In silico analysis of a putative dehalogenase from the genome of halophilic bacterium Halomonas smyrnensis AAD6T (original) (raw)
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We evaluate the applicability of automated molecular docking techniques and quantum mechanical calculations to the construction of a set of structures of enzyme-substrate complexes for use in Comparative binding energy (COMBINE) analysis to obtain 3D structure-activity relationships. The data set studied consists of the complexes of eighteen substrates docked within the active site of haloalkane dehalogenase (DhlA) from Xanthobacter autotrophicus GJ10. The results of the COMBINE analysis are compared with previously reported data obtained for the same dataset from modelled complexes that were based on an experimentally determined structure of the DhlAdichloroethane complex. The quality of fit and the internal predictive power of the two COMBINE models are comparable, but better external predictions are obtained with the new approach. Both models show a similar composition of the principal components. Small differences in the relative contributions that are assigned to important residues for explaining binding affinity differences can be directly linked to structural differences in the modelled enzyme-substrate complexes: (i) rotation of all substrates in the active site about their longitudinal axis, (ii) repositioning of the ring of epihalohydrines and the halogen substituents of 1,2-dihalopropanes, and (iii) altered conformation of the long-chain molecules (halobutanes and halohexanes). For external validation, both a novel substrate not included in the training series and two different mutant proteins were used. The results obtained can be useful in the future to guide the rational engineering of substrate specificity in DhlA and other related enzymes.
Structure-specificity relationships for haloalkane dehalogenases
Environmental Toxicology and Chemistry, 2001
A structural analysis of the substrate specificity of hydrolytic dehalogenases originating from three different bacterial isolates has been performed using the multiple computer-automated structure evaluation methodology. This methodology identifies structural fragments in substrate molecules that either activate or deactivate biological processes. The analysis presented in this contribution is based on newly measured dehalogenation data combined with data from the literature (91 substrates). The enzymes under study represent different specificity classes of haloalkane dehalogenases (haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, Rhodococcus erythropolis Y2, and Sphingomonas paucimobilis UT26). Three sets of structural rules have been identified to explain their substrate specificity and to predict activity for untested substrates. Predictions of activity and inactivity based on the structural rules from this analysis were provided for those compounds that were not yet tested experimentally. Predictions were also made for the compounds with available experimental data not used for the model construction (i.e., the external validation set). Correct predictions were obtained for 28 of 30 compounds in the validation set. Incorrect predictions were noted for two substrates outside the chemical domain of the set of compounds for which the structural rules were generated. A mechanistic interpretation of the structural rules generated provided a fundamental understanding of the structure-specificity relationships for the family of haloalkane dehalogenases.
The FEBS Journal
Reductive dehalogenases (RDases) of organohalide-respiring bacteria are cobamide-containing iron-sulfur proteins that catalyze different reductive dehalogenation reactions. Here, we report a functional analysis of two recombinant RDases, the tetrachloroethene (PCE) reductive dehalogenase (PceA) of Desulfitobacterium hafniense Y51 and the 1,2-dichloroethane (1,2-DCA) reductive dehalogenase (DcaA) of Desulfitobacterium dichloroeliminans DCA1. Both enzymes share 88% protein sequence identity, but appeared to have divergent mechanisms. In this study, the heterologously produced DcaA converted 1,2-DCA and 1,1,2-trichloroethane (1,1,2-TCA) via dihaloelimination to ethene and vinyl chloride, respectively. In addition, halogen substitution at PCE, trichloroethene (TCE) and tribromoethene (TBE) was observed, but only at low rates. In contrast, recombinant PceA exclusively converted halogenated ethenes and showed no dihaloelimination activity. In silico structural analysis of both RDases revealed similar architectures of their active site cavities. Exchange of the highly conserved Tyr298 to Phe led to a complete loss of the PCE, TCE and TBE conversion by both RDases, strengthening the assumption that Tyr298 functions as proton donor in the course of halogen substitution. The exchange did not affect the ability of DcaA to convert 1,2-DCA and 1,1,2-TCA. This result makes the involvement of a proton transfer in the dihaloelimination reaction unlikely and allows for a clear differentiation between two mechanisms working in DcaA and PceA. The analysis of the role of the active site structure for RDase function was extended to the mutations W118F that had a negative effect on DcaA function and W432F or T294V that caused alterations in the substrate specificity of the enzyme. Enzymes Tetrachloroethene reductive dehalogenase (EC 1.21.99.5), DCA-RDase.
