In Silico Biology 6 (2006) 435447 IOS Press

435

In Silico Modeling and Hydrogen Peroxide Binding Study of Rice Catalase

Pagadala Nataraj Sekhara , Polavarapu B. Kavi Kishora , Lakkireddy Ananda Reddya , Prosenjit Mondalb, Ardhendu K. Dashb , Manoranjan Karc , Satya Mohantyd and Surendra C. Sabatb,
a

Department of Genetics, Osmania University, Hyderabad 500 007, India Plant Molecular Biology Division, Institute of Life-Sciences, Bhubaneswar 751 023, India c Department of Botany, Utkal University, Vani Vihar, Bhubaneswar 751 004, India d Distributed Information Sub-Center, Institute of Life-Sciences, Bhubaneswar-751 023, India
b

Edited by H. Michael; received 18 April 2006; revised 18 July, 2006; accepted 4 August 2006; published 24 September 2006

ABSTRACT: Homology modeling of the catalase, CatC cloned and sequenced from rice (Oryza sativa L., cv Ratna an Indica cultivar) has been performed based on the crystal structure of the catalase CatF (PDB code 1m7s) by using the software MODELLER. With the aid of molecular mechanics and molecular dynamics methods, the nal model is obtained and is further assessed by PROCHECK and VERIFY 3D graph, which show that the nal rened model is reliable. With this model, a exible docking study with the hydrogen peroxide, the substrate for catalase, is performed and the results indicate that Arg310, Asp343 and Arg346 in catalase are three important determinant residues in binding as they have strong hydrogen bonding contacts with the substrate. These hydrogen-bonding interactions play an important role for the stability of the complex. Our results may be helpful for further experimental investigations. KEYWORDS: Catalase, docking, molecular dynamics

INTRODUCTION Catalase (EC 1.11.1.6, hydrogenperoxide: hydrogenperoxide oxidoreductase) is one of the most active enzyme catalysts, found in plants, animals and in all aerobic microorganisms. The major function of the enzyme is to decompose hydrogen peroxide (H 2 O2 ), produced by cellular metabolic activities under normal and stressful conditions, to water and oxygen. Besides decomposing H 2 O2 , the enzyme also has the oxidase and peroxidase-like enzyme activities [1]. The plant catalase, unlike the animal catalase, contains multiple isoforms and is encoded by a small family of genes [2]. In rice (Oryza sativa L.), three catalase genes, CatA, CatB and CatC have so far been identied [3]. The expression of these genes is, however, recorded to be highly tissue specic and the expression pattern differs during the ontogenic development of the plant [2]. Recently Dash et al. [4] have sequenced the complete coding region (1500 bp) of CatC from an Indica rice cultivar (Ratna; Accession Number NCBI: DQ118681). An attempt has been made here to develop a
Corresponding author: Institute of Life Sciences Nalco Square, Chandrasekharpur, Bhubaneswar-751023, Orissa, India. E-mail: scsabat@yahoo.com.

Electronic publication can be found in In Silico Biol. 6, 0041 <http://www.bioinfo.de/isb/2006/06/0041/>, 24 September 2006. 1386-6338/06/$17.00 2006 IOS Press and Bioinformation Systems e.V. and the authors. All rights reserved

436

P.N. Sekhar et al. / In Silico Modeling and Hydrogen Peroxide Binding Study of Rice Catalase

