Haloalkane dehalogenase from <i>Xanthobacter autotrophicus</i> GJ10 refined at 1.15 Å resolution

Ridder, Ivo S.; Rozeboom, H.J.; Dijkstra, Bauke W.

doi:10.1107/s090744499900534x

Cited by 41 publications

(33 citation statements)

References 55 publications

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“…In the TS structure, the leaving chloride to HE1 distances of Trp-125 and Trp-175 are almost equivalent. This is consistent with the high-resolution crystal structure of DhlA containing chloride and the molecular dynamics study of DhlA containing the transition state (17,27). When DCE is in the x-ray position (COMF2), interactions between the enzyme with DCE and Asp-124 are very similar in the ground-state and the transition-state structures.…”

Section: Resultssupporting

confidence: 85%

The importance of reactant positioning in enzyme catalysis: A hybrid quantum mechanics/molecular mechanics study of a haloalkane dehalogenase

Lau

Kahn

Bash

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

102

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Hybrid quantum mechanics͞molecular mechanics calculations using Austin Model 1 system-specific parameters were performed to study the S N2 displacement reaction of chloride from 1,2-dichloroethane (DCE) by nucleophilic attack of the carboxylate of acetate in the gas phase and by Asp-124 in the active site of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. The activation barrier for nucleophilic attack of acetate on DCE depends greatly on the reactants having a geometry resembling that in the enzyme or an optimized gas-phase structure. It was found in the gas-phase calculations that the activation barrier is 9 kcal͞mol lower when dihedral constraints are used to restrict the carboxylate nucleophile geometry to that in the enzyme relative to the geometries for the reactants without dihedral constraints. The calculated quantum mechanics͞molecular mechanics activation barriers for the enzymatic reaction are 16.2 and 19.4 kcal͞mol when the geometry of the reactants is in a near attack conformer from molecular dynamics and in a conformer similar to the crystal structure (DCE is gauche), respectively. This haloalkane dehalogenase lowers the activation barrier for dehalogenation of DCE by 2-4 kcal͞mol relative to the single point energies of the enzyme's quantum mechanics atoms in the gas phase. S N2 displacements of this sort in water are infinitely slower than in the gas phase. The modest lowering of the activation barrier by the enzyme relative to the reaction in the gas phase is consistent with mutation experiments.H aloalkane dehalogenases catalyze the hydrolytic cleavage of carbon-halogen bonds in aliphatic and aromatic halogenated compounds. The haloalkane dehalogenase from the nitrogen-fixing hydrogen bacterium Xanthobacter autotrophicus GJ10 (DhlA) prefers 1,2-dichloroethane (DCE) as substrate and converts it to 2-chloroethanol and chloride (1). A catalytic triad consisting of Asp-124, His-289, and Asp-260 is the central residue in the dehalogenation reaction (Scheme 1). On binding DCE in the predominantly hydrophobic active site, it undergoes S N 2 displacement of chloride by nucleophilic attack of Asp-124-COO Ϫ to form an ester intermediate at the rate of 50 Ϯ 10 s Ϫ1(1, 2). The ester intermediate is subsequently hydrolyzed by an activated water molecule. The dyad of His-289 and Asp-260 is thought to be responsible for activating the water molecule (3). This enzyme functions most efficiently at pH 8.2, likely because the imidazole NE2 of His-289 needs to be unprotonated for the hydrolysis reaction to proceed. Dehalogenases are of great interest because they are able to react with halogenated molecules under mild conditions (4). Many halogenated molecules are pollutants, and bioremediation is a highly desirable method for removing these harmful molecules from the environment. Dehalogenases have not existed in nature for a long time andhave not yet evolved into optimal enzymes. For example, the catalytic efficiency for DhlA is 4,550 M Ϫ1 ⅐s Ϫ1 for DCE (5), as compared with 5.6 ϫ 10 7 M Ϫ1 ⅐s Ϫ1for...

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Section: Resultssupporting

confidence: 85%

The importance of reactant positioning in enzyme catalysis: A hybrid quantum mechanics/molecular mechanics study of a haloalkane dehalogenase

Lau

Kahn

Bash

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

102

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“…The two hydrogen bonds are almost equivalent, 2.47 and 2.38 Å, respectively. This is consistent with the high‐resolution crystal structure of DhlA containing chloride 27 and the molecular dynamics study of DhlA containing the TS 36. The difference between E

_{italici → italicj *}^{(2)}

{ n (Cl 1 ) → σ*(NH 3 )} and E

_{italici → italicj *}^{(2)}

{ n (Cl 1 ) → σ*(NH 4 ) } of TS1 and RC1 illuminates that the stabilization effect of Trp125 and Trp175 on Cl 1 atom in TS1 is larger than that of RC1 by 15.67 kcal/mol.…”

Section: Resultssupporting

confidence: 89%

Effect of electrostatic interaction on the mechanism of dehalogenation catalyzed by haloalkane dehalogenase

