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Molecular Modelling and Functional Studies ofthe Non-Stereospecific α-Haloalkanoic AcidDehalogenase (DehE) from Rhizobium SP. RC1 andits Association with 3-Chloropropionic Acid (β-Chlorinated Aliphatic Acid)Azzmer Azzar Abdul Hamida, Ee Lin Wongb, Kwee Hong Joyce-Tanc, Mohd ShahirShamsirb, Tengku Haziyamin Tengku Abdul Hamida & Fahrul Huyopb

a International Islamic University, Malaysia, Faculty of Science, Kuantan, Pahang,Malaysiab University of Technology, Malaysia, Faculty of Biosciences and Bioengineering, JohorBahru, Johor, Malaysiac The National University of Malaysia, Faculty of Science and Technology, Bangi,Selangor, MalaysiaPublished online: 16 Apr 2014.

To cite this article: Azzmer Azzar Abdul Hamid, Ee Lin Wong, Kwee Hong Joyce-Tan, Mohd Shahir Shamsir, TengkuHaziyamin Tengku Abdul Hamid & Fahrul Huyop (2013) Molecular Modelling and Functional Studies of the Non-Stereospecific α-Haloalkanoic Acid Dehalogenase (DehE) from Rhizobium SP. RC1 and its Association with 3-Chloropropionic Acid (β-Chlorinated Aliphatic Acid), Biotechnology & Biotechnological Equipment, 27:2, 3725-3736, DOI:10.5504/BBEQ.2012.0142

To link to this article: http://dx.doi.org/10.5504/BBEQ.2012.0142

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Article http://dx.doi.org/10.5504/bbeq.2012.0142

bioiNForMAticS

Biotechnol. & Biotechnol. eq. 27/2013/2

Biotechnol. & Biotechnol. eq. 2013, 27(2), 3725-3736

Keywords: non-stereospecific haloalkanoic acid, dehalogenase, DehE, Rhizobium sp. RC1, structural model, protein structure and functions, computational analysis

IntroductionThe majority of the environmental pollutions are caused by the abundance of recalcitrant, synthetic organic compounds released into the biosphere. In particular, industrial-scale manufacturing and chemical processing are responsible for the introduction of huge amounts of xenobiotic compounds that are strongly persistent in nature. While the metabolic versatility of microbial communities is enormous, a major part of the xenobiotics possess chemical modifications that can reduce the efficiency of their degradation. For instance, halogen substituents often increase the persistence and toxicity of an organic molecule, and synthetic organohalogen compounds, which represent a significant subset of the industrially important solvents, herbicides, and pesticides, have been studied to investigate their fate in our natural environment (11). For example, D- and L-2-chloropropionic acid are used

as an intermediate compound in the agricultural, chemical and pharmaceutical industries. These chemical compounds were listed as Special Health Hazard Substances because they are corrosive and may affect humans when inhaled.

A dehalogenase is a microbial enzyme that catalyses the critical step in the breakdown of high priority halogenated organic pollutants by cleaving the carbon–halogen bond. Various dehalogenases, e.g. haloalkane dehalogenase (35), haloalkanoic acid dehalogenase (1, 41), and 4-chlorobenzoyl-CoA dehalogenase (49), have been classified and characterised. Haloalkanoic acid dehalogenase is a member of the dehalogenases family, which catalyze the chemical reaction of haloalkanoic acid and water to produce hydroxyacid and a halide. All of these enzymes belong to the hydrolase family (HAD superfamily) (14). Haloalkanoic acid dehalogenase has also been widely confirmed to act on α-halogenated carboxylic acid compounds but not on β-halogenated carboxylic acids. Dehalogenation of β-halogenated carboxylic acids, such as 3-chloropropionate (3CP), is not well studied, and its mechanism is far from clear (27, 50, 51).

MOLECULAR MODELLING AND FUNCTIONAL STUDIES OF THE NON-STEREOSPECIFIC α-HALOALKANOIC ACID DEHALOGENASE (DehE) FROM RHIZOBIUM SP. RC1 AND ITS ASSOCIATION WITH 3-CHLOROPROPIONIC ACID (β-CHLORINATED ALIPHATIC ACID)

Azzmer Azzar Abdul Hamid1, Ee Lin Wong2, Kwee Hong Joyce-Tan3, Mohd Shahir Shamsir2, Tengku Haziyamin Tengku Abdul Hamid1 and Fahrul Huyop2 1International Islamic University, Malaysia, Faculty of Science, Kuantan, Pahang, Malaysia 2University of Technology, Malaysia, Faculty of Biosciences and Bioengineering, Johor Bahru, Johor, Malaysia 3The National University of Malaysia, Faculty of Science and Technology, Bangi, Selangor, Malaysia Correspondence to: Fahrul HuyopE-mail: fzhutm@gmail.com