three-dimensional (3D) structure of this gene product (catalase-C protein; hereafter referred as catalase) using bioinformatics tools. In the process, the active sites of the enzyme and the interaction of the substrate H2 O2 with the enzyme have been deciphered. MATERIALS AND METHODS Total RNA isolation, PCR amplication, cloning and sequencing of CatC Total RNA was extracted from 48-h dark grown rice (Oryza sativa L, Indica cultivar Ratna) embryonic axes using TRIzol (Sigma) following the suppliers instructions. The extracted RNA was submitted for cDNA synthesis using Roche First Strand cDNA Synthesis Kit. The synthesized cDNA was further utilized for RT-PCR using CatC gene specic primers (F-5-TTAATCAGCCATGGATCCT-3; R-5AGCAGATTGCAACGCTGATC-3). The PCR amplied product was ligated into the pGEMT Easy vector (Promega) and transformed to E. coli (TB01 strain). The cloned fragment was subsequently sequenced using vector specic primers (F-(T7)-5-TAATACGACTCACTATAGGG-3; R-(SP6)5TATTTAGGTGACACTATAG-3) in a Beckman Coulter CEQ 8000 Genetic Analysis Sequencing System. The total fragment obtained was of 1500 bp having start and stop codon. The homology search of the inserted fragment was done through BLAST search (NCBI; http://www.ncbi.nlm. nih.gov/BLAST/) and was conrmed as the rice CatC (nucleotide sequence homology 99% and amino acid sequence homology nearly 70%). 3D model building The initial model of catalase was built by using homology-modeling methods and the MODELLER software; a program for comparative protein structure modeling optimally satisfying spatial restraints derived from the alignment and expressed as probability density functions (pdfs) for the features restrained. The pdfs restrain C C distances, main-chain NO distances, main-chain and side-chain dihedral angles. The 3D model of a protein is obtained by optimization of the molecular pdf such that the model violates the input restraints as little as possible. The molecular pdf is derived as a combination of pdfs restraining individual spatial features of the whole molecule. The optimization procedure is a variable target function method that applies the conjugate gradients algorithm to positions of all non hydrogen atoms [5]. The query sequence from Oryza sativa was searched to nd out the related protein structure to be used as a template by the BLAST (Basic Local Alignment Search Tool) [6,7] program against PDB (Protein Databank). Sequences that showed maximum identity with high score and less E -value were aligned (Fig. 1) and were used as a reference structure to build a 3D model for catalase. The co-ordinates for the structurally conserved regions (SCRs) for catalase were assigned from the template using multiple sequence alignment, based on the Needleman-Wunsch algorithm [8,9]. The structure having the least modeler objective function obtained from the modeler was improved by energy minimization. Initial geometric optimizations were carried out using the standard Tripos force eld with 0.05 kcal/mol energy convergence criteria and a distant dependent dielectric constant of 4.0 R to take into account the dielectric shielding in proteins employing Gasteiger-Marshilli charges with non-bonding interaction cut off as 15. All hydrogen atoms were included during the calculation. After undertaking 100 steps of Powell minimization method initially [10,11], a conjugate gradient energy minimization of the full protein was carried out until the root mean-square (r.m.s) gradient was lower than 0.05 kcal mol 1 . Further a molecular dynamic simulation was carried out to examine the quality of the model structures by

P.N. Sekhar et al. / In Silico Modeling and Hydrogen Peroxide Binding Study of Rice Catalase

437

Fig. 1. Sequence alignment of catalase from Oryza sativa with catalase CatF of Pseudomonas syringae (PDB code 1m7s) done using CLUSTALW server that was subsequently submitted to MODELLER. The conserved regions are indicated by *.

checking their stability via performing 1000 femto seconds (fs) simulations at an ensemble of constant number of particles, volume, and temperature of 300K. Boltzmann initial velocities and random number seed with a tolerance of 0.0001 were used for the above calculations. We used the same force eld setup as that used for energy minimization. Snapshots were taken every 5 fs with a coupling phase of 100 fs. No solvent was included in the model [12]. Finally, the average structure was again subjected to energy minimization methods as described above. All calculations are performed on SGI 2000 workstation using the SYBYL software suite implemented on a Silicon Graphics O2+ workstation, operating under IRIX (SYBYL 6.7, Tripos Inc., 1699, South Hanley Rd., St. Louis, Missouri, 63144, USA). In this step, the quality of the initial model was improved. The nal structure obtained was analyzed by Ramachandrans map using PROCHECK (a program to check the stereochemical quality of protein structures) [13] and environment prole using VERIFY-3D graph (structure evaluation server) [14]. This model was used for the identication of active site and for docking of the substrate with the enzyme.

