college, East China University of Science and Technology, Shanghai 200237, China

Abstract: Bacillus pumilus xylanase was cloned and sequenced. Based on the tertiary structure that originated from homology modeling, the potential active pocket was searched and ligand-protein docking was performed using relative softwares. The information extracted from the molecular docking was analyzed; several amino acid residues that may play a vital role in the xylanase catalytic reaction were obtained to instruct the further modification of xylanase directed-evolution. Key Words:

molecular docking; Bacillus pumilus xylanase; active pocket

Xylanase (E.C. 3.2.1.8) is applied to degrade xylan in the process of treating agricultural residues such as rice straw, corn cob etc. The research of microbial xylanase now focuses on the characteristics, enzyme production under different conditions, purification, gene expression and application in paper and pulp industries[1–3]. Thus, it is indispensable to investigate the catalytic mechanism between the action of xylanase and xylan substrate. Molecular docking is defined as docking ligand to the active site of receptor, and searching a reasonable orientation and configuration to make an optimum match of the shape and interaction between the ligand and receptor. According to the lock and key principle, molecular docking can be used to screen compounds that match well with the active site of receptor in consideration of special and electrical characteristics. In drug design, molecular docking is mainly employed to search for molecules that bear relatively good affinity to biomacromolecules from compound database aiming to find new lead compounds. Meanwhile, molecular docking is thought as an effective technology to approach the interaction of proteins. Owing to a global consideration of the matching effect of ligand and receptor, molecular docking

avoids the possibility of good local action but bad global combination as described in other methods[4,5]. In this research, based on the tertiary structure originating from the homology modeling of xylanase from Bacillus pumilus, molecular docking was exploited to model the action of xylanase with xylan; several potential active amino acids were obtained and the catalytic mechanism was discussed, and this established a foundation for further modification of xylanase directed-evolution.

1

Materials and methods

1.1 Materisals Bacillus pumilus, DNA cloning kit, Pentium computer, Bioinformatic databases, Molsoft, visualizational softwares, such as Rasmol, spdbv, and chimera. 1.2 Methods and procedures 1.2.1 Xylanase gene cloning was operated according to molecule biology protocols, and sequencing was executed by Huanuo Company, Beijing. The sequenced nucleotides were translated into amino acids and aligned with different original xylanase sequences.

Received: December 6, 2006; Accepted: January 16, 2007. * Corresponding author. Tel: +86-595-22691560; E-mail: [email protected] This work was supported by the grant from the Innovation Group Development Project of the Ministry of Education of China (No. IRT0435). Copyright © 2007, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.

LIN Jin-Xia et al. / Chinese Journal of Biotechnology, 2007, 23(4): 715–718

1.2.2 Xylanase amino acid sequence was submitted to SWISS-MODEL servicer for homology modeling.1.2.3 IcmPocketFinder program was employed to search for potential substrate binding pockets based on the homology modeling structure. 1.2.3 Structures of xylan and xylanase were edited prior to docking. Docking was performed as per the instruction. 1.2.4 Data analysis.

2

Results and analysis

2.1 Xylanase sequencing and aligning Gene cloning and sequencing results of Bacillus pumilus xylanase are shown in Fig. 1; NCBI ID is EF090270. The result of aligning with different orginal xylanase sequences by Clustal W is indicated in Fig. 2. The scores of Clustal W aligning were 30, 39, 44, 44, 49, 43, 44, and 66, respectively. Two reported conserved sequences, [PSA]-[LQ] -x-E-Y-Y[LIVM](2)-[DE]-x-[FYWHN] and [LIVMF]-x(2)-E-[AG] -[YWG]-[QRFGS]-[SG]-[STAN]-G-xSAF] were closed in two large rectangles while the conservative Glu residues were closed in two small rectangles.

visualize the resulting structure. As presented in Fig. 3, the tertiary structure contained two α-helix, twenty β-sheets, nineteen loops, and the final total energy was -8 600.9 kJ/mol. 2.3 Potential active pocket searching One active pocket binding with substrate was found through the Molsoft programme based on the homology modeling structure of xylanase. As demostrated in Fig. 4, amino acids 23, 25, 50, 52, 54, 84-86, 90, 92, 99, 101, 109, 111,112, 131, 133, 136-139, 145, 147, 149, 188, and 190, denoted by different colors, were located around the active pocket, embodied as the solid.

Fig. 3 The tertiary structure of B. pumilus xylanase from homology modeling lysate

Fig. 1 The sequencing result of B. pumilus xylanase Fig. 4 The structure of active pocket of B. pumilus xylanase The pocket space was represented by the blue solid, and different amino acids appearing around the pocket were denoted by different colors.