ABSTRACTMany environmental pollutions are caused by the abundance of xenobiotic compounds in nature. For instance, halogenated compounds released from chemical industries were proven to be toxic and recalcitrant in the environment. However, haloalkanoic acid dehalogenases can catalyse the removal of halides from organic haloacids and thus have gained interest for bioremediation and synthesis of industrial chemicals. This study presents the first structural model and the key residues of the non-stereospecific haloalkanoic acid dehalogenase, DehE, from Rhizobium sp. RC1. The enzyme was built using a homology modelling technique; the structure of DehI from Pseudomonas putida PP3 was used as a template, because of its homology to DehE. The structure of DehE consists of only α-helices. Twelve conserved residues that line the active site were identified: Trp34, Ala36, Phe37, Asn114, Tyr117 Ala187, Ser188, Asp189, Tyr265, Phe268, Ile269, and Ile272. These residues are consistent with the residues found in the active site of DehI and D,L-DEX 113 from Pseudomonas sp. 113. Asp189 activates the water molecule as a nucleophile to attack the substrate chiral centre, which would result in an inversion of configuration of either D- or L-substrates. Both D- and L-substrates bind to and interact with the enzyme by hydrogen bonding with three residues, Trp34, Phe37, and Ser188. In addition, a putative tunnel was also identified that would provide a channel for the substrate to access the binding site. Based on computational analysis, DehE was proven to have the substrate affinity towards 3-chloropropionic acid (3CP)/β-chlorinated aliphatic acid, however, its dehalogenation process is far from clear. This DehE structural information will allow for rational design of non-stereospecific haloalkanoic acid dehalogenases in the future.

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Previously, a bacterium was isolated from soil by elective culture on 2,2-dichloropropionic acid and was identified as Rhizobium sp. RC1 (3, 15). The Rhizobial produced three haloalkanoic acid dehalogenases (6, 16, 42). The presence of all three dehalogenases was confirmed by mutational analysis, and all three enzymes could utilize 2-chloropropionic acid as a substrate. DehL is specific for L-2-chloropropionic acid, DehD is specific for D-2-chloropropionic acid, and DehE acts on both stereoisomers (24). DehE is grouped into class 2I because it is a non-stereospecific halkoalkanoic acid dehalogenase that inverts the product configuration (40). Other non-stereospecific haloalkanoic acid dehalogenases in the same class as DehE are DehI from Pseudomonas putida PP3 (33, 43) and D,L-DEX113 from Pseudomonas sp. 113 (29, 31). D,L-DEX113 was functionally characterised in terms of its reaction mechanism and active site residues (31), whereas DehI was crystallised and information has been gained as to how the enzyme facilitates the process of dehalogenation (38). To the best of our knowledge, the three-dimensional structure of DehE from Rhizobium sp. RC1 and its active mechanism have not been reported. The absence of this information prevented us from understanding the structure, specificity and selectivity of this non-stereospecific haloalkanoic acid dehalogenase.

In this study, we built a structural model and identified the key active site residues of DehE, which is a representative member of the group I α-haloalkanoic acid dehalogenases as described by Hill et al. (14). The amino acid sequence of DehE, was retrieved and analysed using in silico methods to model its three-dimensional structure and to understand the active site formation. Homology or comparative modelling was used to determine the tertiary structure of DehE because it has a high similarity to the crystal structure of DehI from Pseudomonas putida PP3. The structural model of DehE provides details of a new fold and also recognition of the substrate-binding location, which allows for the role of key functional amino acids to be resolved. This study aspired to contribute to the knowledge of the molecular structure of the non-stereospecific haloalkanoic acid dehalogenase (configuration-converting) (EC3.8.1.10) of Rhizobium sp. RC1. The results are useful for the comprehensive understanding on the biodegradation facilitated by the non-stereospecific haloalkanoic acid dehalogenases.

Materials and MethodsPrimary structural analysis of DehEBefore applying any modelling technique, the DehE sequence must be searched for sequence identity with an experimentally determined structure. The amino acid sequence of the non-stereospecific haloalkanoic acid dehalogenase (DehE) from Rhizobium sp. RC1 was obtained from the National Centre for Biotechnology Information (NCBI) database (GenBank ID: CAA75671). The entry name was used in BRENDA (The Comprehensive Enzyme Information System) to identify the Enzyme Commission number for DehE (37). The original amino acid sequence of DehE from the NCBI database was

obtained in the FASTA format and subjected to the ProtParam and ColorSeq web servers via the Expasy (Expert Protein Analysis System) Proteomic Server for characterization (12). The DehE sequence was also searched by Basic Local Alignment Search Tool (BLAST) against the non-redundant (nr) protein sequences database (2). The sequence was further searched by BLAST for structural homologs in the RCSB Protein Data Bank (PDB). There were high identity and similarity between the sequences of Rhizobium sp. RC1 and Pseudomonas putida PP3 haloalkanoic acid dehalogenases. Therefore, we used homology modelling to construct the DehE structure.