Zhang

Chen

Zhang

et al. 2011

Int J of Quantum Chemistry

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Theoretical calculation has been carried out for the nucleophilic displacement reaction of 1,2-dichloroethane catalyzed by haloalkane dehalogenase. The results indicate that different hydrogen bond patterns of the oxyanion hole and the halide-stabilizing residues play an important role in the dehalogenation reaction. They cause concertedly an earlier transition state (TS) with the activation barrier of 16.60 kcal/mol. The stabilization effect of Trp125 and Trp175 on chlorine atom in the TS is larger than that of the reactant complex by 15.67 kcal/mol so that, they make contribution to the stabilization of the TS. Moreover, the reaction shows the enzymatic action can be attributed to a combination of reactant-state destabilization and transition-state electrostatic stabilization.

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“…Hence, the CNS topology and parameter files were adjusted such that the restraint on the main chain dihedral angle was relaxed to 20% of its original value without changing the restraints on the other X-N-C-X dihedral angles (X ϭ any atom). This was done to allow for the large variability of the angle observed in atomic resolution structures (25,26). After the adjustment, no deviating bond angles were flagged anymore.…”

Section: Methodsmentioning

confidence: 99%

Crystal Structures of Intermediates in the Dehalogenation of Haloalkanoates by l-2-Haloacid Dehalogenase

Ridder

Rozeboom

Kalk

et al. 1999

Journal of Biological Chemistry

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The L-2-haloacid dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic dehalogenation of small L-2-haloalkanoates to their corresponding D-2-hydroxyalkanoates, with inversion of the configuration at the C 2 atom. The structure of the apoenzyme at pH 8 was refined at 1.5-Å resolution. By lowering the pH, the catalytic activity of the enzyme was considerably reduced, allowing the crystal structure determination of the complexes with L-2-monochloropropionate and monochloroacetate at 1.7 and 2.1 Å resolution, respectively. Both complexes showed unambiguous electron density extending from the nucleophile Asp 8 to the C 2 atom of the dechlorinated substrates corresponding to a covalent enzyme-ester reaction intermediate. L-2-Haloacid dehalogenase (L-DEX) 1 catalyzes the hydrolytic dehalogenation of L-2-haloalkanoates to the corresponding D-2-hydroxyalkanoates with inversion of the configuration at the C 2 atom. Several homologous L-DEXs have been found in various Pseudomonas species and in Xanthobacter autotrophicus GJ10, a bacterium that is able to degrade the xenobiotic compound 1,2-dichloroethane (1, 2). This halogenated hydrocarbon is industrially produced in large quantities and is applied as a solvent and as an intermediate in the production of plastics (3). Because microorganisms that contain dehalogenases can be used in a biotechnological approach to detoxify halogenated aliphatics (4), such enzymes are a fascinating target for research. In addition, the stereospecificity of L-DEXs could make them useful for the biosynthesis of chiral 2-hydroxyalkanoic acids. Furthermore, L-2-haloacid dehalogenase is the prototypical member of a large superfamily of hydrolases, the haloacid dehalogenase (HAD) superfamily identified by Koonin and coworkers (5, 6). Based on three conserved sequence motifs, the L-DEXs, epoxide hydrolases, P-type ATPases, and a variety of phosphatases are recognized as members of this superfamily. Detailed information on L-DEXs is of interest as the enzyme is the only member of the HAD superfamily that has been structurally characterized so far.The x-ray structures of two L-2-haloacid dehalogenases have been reported, L-DEX YL from Pseudomonas sp. YL (Protein Data Bank code 1JUD (7)) and DhlB from X. autotrophicus GJ10 (Protein Data Bank code 1AQ6 (8)). The enzymes share a sequence identity of 40%, and their structures are closely related. Both enzymes have a mixed ␣/␤ core domain in a Rossmann fold with a four-helix bundle subdomain insertion. DhlB is somewhat larger, and the 21 extra residues form a two-helix excursion from the ␣/␤ core domain on the same side as the four-helix bundle. Together these helical domains provide a tight dimer interface and limit the substrate specificity of the X. autotrophicus enzyme to short substrates such as haloacetates and halopropionates (8,9).Comprehensive biochemical data have been obtained for the Pseudomonas enzyme (1, 10, 11).2 Asp 8 was identified as the nucleophile in the first step of the ...

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Haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 refined at 1.15 Å resolution

Cited by 41 publications

References 55 publications

The importance of reactant positioning in enzyme catalysis: A hybrid quantum mechanics/molecular mechanics study of a haloalkane dehalogenase

The importance of reactant positioning in enzyme catalysis: A hybrid quantum mechanics/molecular mechanics study of a haloalkane dehalogenase

Effect of electrostatic interaction on the mechanism of dehalogenation catalyzed by haloalkane dehalogenase

Crystal Structures of Intermediates in the Dehalogenation of Haloalkanoates by l-2-Haloacid Dehalogenase

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