Fig. 2 The multi-alignment of xylanase from different origins

2.2 Homology modeling Bacillus pumilus xylanase sequence was submitted to the Swiss-model server, and the rasmol software was used to

2.4 Molecular docking Although there are still no reports about the crystal structure of Bacillus pumilus xylanase in PDB, the crystal structure of Bacillus circulans xylanase was reported with PDB id 1BCX. It was indicated that during the catalytic process, only two xylose residues could be fitted into the pocket while the rest of the xylan substrate extended beyond the active site cleft, waiting to enter the pocket two by two rings. In the ground of this cyatlytic mechanism, and for the sake of reducing computational complexity, only two xylose residues (Fig. 5) were taken as the docking ligand, and the structure was extracted from the 1BCX crystal structure, and edited with the spdbv software. Bacillus pumilus xylanase docking was executed as the protocols and the consequential structure was

LIN Jin-Xia et al. / Chinese Journal of Biotechnology, 2007, 23(4): 715–718

edited with chimera, spdbv, rasmol, and molsoft softwares, followed by Fig. 6. Bacillus circulans xylanase docking was also done for comparison.

Table 2 The residues and bonds’ length of B. circulans xylanase from the docking results Substrate ring Xyl 1

Xyl 2

Fig. 5 The structure of xylan substrate (two xylose residues)

Amino acid Tyr 166 Tyr 69 Trp 71 Tyr 69 Tyr 80 Arg 112 Arg 112 Arg 112 Pro 116

Distance/Å 2.15 1.98 2.53 2.43 2.62 2.06 2.07 2.52 1.89

2.5.2 B. pumilus xylanase docking: As concluded from Fig. 2 and Table 1, Trp25, Tyr90, Glu99, Tyr101, Arg133, Pro137, and Glu188 of B. pumilus xylanase, which also showed around the active pocket were possibly involved in the catalytic reaction. From the docking result of B. pumilus xylanase, six amino acid residues linked by nine bonds reacted with the xylan substrate, given in Table 3. Three amino acid residues (Glu99, Arg133 and Pro137) were consistent with the above-mentioned potential residues. Owing to the adjoining location of Tyr86 with Tyr90 and Tyr101, it is presumed that Tyr may play a critical role in the catalytic process. The resulting locational relationship of the different active amino acids is depicted in Fig. 7. Table 3 The residues and bonds’ length of Bacillus pumilus xylanase from the docking results

Fig. 6 The docking structure of xylanase and xylan

2.5 Docking analysis 2.5.1 B. circulans xylanase docking: As reported in reference 6, nine amino acid residues of B. circulans xylanase were included in the catalytic process linked by eleven bonds, as given in Table 1. According to the docking result of B. circulans xylanase, given in Table 2, seven amino acid residues linked by nine bonds were involved. Except Trp71, the residues shown in Table 2 were contained in Table 1, indicating relative accuracy of molecular docking. Table 1 The residues and bonds’ length of B. circulans xylanase from the reported crystal structure Substrate ring

Amino acid

Distance/Å

Xyl 1

Tyr 166

2.82

Xyl 2

Tyr 69

2.88

Trp 9

3.10

Trp 9

3.37

Glu 78

3.00

Glu 78

3.37

Glu 172

2.15

Tyr 80

3.57

Arg 112

2.97

Arg 112

3.15

Pro 116

2.56

Substrate ring

Amino acid

Xyl 1

Asn 84

2.51

Asn 84

2.21

Xyl 2

Distance/Å

Tyr 86

2.67

Glu 99

2.50

Arg 133

2.70

Arg 133

2.78

Gln 147

2.14

Gln 147

2.56

Pro 137

2.41

Based on our research the tested Bacillus pumius had relative good alkali-tolerance, and combined with the docking information that Asn84 formed two hydrogen bonds with O1 and O2 of xylan (Table 3), it can be concluded that Asn84 may play the role of catalytic residue.

Fig. 7 The detailed docking structure of xylanase and xylan The active residues were marked by colors.

LIN Jin-Xia et al. / Chinese Journal of Biotechnology, 2007, 23(4): 715–718

3

Conclusion

Molecular docking is a key ingredient of computer-aided design, an effective method applied to study the interaction and binding mode of ligand and receptor. It is required to place the ligand next to the active site of receptor, and evaluate the effect of interaction according to the principle of geometric, energy and chemical environmental complementations[7]. Scoring function and global optimization are two vital problems in the process of docking; however, the paramount and predominant factor is the amino acids involved in the several Å scope, that is, the active site of receptor directly effects the docking result, and therefore it is necessary to determine the active pocket before performing molecular docking [8]. In this study, residues in 5 Å scope of the ligand were selected to execute docking, and the consequent RMSD values were better than that in the 3 or 7 Å scope. It was concluded that the former (5 Å scope) contained more key residues, and was more reliable. In the absence of the crystal structure of B. pumilus xylanse in PDB, molecular docking of xylanase with xylan may obtain some key residues’ information and may lay a theoretical foundation for further modification of xylanase.

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