Secondary structural analysis of DehEThe secondary structure prediction was performed using the Psipred, Jnet and SSpro servers integrated via the PHYRE (Protein Homology/Analogy Recognition Engine) web server (19). The server analysed the submitted sequence of DehE and illustrated the helix and sheet regions.

Construction of the DehE structure by homology modellingThe amino acid sequence of DehE was submitted to the SWISS-MODEL comparative protein modelling server for three-dimensional structure building (39). The resulting structure of DehE was generated as a PDB output file and the model was subjected to energy minimisation by integrated tools in the Swiss-Pdb viewer (13).

Model refinement. The initial model was refined with a molecular dynamics simulation that was carried out with the parallel version of GROMACS 4.5.1 (44) using the Gromos96 53a6 force field. The protein was put into a suitably sized simulation cubic box and solvated with 18009 SPC/E water molecules. In addition, 7 Na+ counter ions were added to neutralise the negative charge. The entire system was minimised using the steepest descent of 515 steps. To obtain the equilibrium geometry at 300 K and 1 atm, the system was heated at a weak temperature (t = 0.1 ps) and pressure (t = 0.5 ps) coupling by taking advantage of the Berendsen algorithms. All simulations were performed at a constant temperature and pressure with a non-bonded cut-off of 1.4 Å . The molecular dynamics simulation was carried out for 5 ns at 300 K, LINCS was used to constrain the bond length, and the particle mesh Ewald method was employed for the electrostatic interactions. The integration time step was 2 fs, and the neighbour list was updated every fifth step using the grid option and a cut-off distance of 1.4 Å. A periodic boundary condition was used with a constant number of particles in the systems, pressure, and temperature simulation criteria (NPT). During the simulation, every 1.0 ps of the actual frame was stored. The stabilised structure was taken from the trajectory system for the quality of the determination of the protein geometry and the structure folding reliability. Subsequently, the dynamic behaviour and the structural changes of the protein were analysed by the calculation of the root mean square deviation (RMSD).

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Structural assessment of the DehE model. The quality of the protein geometry was verified by employing Anolea (26), GROMOS (45), and Verify 3D (9) via the SWISS–MODEL Structure Assessment tools. PROCHECK analysis was also applied to generate a Ramachandran plot to view how well the dihedral angles are within the allowed conformations (36). After verification, the three-dimensional model of DehE was aligned with its template structure using Pymol software, and a RMSD value between the superimposed structures was obtained. The final structure of DehE was visualised using the same software.

DehE catalytic site predictions and tunnel construction. The active site and key catalytic residues of DehE were predicted by multiple sequence alignment using Multalin (8) and further viewed by Espript. The Q-SiteFinder server was used for pocket detection analysis and it located a few probes to identify interactions at the cavity within the DehE model. From the server analysis, the Q-SiteFinder predicted ten sites with different colour probabilities of being a catalytic site (32). The green spot is the most favourable of the predicted active sites. The DehE tunnel construction was built using the program CAVER (http://www.caver.cz/) by localising a starting point in the centre coordinate of the active site (34). The tunnel was visualised using the program PyMOL.

Validation of DehE by docking analysis. Docking of D- and L-2-chloropropionic acid, to the binding site of DehE was performed by AutoDock version 4.2 (28). AutoDockTools (ADT) was used to add polar hydrogen atoms and partial charges, and to assign Kollman charges to DehE and the ligands. To assess the binding site of 2-chloropropionic acid, the search space included only the active site region with the grid maps 62×52×52 points and a spacing of 0.375 Å using AutoGrid. The molecular docking was performed employing a Lamarckian Genetic Algorithm (LGA) with a total of 100 docking runs. The final structures were clustered and ranked according to the native Autodock scoring function. The top-ranked conformation of each ligand was selected. The same docking simulation approach was performed with the single point mutants of DehE, Trp34Ala, Phe37Ala and Ser188Ala, and with a triple mutant of these residues. Each mutated DehE was generated by changing a specific residue in the DehE model using the Pymol software.

Binding analysis of DehE with 3-chloropropionic acid3-chloropropionic acid, as a β-halogenated carboxylic acid compound, was obtained from Pubchem database (http://pubchem.ncbi.nlm.nih.gov/). The molecule was then analysed together with the DehE model by Autodock4.2 software in order to determine the binding affinity of the receptor towards the substance β-halogenated acid. The scale of the binding region was specified at 46×52×54 points of grid map and a spacing of 0.375 Å, using Autogrid. The Lamarckian Genetic Algorithm (LGA) was chosen for molecular docking analysis. The highest cluster of conformation with the lowest binding energy was selected to get the best docking result. The complex

molecule was visualised using Pymol visualization software. The observation included the docked receptor with a water molecule for a general view of binding that occurred in the active site of the DehE model.