438

P.N. Sekhar et al. / In Silico Modeling and Hydrogen Peroxide Binding Study of Rice Catalase

Active site identication of catalase enzyme The active site of catalase from rice was identied using SiteID of SYBYL software suite (Fig. 4). This method generated many possible spheres of the radius of water inside the protein molecules in search of the largest space or cluster available, which could be identied as active site. A ood ll algorithm, similar to one implemented in CAVITY, was used [15]. For each solvent molecule in the pocket, all atoms in the protein lying within a specic distance of 5 are considered. The unions of all such atoms are considered as belonging to the active site. In addition to this, an electrostatic or polar surface identication method was used to distinguish between the actual site and the other smaller cluster of sites. Docking studies The ligands, including all hydrogen atoms, were built and minimized with SYBYL software suite as described above. Genetic optimization for ligand docking (GOLD) version 2.1.2 (Cambridge Crystallographic Data Center, Cambridge, United Kingdom) was used for docking catalase enzyme for 50 times [16]. The active site was dened as the collection of protein residues enclosed within a 15 radius sphere. The annealing parameters for Van der Waals and hydrogen bonding were set to 4.0 and 2.5 respectively. The parameters used for genetic algorithm were population size (100), selection pressure (1.1), number of operations (100,000), number of islands (5), niche size (2), migrate (10), mutate (95) and cross-over (95). The default speed selection was used to avoid a potential reduction in docking accuracy. Fifty genetic algorithm runs with default parameter settings were performed without early termination. To estimate the protein-ligand complexes, the scoring function, GOLD SCORE was employed [16].

RESULTS Homology modeling of catalase enzyme A high level of sequence identity should guarantee a more accurate alignment between the target sequence and template structure. In the results of BLAST search against PDB, only three reference proteins, including catalase CatF of Pseudomonas syringae [17], catalase 1 from Neurospora crassa [18] and mutant His392Gln catalase HPII from E. coli [19] have a high level of sequence identity and the identity of these three reference proteins with the catalase enzyme are 37%, 36% and 40% respectively (Table 1). Structurally conserved regions (SCRs) for the model and the template were determined by superimposition of the two structures and multiple sequence alignment (Fig. 1). In the following study, we have chosen 1m7s as a reference structure for modeling catalase enzyme. Coordinates from the reference protein (1m7s) to the SCRs, structurally variable regions (SVRs), N-termini and C-termini were assigned to the target sequence based on the satisfaction of spatial restraints. All side chains of the model protein were set by rotamers. The initial model was thus generated with the above procedure. After the initial model was complete, we searched the possible active sites using the SYBYL SiteID module and compared these results with the active site of 1m7s. Thus we know that residues 114 do not locate near the active site. In our study, residues 114 are removed from the model because no homologous region occurs in 1m7s and these residues do not locate near the active site. Thus the model is made up of residues 15500. The options used for running the modeler are shown in Table 2. This

P.N. Sekhar et al. / In Silico Modeling and Hydrogen Peroxide Binding Study of Rice Catalase

439

Table 1 Data for closest homologue for catalase enzyme from Oryza sativa with known 3D structure obtained with the blast server against PDB PDB 1M7S 1SY7 1QF7 Protein Crystal Structure analysis of catalase CatF of Pseudomonas syringae Crystal structure of catalase-1 from Neurospora crassa Structure of the mutant His392Gln of catalase HPII from E. coli Table 2 Options used in MODELLER program INCLUDE SET ALNFILE SET KNOWNS SET SEQUENCE SET HETATM IO SET WATR IO SET HYDROGEN SET STARTING MODEL SET ENDING MODEL CALL ROUTINE # = = = = = = = = = Include the predened TOP routines. alignment1 m7s query on off off 1 20 model Chain A, B, C, D A, B A, B, C, D Reference [17] [18] [19] Identity to catalase (%) 37% 36% 40%