PLOS ONE, 2015
The non-stereospecific α-haloalkanoic acid dehalogenase E (DehE) degrades many halogenated compounds but is ineffective against β-halogenated compounds such as 3chloropropionic acid (3CP). Using molecular dynamics (MD) simulations and site-directed mutagenesis we show here that introducing the mutation S188V into DehE improves substrate specificity towards 3CP. MD simulations showed that residues W34, F37, and S188 of DehE were crucial for substrate binding. DehE showed strong binding ability for D-2chloropropionic acid (D-2CP) and L-2-chloropropionic acid (L-2CP) but less affinity for 3CP. This reduced affinity was attributed to weak hydrogen bonding between 3CP and residue S188, as the carboxylate of 3CP forms rapidly interconverting hydrogen bonds with the backbone amide and side chain hydroxyl group of S188. By replacing S188 with a valine residue, we reduced the inter-molecular distance and stabilised bonding of the carboxylate of 3CP to hydrogens of the substrate-binding residues. Therefore, the S188V can act on 3CP, although its affinity is less strong than for D-2CP and L-2CP as assessed by K m . This successful alteration of DehE substrate specificity may promote the application of protein engineering strategies to other dehalogenases, thereby generating valuable tools for future bioremediation technologies.
Substrate specificity of haloalkane dehalogenases
Biochemical Journal, 2011
An enzyme's substrate specificity is one of its most important characteristics. The quantitative comparison of broad-specificity enzymes requires the selection of a homogenous set of substrates for experimental testing, determination of substrate-specificity data and analysis using multivariate statistics. We describe a systematic analysis of the substrate specificities of nine wild-type and four engineered haloalkane dehalogenases. The enzymes were characterized experimentally using a set of 30 substrates selected using statistical experimental design from a set of nearly 200 halogenated compounds. Analysis of the activity data showed that the most universally useful substrates in the assessment of haloalkane dehalogenase activity are 1bromobutane, 1-iodopropane, 1-iodobutane, 1,2-dibromoethane and 4-bromobutanenitrile. Functional relationships among the enzymes were explored using principal component analysis. Analysis of the untransformed specific activity data revealed that the overall activity of wild-type haloalkane dehalogenases decreases in the following order: LinB∼DbjA > DhlA∼DhaA∼DbeA∼ DmbA > DatA∼DmbC∼DrbA. After transforming the data, we were able to classify haloalkane dehalogenases into four SSGs (substrate-specificity groups). These functional groups are clearly distinct from the evolutionary subfamilies, suggesting that phylogenetic analysis cannot be used to predict the substrate specificity of individual haloalkane dehalogenases. Structural and functional comparisons of wild-type and mutant enzymes revealed that the architecture of the active site and the main access tunnel significantly influences the substrate specificity of these enzymes, but is not its only determinant. The identification of other structural determinants of the substrate specificity remains a challenge for further research on haloalkane dehalogenases.