Results and DiscussionPrimary structural analysis of DehE from Rhizobium sp. RC1Dehalogenase E (DehE) is a non-stereospecific α-haloalkanoic acid dehalogenase enzyme that belongs to the well-characterised HAD superfamily (17, 23, 42). Based on the BRENDA database analysis (comprehensive enzyme information system), DehE was clustered with the non-stereospecific haloalkanoic acid dehalogenases, EC 3.8.1.10 (2-haloacid dehalogenase, configuration-inverting). The DehE from Rhizobium sp. RC1 consists of 296 amino acid residues.

Twenty-five years ago, DehE was initially found to degrade both enantiomers of halogenated compounds that were introduced to Rhizobium sp. RC1. Since the discovery of this novel enzyme, DehE was studied until its kinetic activity with various kinds of halocompounds was determined. Unfortunately, structural information about the enzyme is still lacking, leading to difficulties for researchers who investigate the physical function of DehE. In this study, a structural prediction of DehE was proposed, and this is the first reported model of the dehalogenase produced by Rhizobium sp. Rc1. DehE was chosen in this study because it contains a unique criterion in that it can react with both stereoisomers of the substrate. Moreover, details of the other non-stereospecific haloalkanoic dehalogenase structures with homology to DehE have been characterised (31, 38).

Analysis by the program ProtParam (12) indicated that DehE has a molecular weight of 32675.4 Da and a thereotical pI of 5.29. DehE was found to have a negative GRAVY value (grand average of hydropathicity) of -0.109, which is generally hydrophilic. There were a total of 37 negatively charged and 29 positively charged residues in the amino acid sequence. Furthermore, the total number of atoms was 4,600 with a molecular formula of C1455h2302n402o429S12 and an Aliphatic Index of 93.01. From the Color Protein Sequence analysis provided by Integrated Webware (NPSA) in Pole Bioinformatic Lyonnais-Gerland (7), DehE was found to have less non-polar residues (48.3 %) than polar residues. In addition, among the polar residues, the frequency of polar uncharged residues was greater than that of polar charged residues.

Using in silico techniques, DehE was determined to have a molecular weight of 32 kDa which corresponds to the data gained from the technical study (17) and is very similar to most of the non-stereospecific haloalkanoic acid dehalogenases (4, 29, 48). In addition, DehE was found to achieve equilibrium at pH 5.29 and is described as a soluble protein that has many charged and polar side groups on its surface that provide affinity to water molecules. The large amount of the relative volume occupied by the aliphatic amino acid side chains of DehE

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may contribute to the thermostability of the enzyme. Disulfide bonds are believed to increase the protein stability and studies have demonstrated that introduction of disulfide bonds to the xylanase improved the thermostability by 5 °C but not the optimum temperature (18, 46). However, cysteine residues are not very common in dehalogenases, and DehE only contains four cysteine residues. The wild-type dehalogenase or DehE was thought to achieve its stability through other structural conformations such as hydrogen bonds and electrostatic interactions.

BLAST is a web-based program that has been extensively used to find regions of local similarity between sequences. The role of an unknown protein can be inferred from its sequence and structural homology to other known proteins. Using the Basic Local Alignment Search Tool (http://blast.ncbi.nlm.nih.gov/) against the non-redundant protein database, the DehE sequence was found to have at least 38 % identity and 58 % similarity, with the haloalkanoic acid dehalogenase from Alcaligenes xylosoxidans ssp. denitrificans ABiV (5), Pseudomonas putida PP3 (47), and Pseudomonas sp. 113. From the findings, DehE shares very high sequence identity to D,L-haloalkanoic acid dehalogenase from ABIV (Q59168) and DehI from PP3 (AAN60470) with an expected value of 6e-126 and 5e-125, respectively. The structure of DehI from Pseudomonas putida PP3 (PDB ID: 3BJXA) is the most closely related structure with DehE (72 % identity). The results from BLAST analyses indicate that the 32 kDa DehE has high sequence identity and similarity with non-stereospecific haloalkanoic acid dehalogenases from the HAD superfamily, and therefore may have an analogous fold and substrate specificity.