Fig. 2. The nal 3D structure of catalase enzyme. The structure is obtained by energy minimizing the average conformation over the last 1000 femto seconds of molecular dynamics simulation. The -helix is represented by cylinders and -sheet by arrows.

model was rened by molecular dynamics method and the nal stable structure of the catalase enzyme obtained is shown in Fig. 2. From Fig. 2 it is evident that this enzyme has 13 helices and 11 sheets. The nal structure was further checked by VERIFY-3D graph and the results are shown in Fig. 3. The compatibility score above zero in

440

P.N. Sekhar et al. / In Silico Modeling and Hydrogen Peroxide Binding Study of Rice Catalase Table 3 Ramachandran plot calculations on 3D model of catalase computed with the PROCHECK program % of residue in most favored regions % of residue in the additionally allowed zones % of residue in the generously regions % of residue in disallowed regions % of non-glycine and non-proline residues 70.2 26.2 2.3 1.3 100.0

Fig. 3. The 3D proles veried results of catalase model, residues with positive compatibility score are reasonably folded.

the VERIFY-3D graph (Fig. 3) corresponded to acceptable side chain environments. The energy prole obtained from the VERIFY-3D program showed all residues expect 2535 and 365375, which were not satisfactory and hence considered for loop modeling. These regions do not have template structural fragments, and additionally when the modeled loop was inserted, it affected the complete structure and conformation of the molecule. Further no suitable structural fragment was available to model 154164 residue regions, from the family of catalase enzymes. Therefore, a fragment outside the family was explored. The search was made using LOOP DATA BASE option in the SWISS PROTEIN DATABANK VIEWER software suite; http://www.expasy.org/spdbv [20]. This database has been built by a large set of high-resolution protein structural fragments and the best segment was 1PFK (phosphofructokinase) with root mean square deviation of 2.40 and the fragment selected was tted to the model and visually inspected for stereo chemical compatibility. This model was again subjected to energy minimization described above. Validation of catalase enzyme After the renement process, validation of the model was carried out using Ramachandran plot calculations computed with the PROCHECK program. The and distributions of the Ramachandran plots of non-glycine, non-proline residues are summarized in Fig. 4 and Table 3. The RMSD (Root Mean Square deviation) deviation for covalent bonds relative to the standard dictionary was 0.39 and for the covalent angles was 0.53 . Altogether 96.4% of the residues was in favored and allowed regions. The overall PROCHECK G-factor was 0.46 and VERIFY-3D environment prole was good. Superimposition of 1m7s with catalase enzyme The structural superimposition of C trace of template and catalase enzyme is shown in Fig. 5. The weighted root mean square deviation of C trace between the template and nal rened model was 3.1

P.N. Sekhar et al. / In Silico Modeling and Hydrogen Peroxide Binding Study of Rice Catalase

441

Fig. 4. Ramachandrans map of catalase enzyme built using MODELLER software. The plot calculations on the 3D model of catalase enzyme were computed with the PROCHECK program.

Fig. 5. Superimposition of C trace of catalase (represented in dark gray) and 1m7s (represented in light gray).

with a signicance score of 27.2. This model was used for the identication of active site and for docking of the substrate with the enzyme catalase. Secondary structure prediction The amino acid sequences of template, initial (structure generated from the modeller) and nal structures are generated using JOY server (protein sequence-structure representation and analysis [21]), were aligned using CLUSTALW (not shown here). Given their PDB les, secondary structures were

442

P.N. Sekhar et al. / In Silico Modeling and Hydrogen Peroxide Binding Study of Rice Catalase