Biochemistry, 2002
Haloalkane dehalogenases catalyze cleavage of the carbon-halogen bond in halogenated aliphatic compounds, resulting in the formation of an alcohol, a halide, and a proton as the reaction products. Three structural features of haloalkane dehalogenases are essential for their catalytic performance: (i) a catalytic triad, (ii) an oxyanion hole, and (iii) the halide-stabilizing residues. Halide-stabilizing residues are not structurally conserved among different haloalkane dehalogenases. The level of stabilization of the transition state structure of S N 2 reaction and halide ion provided by each of the active site residues in the enzymes DhlA, LinB, and DhaA was quantified by quantum mechanic calculations. The residues that significantly stabilize the halide ion were assigned as the primary (essential) or the secondary (less important) halide-stabilizing residues. Site-directed mutagenesis was conducted with LinB enzyme to confirm location of its primary halide-stabilizing residues. Asn38Asp, Asn38Glu, Asn38Phe, Asn38Gln, Trp109Leu, Phe151Leu, Phe151Trp, Phe151Tyr, and Phe169Leu mutants of LinB were constructed, purified, and kinetically characterized. The following active site residues were classified as the primary halide-stabilizing residues: Trp125 and Trp175 of DhlA; Asn38 and Trp109 of LinB; and Asn41 and Trp107 of DhaA. All these residues make a hydrogen bond with the halide ion released from the substrate molecule, and their substitution results in enzymes with significantly modified catalytic properties. The following active site residues were classified as the secondary halide-stabilizing residues: Phe172, Pro223, and Val226 of DhlA; Trp207, Pro208, and Ile211 of LinB; and Phe205, Pro206, and Ile209 of DhaA. The differences in the halide stabilizing residues of three haloalkane dehalogenases are discussed in the light of molecular adaptation of these enzymes to their substrates. haloalkane dehalogenase from Xanthobacter autotrophicus GJ10; DhaA, haloalkane dehalogenase from Rhodococcus erythropolis strains Y2, m15-3, HA1, GJ70, NCIMB13064, and TB2; LinB, haloalkane dehalogenase from Sphingomonas paucimobilis UT26; CBT, 1-chlorobutane; ∆Eelst(TS-ES), difference in electrostatic interaction energy between an amino acid residue and halogen in transition state and enzyme-substrate structures; ∆E elst(EP-ES), difference in electrostatic interaction energy between an amino acid residue and halogen/halide in enzyme-product and enzyme-substrate
Biochemistry, 2001
Comparative binding energy (COMBINE) analysis was conducted for 18 substrates of the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA): 1-chlorobutane, 1-chlorohexane, dichloromethane, 1,2-dichloroethane, 1,2-dichloropropane, 2-chloroethanol, epichlorohydrine, 2-chloroacetonitrile, 2-chloroacetamide, and their brominated analogues. The purpose of the COMBINE analysis was to identify the amino acid residues determining the substrate specificity of the haloalkane dehalogenase. This knowledge is essential for the tailoring of this enzyme for biotechnological applications. Complexes of the enzyme with these substrates were modeled and then refined by molecular mechanics energy minimization. The intermolecular enzyme-substrate energy was decomposed into residue-wise van der Waals and electrostatic contributions and complemented by surface area dependent and electrostatic desolvation terms. Partial least-squares projection to latent structures analysis was then used to establish relationships between the energy contributions and the experimental apparent dissociation constants. A model containing van der Waals and electrostatic intermolecular interaction energy contributions calculated using the AMBER force field explained 91% (73% cross-validated) of the quantitative variance in the apparent dissociation constants. A model based on van der Waals intermolecular contributions from AMBER and electrostatic interactions derived from the Poisson-Boltzmann equation explained 93% (74% crossvalidated) of the quantitative variance. COMBINE models predicted correctly the change in apparent dissociation constants upon single-point mutation of DhlA for six enzyme-substrate complexes. The amino acid residues contributing most significantly to the substrate specificity of DhlA were identified; they include Asp124, Trp125, Phe164, Phe172, Trp175, Phe222, Pro223, and Leu263. These residues are suitable targets for modification by site-directed mutagenesis. † Supported by NATO Linkage Grant MTECH.LG.974701. 1 Abbreviations: BUW, block unscaled weights; COMBINE, comparative binding energy; DhlA, haloalkane dehalogenase from X. autotrophicus GJ10; Eele ES , enzyme-substrate electrostatic interaction energy in the presence of the surrounding solvent derived from the Poisson-Boltzmann equation; Edesolv-sur, surface area dependent term of the desolvation energy; Edesolv S , desolvation energy of a substrate; Edesolv E , desolvation energy of an enzyme; FFD, fractional factorial design; ∆Gele, overall electrostatic free energy change upon binding; Gele ES , electrostatic energy for all atoms in the enzyme-substrate complex; Gele S , electrostatic energy for substrate atoms; Gele E , electrostatic energy for enzyme atoms; ∆Gdesolv S , change in desolvation energy of the substrate upon binding; ∆Gdesolv E , change in desolvation energy of the enzyme upon binding; ∆Hvap, enthalpy of vaporization; INTCQ 2 , intercept of the permutation plot for Q 2 ; Km, enzyme-substrate dissociation constant; PLS, partial least-squares; Q 2 , cross-validated correlation coefficient; R 2 , correlation coefficient; SDEC, standard deviation of error of calculation for the working or training set; SDEPint, standard deviation of error of (internal) predictions; SDEPext, standard deviation of error of (external) predictions; UV, scaling to unit variance; ∆U, total binding energy.