Secondary structure of DehEIn protein, the secondary structure is defined by the patterns of hydrogen bonds between the amide and carboxyl groups in the invariant parts of the amino acids in the polypeptide backbone or main chain. Table 1 lists the secondary structure predictions for DehE from Rhizobium sp. RC1, using three integrated software packages via the Protein Homology/anologY

Recognition Engine (Phyre) server (19). All of the predictions had similar results. The predicted quantity of helices and sheets was between 16 and 0, respectively, from 3 different consensus prediction servers. A majority of the predicted secondary structures had a high consensus probability. The longest α-helix for DehE was the fifteenth helix, and the shortest α-helices were both the first helix and the tenth helix. For the secondary structure predictions, DehE illustrates similar local contacts to its closely related enzyme, the crystallised structure of DehI from Pseudomonas putida PP3. Both enzymes contain mostly α-helices, and no β-strand formation was observed in any segments of the structure.

Homology modelling of DehEWe design one monomer of DehE (residues 1–296) in the apo-form. In this study, the three-dimensional model of DehE was constructed through template-based homology modelling using Swiss-Model, a protein modelling server. The Swiss-Model server chose DehI (PDB ID: 3bjx) as a template for homology modelling of DehE because it had the lowest E-value (9.43 e-125) and had the closest sequence identity (72.3 %). As DehE consisted of only loop regions and α-helices, it is considered an α-hydrolase enzyme. The final total energy of the predicted model was -14186.647 kJ/mol. By performing energy minimisation with the GROMOS96 software package implemented by the Swiss-PDB Viewer, the total energy of the DehE model reduced to -15230.833 kJ/mol.

In terms of predicted structure, the high sequence identity hinted at the possibility that DehE possessed a DehI-like fold. The basis of comparative or homology model building is that similar sequences adopt similar protein structures. As a majority of the newly sequenced proteins are likely to share similar structures with one that has already been experimentally determined, template-based protein structure prediction is considerably constructive and convenient (25). Energy minimisation was carried out for the model and resulted in a model with reduced energy. The energy minimisation step was utilised in an attempt to reach a more preferential global minimum level of total

TABLE 1 Consensus prediction on the whole residues of DehE by Psipred, Jnet and Sspro.

h: helix; e: sheet; c: coil

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energy as protein structures are more stable at low energy levels. According to the thermodynamic hypothesis, the native conformation of proteins corresponds to global minima of their free energy (10). This fold may be the native conformation of DehE, which is trying to follow the thermodynamic hypothesis. Proteins, described as the servants of life, can perform their function efficiently only in their native state.

Refinement of the initial DehE modelTo improve and verify the stability of the initial Swiss-Model structure, a molecular dynamics calculation was carried out for 5 ns. It is noteworthy that the RMSD of the resulting structures relative to the starting structures were ~1.7 Å after 2.8 ns, and the RMSD value did not change significantly after 2.8 ns of simulations as is illustrated in Fig. 1A. These RMSD values indicate that the employed simulation time was long enough to obtain an equilibrium structure of DehE. Thus, the applied molecular dynamics was necessary to specify the geometry of DehE. In addition, an average temperature of 5 ns simulation at 300 K for the studied system was equal to 300 ± 0.5 K (Fig. 1B). Therefore, the extracted equilibrium structure at 300 K for DehE was obtained under stable temperature conditions.

A

BFig. 1. Molecular dynamics simulation of DehE at 5 ns. Backbone root mean square deviation (RMSD) of the model during the simulation (A). Variation of the temperature during molecular simulation (B).

Fig. 2. Evaluation of DehE structure using GROMOS and Anolea.

The verification of DehE structureThe DehE model was verified after a molecular dynamics simulation using Anolea, GROMOS (Fig. 2) and Verify 3D (Fig. 3). For Anolea and GROMOS, negative values (green) represent a favourable energy environment for the amino acids, while positive values (red) correspond to an unfavourable energy environment. The vertical axis of Verify 3D shows the average 3D-1D profile score for each residue in a 21-residue sliding window from -1 (bad score) to 1+ (good score). In the Anolea and GROMOS validation results, a majority of the bars for the DehE structure were green, whereas the score is between 0.2 and 0.7 for Verify 3D. According to the structure verification analysis using Anolea, GROMOS and Verify 3D, the environment profile of the model was good. Residues with a score over 0.2 in Verify 3D should be considered reliable.

The Ramachandran plot is a two-dimensional plot of the Phi (φ) – Psi (ψ) torsion angles of the protein backbone that

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Fig. 3. Validation of DehE structure using Verify 3D.

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provides a simple view of the conformation of a protein. The three-dimensional structure of DehE was analysed using a Ramachandran plot by the program PROCHECK (20, 21). Most of the amino acids except for glycine and proline, were located in allowed regions. The φ and ψ distribution values of the Ramachandran plot of the non-glycine and non-proline residues after energy minimisation are presented in Fig. 4. Altogether 100.0 % of the residues were in favoured and allowed regions. The total quality of the G-factors was 0.13, which indicates that the designed model was a good quality structure.

Fig. 4. Ramachandran plot of the three-dimensional model of DehE.