Fig. 6. Structure based sequence alignment of template, initial and nal structures of the catalase enzyme using JOY program. The key to the JOY annotation is as follows: -helix residues are labeled with a, -strand residues with b, residues with 310 -helix conformation with 3; uppercase letter indicate solvent-inaccessible residues, lowercase letters solvent-accessible residue; italic lowercase letter, positive ; bold lowercase letter, hydrogen bond to main-chain amide; underlined lowercase letter, hydrogen bond to main-chain carbonyl; conserved secondary structure elements are framed by large boxes, within which conserved active site residues are framed by smaller boxes.

also analyzed and compared by the JOY program. The secondary structures of template, initial and nal catalase enzymes are highly conserved except in two regions between 310320 and 360370, which showed that nal structure is highly reliable as shown in Fig. 6 and further used for active site identication. Active site identication of catalase enzyme After the nal model was built, the possible binding sites of catalase were searched using the Site ID module. Twenty-three possible binding sites are obtained using SYBYL Site ID module and are shown in Fig. 7. These pockets were compared with the active site of the template and were found that site 12 (yellow region) is highly conserved. Catalase from Oryza and 1m7s are well conserved in both sequence and structure, their biological function should be identical. In fact from the structure-structure comparison of template, initial and nal rened models of catalase enzyme using JOY program [21], it was found that secondary structures are highly conserved and the residues in the site 12, Lys58, Pro83,

P.N. Sekhar et al. / In Silico Modeling and Hydrogen Peroxide Binding Study of Rice Catalase

443

Fig. 7. The possible binding-sites of catalase enzyme from Oryza sativa model. The different sites are displayed in different grey shades (more detailed color version available online under http://www.bioinfo.de/isb/2006/06/0041).

Gln110, Val111, Gln117, Leu118, Gly119, Ala308, Arg310, Asp343, Arg346, Pro358, and Asn359, are conserved with the active site of template and was shown in Fig. 6. By considering the experimental fact that the active site of 1m7s [17] includes all residues as mentioned above, and on the other hand, among the twelve sites, the shape of site 12 (Fig. 7, yellow region) in catalase of rice is similar to that of the catalytic site in Pseudomonas syringae, site 12 is chosen in this study as the most favorable site to dock the ligand and the other sites are not discussed further. Thus, in this study site 12 is chosen as the more favorable binding site to dock the substrate, and the other twenty-two sites are not discussed further. Thus we suggest that H 2 O2 binds in a similar manner for both catalase enzyme and 1m7s. The nal stable structure of catalase enzyme obtained was shown in Fig. 2. Docking of substrate with the active site of catalase Docking of the substrate with the enzyme catalase was performed using GOLD, which was set to 50 cycles of run. The docking procedure proceeds in several steps. First, the protein-ligand complex is generated using the GOLD package [16] without constraints between the ligand and the specic amino acids of the pocket. The algorithm exhaustively searches the entire rotational and translational space of the ligand with respect to the receptors. The exibility of the ligand is given by dihedral angle variations. The various solutions evaluated by a score, which is equivalent to the absolute value of the total energy of the ligand in the protein environment. The best ve docking solutions GOLD score difference is given in Table 4. It can be seen that the GOLD scores are very similar in all the ve top solutions. The

444

P.N. Sekhar et al. / In Silico Modeling and Hydrogen Peroxide Binding Study of Rice Catalase Table 4 GOLD score, GOLD score difference () and RMSD deviation of C trace of top ve docked conformations of the substrate GOLD score RMSDd 0.00 12 0.00 12 0.00 23 0.00 23 0.00 34 0.00 34 0.00 45 0.00 45 The subscript indicates RMSD deviation between top ve gold scores between 1 and 2, 2 and 3, 3 and 4, and 4 and 5. Substrate H2 O2 GOLD score 14.34

Fig. 8. Binding of H2 O2 substrate in the active site of catalase enzyme. Active site residues are represented in light gray and the substrate is represented in dark gray.