FEBS Journal, 2013
Haloalkane dehalogenases catalyze the hydrolysis of carbon-halogen bonds in various chlorinated, brominated and iodinated compounds. These enzymes have a conserved pair of halide-stabilizing residues that are important in substrate binding and stabilization of the transition state and the halide ion product via hydrogen bonding. In all previously known haloalkane dehalogenases, these residues are either a pair of tryptophans or a tryptophan-asparagine pair. The newly-isolated haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 (EC 3.8.1.5) possesses a unique halide-stabilizing tyrosine residue, Y109, in place of the conventional tryptophan. A variant of DatA with the Y109W mutation was created and the effects of this mutation on the structure and catalytic properties of the enzyme were studied using spectroscopy and pre-steady-state kinetic experiments. Quantum mechanical and molecular dynamics calculations were used to obtain a detailed analysis of the hydrogen-bonding patterns within the active sites of the wild-type and the mutant, as well as of the stabilization of the ligands as the reaction proceeds. Fluorescence quenching experiments suggested that replacing the tyrosine with tryptophan improves halide binding by 3.7-fold, presumably as a result of the introduction of an additional hydrogen bond. Kinetic analysis revealed that the mutation affected the substrate specificity of the enzyme and reduced its K 0.5 for selected halogenated substrates by a factor of 2-4, without impacting the rate-determining hydrolytic step. We conclude that DatA is the first natural haloalkane dehalogenase that stabilizes its substrate in the active site using only a single hydrogen bond, which is a new paradigm in catalysis by this enzyme family.
Structural and functional analysis of a novel haloalkane dehalogenase with two halide-binding sites
Acta Crystallographica Section D Biological Crystallography, 2014
The crystal structure of the novel haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 revealed the presence of two chloride ions buried in the protein interior. The first halide-binding site is involved in substrate binding and is present in all structurally characterized haloalkane dehalogenases. The second halide-binding site is unique to DbeA. To elucidate the role of the second halide-binding site in enzyme functionality, a two-point mutant lacking this site was constructed and characterized. These substitutions resulted in a shift in the substrate-specificity class and were accompanied by a decrease in enzyme activity, stability and the elimination of substrate inhibition. The changes in enzyme catalytic activity were attributed to deceleration of the ratelimiting hydrolytic step mediated by the lower basicity of the catalytic histidine.
The Scientific World Journal
Recently, haloacid dehalogenases have gained a lot of interest because of their potential applications in bioremediation and synthesis of chemical products. The haloacid dehalogenase gene from Bacillus cereus IndB1 (bcfd1) has been isolated, expressed, and Bcfd1 enzyme activity towards monochloroacetic acid has been successfully studied. However, the structure, enantioselectivity, substrate range, and essential residues of Bcfd1 have not been elucidated. This research performed computational studies to predict the Bcfd1 protein structure and analyse the interaction of Bcfd1 towards several haloacid substrates to comprehend their enantioselectivity and substrates’ range. Structure prediction revealed that Bcfd1 protein consist of two domains. The main domain consists of seven β-sheets connected by six α-helices and four 310-helices forming a Rossmannoid fold. On the other hand, the cap domain consists of five β-sheets connected by five α-helices. The docking simulation showed that 2-...