Fig. 5. Three-dimensional structure of DehE coloured as a spectrum from N-terminus (blue) to C- terminus (red).

The analysis by PROCHECK reveals that all the residues are within the allowed limits of the Ramachandran plot. The model can be considered good when the values of the dihedral angles agree with the values of allowed conformations. The Ramachandran plot shows that the areas correspond to the conformations of the amino acids in the proteins to their van der Waals radii. The DehE structure was considered a good predicted model because the G-factor value in the PROCHECK analysis was close to zero. Only two glutamic acids were found in generously allowed regions of the Ramachandran graph, and this can be ignored because these positions do not interfere with the catalytic site of DehE as their locations are far from the domain site.

Three-dimensional (3D) structure of DehEThe three-dimensional structure of Rhizobial DehE was successfully depicted by the homology modelling server, Swiss-Model and was further optimised using molecular dynamics simulation and visualised with PyMOL software. The tertiary structure of DehE is illustrated in Fig. 5. Currently, the final model consists of a single polypeptide chain of DehE (32.7 kDa), with the major secondary structural elements being α-helices. This is the first structural model of a dehalogenase expressed in Rhizobium sp. RC1. In detail, DehE is comprised of 14 α-helices according to the Swiss-Model analysis (contradict to PHYRE which predicts 16 α-helices) that are connected together by a single loop. There are approximately 194 residues involved in α-helices while the rest are in loop regions. Obviously, a large amount of the α-helices detected in the DehE representation were similar to the crystallised structure of DehI which had been used as the template. Furthermore, the longest α-helices in the DehE structure were α6 and α13 and the shortest one was α7. Based on a conserved domain analysis via NCBI, DehE is composed of only one domain (residues 34 to 190) that hits to the DehI superfamily with the expected E-value of 1.31e-52.The structural prediction of DehE revealed a similar conformation to DehI. Furthermore, DehI also belongs to the HAD superfamily and is classified as a non-stereospecific dehalogenase. Although both structures contain only α-helices and loops, DehE has more α-helices than DehI. However, they both have the same long sixth α-helix. Additional confirmatory evidence was obtained by the overall superposition of backbone atoms of the enzyme with a very small average RMSD. Referring to the Pymol analysis, the overall superposition of the backbone atoms of DehE to the structure of DehI (template) had an average RMSD of 0.17 Å. This finding indicates that the molecular topologies of both enzymes are globally similar.

Study of conserved residuesWe used our homology modelled structure to search for conserved residues for non-stereospecific haloalkanoic acid dehalogenases. Analysis using a multiple sequence alignment program reveals a striking conservatism involving twelve active-site residues that line the active site of DehE: Trp34, Ala36, Phe37, Asn114, Tyr117 Ala187, Ser188, Asp189,

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Tyr265, Phe268, Ile269, and Ile272. These residues, were all identical in DL-DEX 113 of Pseudomonas sp. 113 and DehI of Pseudomonas putida PP3 (Fig. 6). Fig. 7 shows the cluster of the predicted amino acids within the active site. These conserved residues were previously analysed to form the active site for DehI (38). The superimposed DehE and DehI active sites have an RMSD value of 0.11 Å. Therefore, the expected active site and catalytic residues of DehE are parallel to DehI. Asp189 is expected to be involved in the mechanism of degradation by the Rhizobium sp. RC1 DehE since it is located among the conserved residues. Aspartate has been identified as an important nucleophile for the dehalogenation process. For instance, Asp194 in DL-DEX113 is responsible for the Sn2 direct attack mechanism, while Asp10 from L-2-haloacid dehalogenase (Pseudomonas sp. YL) and Asp105 from fluoroacetate dehalogenase (Moraxella sp. B) are accountable for Sn2 mechanism involving an ester intermediate.

The Q-SiteFinder was employed to support the prediction of the amino acids located in the active site of DehE (22). In a ligand-binding site analysis, the active site of DehE was located, and several residues were examined to act as a cluster for ligand attachment. Similarly, approximately 15 residues were involved in contributing a single exact binding site for a ligand to interact with. Furthermore, the predicted binding site had a volume of 114 cubic out of the 27,798 cubic for the entire protein. All of the binding-site residues suggested by the

server matched the prediction, except for Ala38, Cys39, and Phe110. In this case, 12 residues out of 15 corresponded to the conserved residues found in the active site analysis.

Fig. 7. The residues lining the active site of DehE (shown in skyblue colour).

After a series of analyses conducted to find the substrate binding site of DehE, a monomer structure was characterised to have a single substrate binding site, which was identified as a binding pocket lined by the following residues: Trp34, Ala36, Phe37, Asn114, Tyr117, Ala187, Ser188, Asp189, Tyr265, Phe268, Ile269, and Ile272. These residues are all identical in DehI and DL-DEX of Pseudomonas sp. 113 (30). the

Fig. 6. Multiple sequence alignment of the non-stereospecific haloalkanoic acid dehalogenases. Green boxes indicate conserve regions for active site residues.