GOLD score and RMSD differences between the consecutive top ve runs of the substrate is zero and this indicates the stabilization of particular substrate at the active site and was shown in Table 4. To understand the interaction between catalase enzyme and H 2 O2 , the substrate-catalase complex was generated using the SYBYL software suite (Fig. 8). It is evident from the gure that H 2 O2 substrate is located in the center of the active site, and is stabilized by hydrogen bonding interactions. It is also evident from the gure that H2 O2 is binding in between the 1st, 2nd, 3rd, 4th -helices and the 7th -sheet as shown in Fig. 9. The hydrogen bonds present in the enzyme-substrate complex along with their distances and angles are listed in Table 5. Signicant binding key residues in the active site of the model were determined based on the interaction energies of the substrate with residues in the active site of the enzyme catalase. This identication, compared with a denition based on the distance from the substrate can clearly show the relative

P.N. Sekhar et al. / In Silico Modeling and Hydrogen Peroxide Binding Study of Rice Catalase Table 5 Hydrogen bonds along with their distances and angles between the H2 O2 substrate and active site residues of catalase as deciphered using SYBLY Software suite Catalase enzyme Residue Atom Asp343 OD1 Arg346 HH22 Arg346 HH22 Arg346 HE H2 O2 atom hydrogen peroxide H hydrogen peroxide O hydrogen peroxide O hydrogen peroxide O Distance 1.854 2.244 1.813 2.504 Angle 172.84 Deg 56.17 Deg 164.47 Deg 60.95 Deg

445

Fig. 9. Binding of the H2 O2 substrate in the catalase enzyme between the alpha helices and beta sheets. The -helix is represented by cylinders and -sheet by arrows; the substrate appears in dark gray behind PRO358.

signicance for every residue. Table 6 shows the interaction energies including the total, electrostatic and steric energies for all the residues in the active site of enzyme-substrate complex. From Table 6, it is evident that the enzyme substrate complex has large favorable total interaction energy of 11.274 kcalmol 1 . The electrostatic and steric energies are 18.140 and 6.866 kcalmol 1 , respectively. These results indicate that both the electrostatic and steric interactions are important for the protein-substrate complex interaction. Through the interaction analysis, we know that Arg310, Asp343, and Arg346 are important anchoring residues for the substrate and are the main contributors to the substrate interaction. Though the interaction energy does not include the contribution from the water or the extended enzyme structure, this preliminary data along with the list of hydrogen bond interactions

446

P.N. Sekhar et al. / In Silico Modeling and Hydrogen Peroxide Binding Study of Rice Catalase Table 6 The total energy (Etotal ), electrostatic energy (Eele ), steric energy (Este ) of the best-docked conformations Substrate H2 O2 Etotal (kcal/mol) 11.247 Eele (kcal/mol) 18.140 Este (kcal/mol) 6.866

between the enzyme and the active site residues clearly supports that Arg310, Asp343, and Arg346 are more preferred residues in the H 2 O2 binding. DISCUSSION Catalase is one of the most potent enzyme catalysts that convert a potentially harmful oxidant H 2 O2 to water and molecular oxygen. In this work, we have constructed a 3D model of catalase enzyme (CatC product), cloned and sequenced by us from Oryza sativa (Indica cultivar Ratna), using the MODELLER software and obtained a rened model after energy minimization. The nal rened model was further assessed by VERIFY-3D and PROCHECK program, and the results show that this model is reliable. The stable structure is further used for docking of substrate. Docking results indicate that conserved amino-acid residues in catalase enzyme play an important role in maintaining a functional conformation and are directly involved in donor substrate binding. The interaction between the enzyme and the substrates proposed in this study are useful for understanding the potential mechanism of enzyme and the substrate binding. As is well known, hydrogen bonds play an important role for the structure and function of biological molecules, especially for the enzyme catalysis. In this study it was found that Arg310, Asp343, and Arg346 are important for strong hydrogen bonding interaction with the substrates. To the best of our knowledge Arg310, Asp343, and Arg346 are conserved in these two enzymes and may be important for structural integrity or maintaining the hydrophobicity of the substrate-binding pocket. ACKNOWLEDGEMENTS The authors are thankful to the Director, Center for Distance Education, Osmania University, for his kind support for providing the software for completion of the work. Thanks are due to the Director, Institute of Life sciences for providing nancial support to PM and AKD. MK gratefully acknowledges the co-operation of the Head, Department of Botany Utkal University. REFERENCES
[1] [2] [3] [4] [5] [6] Vetrano, A. M., Heck, D. E., Mariano, T. M., Mishin, V., Laskin, D. L. and Laskin, J. D. (2005). Characterization of the oxidase activity in mammalian catalase. J. Biol. Chem. 280, 35372-35381. Iwamoto, M., Higo, H. and Higo, K. (2000). Differential diurnal expression of rice catalase genes: the 5-anking region of CatA is not sufcient for circadian control. Plant Sci. 151, 39-46. Iwamoto, M., Higo, H. and Higo, K. (2004). Strong expression of the rice catalase gene CatB promoter in protoplasts and roots of both a monocot and dicots. Plant Physiol. Biochem. 42, 241-249. Dash, A. K., Mondal, P., Mandal, S., Kar, M. and Sabat, S. C. (2005). Oryza sativa (indica cultivar-group) catalase-C mRNA, complete cds. NCBI Accession number DQ118681. Sali, A. and Blundell, T. L. (1993). Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779-815. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. (1990) A basic local alignment search tool. J. Mol. Biol. 215, 403-410.

P.N. Sekhar et al. / In Silico Modeling and Hydrogen Peroxide Binding Study of Rice Catalase [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21]

447

Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389-3402. Needleman, S. B. and Wunsch, C. D. (1970). A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol. 48, 443-453. Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specic gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680. Powell, M. J. D. (1977). Restart procedures for the conjugate gradient method. Math. Program. 12, 241-254. Press, W. H., Flannery, B. P., Teukolsky, S. A. and Vetterling, W. T. (1998). Numerical recipes in C, the art of scientic computing. Cambridge University Press, New York. Alder, B. J. and Wainwright, T. E. (1959). Studies in molecular dynamics. I. General method. J. Chem. Phys. 31, 459-466. Laskoswki, R. A., MacArthur, M. W., Moss, D. S. and Thornton, J. M. (1993). PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283-291. L thy, R., Bowie, J. U. and Eisenberg, D. (1992). Assessment of protein models with three-dimensional proles. Nature u 356, 83-85. Ho, C. and Marshall, G. (1990). Cavity search: an algorithm for isolation and display of cavity-like binding regions. J. Comput. Aided Mol. Des. 4, 337-354. Jones, G., Willett, P., Glen, R. C., Leach, A. R. and Taylor, R. (1997). Development and validation of a genetic algorithm for exible docking. J. Mol. Biol. 267, 727-748. Carpena, X., Soriano, M., Klotz, M. G., Duckworth, H. W., Donald, L. J., Melik-Adamyan, W., Fita, I. and Loewen, P. C. (2003). Structure of the Clade 1 catalase, CatF of Pseudomonas syringae, at 1.8 resolution. Proteins 50, 423-436. Daz, A., Horjales, E., Rudino-Pi era, E., Arreola, R. and Hansberg, W. (2004). Unusual Cys-Tyr covalent bond in a n large catalase. J. Mol. Biol. 342, 971-985. Mat , M. J., Sevinc, M. S., Hu, B., Bujons J., Bravo, J., Switala, J., Ens, W., Loewen, P. C. and Fita, I. (1999). Mutants e that alter the covalent structure of catalase HPII from Escherichia coli. J. Biol. Chem. 274, 27717-27725. Guex, N. and Peitsch, M. C. (1997). SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714-2723. Mizuguchi, K., Deane, C. M., Blundell, T. L., Johnson, M. S. and Overington, J. P. (1998). JOY: protein sequence-structure representation and analysis. Bioinformatics 14, 617-623.