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prediction of the active site of DehE reveals new information for future rational design of DehE studies. Currently, 12 residues in the active site of DehE were established through multiple sequence alignment, as the catalytic site for dehalogenation of D- and L-Haloalkanoic acids. These predicted residues were also supported by the results from the Q-SiteFinder server. The DehE structure reveals which of the residues identified in a study of the related DehI are likely to represent the key catalytic residues. Based on the residue conservation across DehE homologues and the one predicted using Q-SiteFinder, Asp189 was proposed to be involved in the mechanism of dehalogenation. Aspartate is an important residue at the active site that activates a water molecule to attack the α-Carbon of the substrate in an Sn2 displacement reaction (30, 38). Asp189 was proved to activate a water molecule for nucleophilic attack of the substrate chiral centre that results in an inversion of configuration of either the L- or D-substrates (Fig. 8) (38). This information will also lead to the future site-directed mutagenesis study of DehE.

DehE tunnelThe active site of DehE is inaccessible to substrates except for an internal narrow channel, which was located by the program CAVER. This tunnel is ~41.7 Å in length and extends from the interior substrate-binding site to the protein surface (Fig. 9A). The average gate diameter of the channel is 2.68 Å. According to the analysis, 22 residues were predicted to line the channel. The residues that were located at the entrance of the channel on the protein surface were both uncharged and basic polar residues (Fig. 9B). From the vacuum electrostatics view using PyMOL, the entrance of the tunnel was located at

the site indicated in blue colour (Basic region) (Fig. 9C). D- or L-2-chloropropionic acid could bind to the active site of DehE via the tunnel. The electrostatic surface of DehE at the tunnel entrance is highly basic because of the lysine, when compared to the rest of the molecular surface. This may be the method of attracting acidic substrates such as D- and L-2-chloropropionic acids to access the tunnel. DehI also has a tunnel that extends from the exterior towards the binding location. Both DehI and DehE tunnels are basic at the entrance, and so far there is no information about which amino acid plays an important role in DehI’s entry gate. Therefore, the presence of Lysine at the door tunnel of DehE is important.

Substrate orientation in the DehE active site A docking simulation illustrated that the binding site of D- or L-2-chloropropionic acid is located in the predicted active site of DehE, which is specifically at the buried surface area of the enzyme itself. The carbonyl oxygen atoms of L-2-chloropropionic acid were 1.7 Å to 1.8 Å from the hydrogen atoms of the side chains Ser188, Phe37 and Trp34 of DehE (Fig. 10A). The same residues interact with D-2-chloropropionic acid, but at distances of 1.7 Å to 2.2 Å from the hydrogen atoms (Fig. 10B). Both ranges of values are within the distance for hydrogen bonding to occur. The results from a multiple sequence alignment with other non-stereospecific haloalkanoic acid dehalogenases indicated that these residues are highly conserved (Fig. 10C).

There are several regions for the L- or D-2-chloropropionic acids to be oriented in the active site. The carboxyl group (COOH) of L-2-chloropropionic acid points at Ala36, Phe 37, Trp34, Ser188, and Ala187, and its chloride atom is directed

CH3

O- O

- O

H

H

R

COO-

ClH CH

R

COO-

Cl-OH

-

H

CH3

O- OH

R

COO-

HOH

Cl-

Fig. 8. Aspartate activates the water molecule for a nucleophilic attack displacing a Chloride ion via an Sn2-displacement reaction.

A B CFig. 9. Susbtrate access to the active site. Substrate channel (Mesh representation) towards the active site of DehE (A). Residues lining the channel entrance (B). Water-accessible surface of the DehE monomer (C) coloured according to electrostatic potential: red is negative, blue is positive; black mesh is the entrance to the channel.

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A B

C DFig. 10. In silico substrate binding, interacting residues and degradation step. Polar interactions between interacting residues and L-2-chloropropionic acid (A) or D-2-chloropropionic acid (B) in the active site of DehE. (C) Multiple sequence alignment of the non-stereospecific haloalkanoic acid dehalogenases of different bacteria: Trp34, Phe37, and Ser188 of DehE (red) are highly conserved among the other non-stereospecific haloalkanoic acid dehalogenases; conserved regions (*). (D) Interaction in the DehE active site: active site residues (green), L 2-chloropropionic acid (yellow), D-2-chloropropionic acid (orange), catalytic residue (red); carbons labelled on the substrates are α-Carbons.

at Ile269 and Asn114. Meanwhile, D-2-chloropropionic acid has a similar orientation with its carboxyl group and chloride atom facing Trp34, Ala36, Phe37, and Ser188 and Phe268, Ile269, and Tyr265, respectively. The binding affinity was also calculated using Autodock4.2; the results indicate that the free energy of binding for DehE towards L-2-chloropropionic acid is -4.68 kcal/mol, whereas the energy of binding for D-2-chloropropionic acid is -4.69 kcal/mol. The molecular docking simulation of D- and L-2-chloropropionic acid on the mutant type DehE (single point mutation: Trp34, Phe37 and Ser188, and the triple point mutant) showed that the mutation significantly decreases the binding affinity towards the substrates. The free binding energies of the DehE mutants were increased to the range of -3.75 kcal/mol to -3.29 kcal/mol. However, the binding site of the substrates in the single and triple point mutants of DehE is the same area as in the non-mutated type. Visualisation of the docking results indicated that the position of the chiral carbon of the L- and D-2-chloropropionic acid substrates (the focus of the enzyme catalysis) was approximately 3.9 Å from the oxygen atom of the acidic side chain of Asp189, which is consistent with the

finding that Asp189 is the key catalytic residue. The empty space between the substrate and Aspartate is probably for an inversion process. A water molecule was docked into the active site in the absence of a ligand. The only binding site for the water molecule was directly adjacent to Asn114 and Asp189, which is positioned appropriately for the nucleophilic attack of the chiral carbon of the docked substrates (Fig. 10D). It is clear that the nucleophile, which is the hydroxide anion (oh-) from the water molecule, is easy to directly attack the α-carbon without any obstacles. Here, the position of the nucleophile and a leaving group which is chloride, in the active site of DehE is crucial and proven to be suitable for an Sn2 mechanism to occur.

The DehE residues in the binding site that interact and bind the haloalkanoic acids substrates for a nucleophilic attack were similar to residues in DehI that were confirmed to grip the same substrates; however Tryptophan was not seen to form a hydrogen bond when a docking simulation was applied to DehE. When all the interacting residues were mutated to Alanine, the binding affinity was reduced.

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Substrate affinity to 3-chloropropionic acidA docking analysis demonstrated that the binding site of 3-chloropropionic acid was 1.6 Å to 2.1 Å from the hydrogen atoms of the side chains Ser188, Phe37, and Trp34 of DehE (Fig. 11). There was a specific region for 3-chloropropionic acid to be oriented in the active site. The carboxyl group (COOH) of 3-chloropropionic acid points at these interacting residues, and its chloride atom was directed at Asn114 and Tyr117. The binding affinity was also calculated using Autodock4.2; the results indicated that the free energy of binding for DehE towards 3-chloropropionic acid was -4.48 kcal/mol. A water molecule was docked into the active site in the absence of ligand. The only binding site for the water molecule was directly adjacent to Asn114 and Asp189, which was positioned appropriately for the nucleophilic attack of the β-carbon of the docked substrates. However, the β-carbon in this position was resistant to the nucleophilic attack.

Fig. 11. Substrate binding of 3-chloropropionic acid into the active site of DehE.

Currently, β-haloacid dehalogenases have become a topic of interest. Previous research found that many haloalkanoic acid dehalogenases were not able to facilitate dehalogenation towards the chloride located at the β-carbon (i.e. 3-chloropropionic acid or 3CP), including DehE. In this study, DehE was proven to bind with 3CP within the same interacting residues as D- or L-2-chloropropionic acid. The binding energy also indicates a favourable condition for DehE to bind to the substrate. However, the nucleophile (OH-) is unable to attack the chloride at the β-carbon possibly because of its location, which was slightly distant from substrate binding. However, the actual mechanism of enzyme action is not clear.

ConclusionsThis is the first model of DehE from Rhizobium sp. RC1 and its fold is similar to that of DehI, which is a member of the same class of enzymes. DehE contains an only α-helical motif, a domain structure that is conserved among non-stereospecific haloalkanoic acid dehalogenases. Twelve residues act as active site residues, of which three (Trp34, Phe37, and Ser188) are the interacting residues that hold D- and L-2-chloropropionic acid for nucleophilic attack. Asp189 was predicted to be an important catalytic residue that activates a water molecule to hydrolyse the substrate with the subsequent inversion,

followed by the release of a chloride ion. Surprisingly, based on computational analysis, 3CP, which is not a substrate for Group I and II dehalogenases, can bind to the DehE enzyme at the active site but is naturally resistant to a nucleophilic attack. All of this structural information gained about DehE can contribute to future rational design studies.

AcknowledgementsThe authors would like to thank the Malaysian Government and University of Technology, Malaysia (Universiti Teknologi Malaysia) – GUP Q.J130000.7135.00H34&FRGS-MOHE Vot No. 78354F008, for partially sponsoring this work.

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