Influence of mutations of Val226 on the catalytic rate of haloalkane dehalogenase

Haloalkane dehalogenase converts haloalkanes to their corresponding alcohols. The 3D structure, reaction mechanism and kinetic mechanism have been studied. The steady state kcat with 1,2-dichloroethane and 1,2-dibromoethane is limited mainly by the rate of release of the halide ion from the buried active-site cavity. During catalysis, the halogen that is cleaved off (Clα) from 1,2-dichloroethane interacts with Trp125 and the Clβ interacts with Phe172. Both these residues have van der Waals contacts with Val226. To establish the effect of these interactions on catalysis, and in an attempt to change enzyme activity without directly mutating residues involved in catalysis, we mutated Val226 to Gly, Ala and Leu. The Val226Ala and Val226Leu mutants had a 2.5-fold higher catalytic rate for 1,2-dibromoethane than the wild-type enzyme. A pre-steady state kinetic analysis of the Val226Ala mutant enzyme showed that the increase in kcat could be attributed to an increase in the rate of a conformational change that precedes halide release, causing a faster overall rate of halide dissociation. The kcat for 1,2-dichloroethane conversion was not elevated, although the rate of chloride release was also faster than in the wild-type enzyme. This was caused by a 3-fold decrease in the rate of formation of the alkyl-enzyme intermediate for 1,2-dichloroethane. Val226 seems to contribute to leaving group (Clα or Brα) stabilization via Trp125, and can influence halide release and substrate binding via an interaction with Phe172. These studies indicate that wild-type haloalkane dehalogenase is optimized for 1,2-dichloroethane, although 1,2-dibromoethane is a better substrate.

Specificity and Kinetics of Haloalkane Dehalogenase

Haloalkane dehalogenase converts halogenated alkanes to their corresponding alcohols. The active site is buried inside the protein and lined with hydrophobic residues. The reaction proceeds via a covalent substrate-enzyme complex. This paper describes a steady-state and pre-steady-state kinetic analysis of the conversion of a number of substrates of the dehalogenase. The kinetic mechanism for the “natural” substrate 1,2-dichloroethane and for the brominated analog and nematocide 1,2-dibromoethane are given. In general, brominated substrates had a lower Km, but a similar kcat than the chlorinated analogs. The rate of C-Br bond cleavage was higher than the rate of C-Cl bond cleavage, which is in agreement with the leaving group abilities of these halogens. The lower Km for brominated compounds therefore originates both from the higher rate of C-Br bond cleavage and from a lower Ks for bromo-compounds. However, the rate-determining step in the conversion (kcat) of 1,2-dibromoethane and 1,2-dichloroethane was found to be release of the charged halide ion out of the active site cavity, explaining the different Km but similar kcat values for these compounds. The study provides a basis for the analysis of rate-determining steps in the hydrolysis of various environmentally important substrates.

Kinetic Characterization and X-ray Structure of a Mutant of Haloalkane Dehalogenase with Higher Catalytic Activity and Modified Substrate Range

Conversion of halogenated aliphatics by haloalkane dehalogenase proceeds via the formation of a covalent alkyl-enzyme intermediate which is subsequently hydrolyzed by water. In the wild type enzyme, the slowest step for both 1,2-dichloroethane and 1,2-dibromoethane conversion is a unimolecular enzyme isomerization preceding rapid halide dissociation. Phenylalanine 172 is located in a helix-loop-helix structure that covers the active site cavity of the enzyme, interacts with the Clβ of 1,2-dichloroethane during catalysis, and could be involved in stabilization of this helix-loop-helix region of the cap domain of the enzyme. To obtain more information about the role of this residue in dehalogenase function, we performed a mutational analysis of position 172 and studied the kinetics and X-ray structure of the Phe172Trp enzyme. The Phe172Trp mutant had a 10-fold higher kcat/Km for 1-chlorohexane and a 2-fold higher kcat for 1,2-dibromoethane than the wild-type enzyme. The X-ray structure of the Phe172Trp enzyme showed a local conformational change in the helix-loop-helix region that covers the active site. This could explain the elevated activity for 1-chlorohexane of the Phe172Trp enzyme, since it allows this large substrate to bind more easily in the active site cavity. Pre-steady-state kinetic analysis showed that the increase in kcat found for 1,2-dibromoethane conversion could be attributed to an increase in the rate of an enzyme isomerization step that preceeds halide release. The observed conformational difference between the helix-loop-helix structures of the wild-type enzyme and the faster mutant suggests that the isomerization required for halide release could be a conformational change that takes place in this region of the cap domain of the dehalogenase. It is proposed that Phe172 is involved in stabilization of the helix-loop-helix structure that covers the active site of the enzyme and creates a rigid hydrophobic cavity for small apolar halogenated alkanes.

Far-infrared spectroelectrochemistry: a study of linear molybdenum/iron/sulfur clusters

The far-infrared spectroelectrochemistry of linear M/Fe/S (M=Mo, W) complexes was investigated in methylene chloride and dichloroethane. With CsI as spectral windows, bands above 200 cm−1 can be observed in methylene chloride, except for a weak methylene chloride band at 450 cm−1. Substitution of dichloroethane for methylene chloride, solvents of nearly identical electrochemical properties, allows one to observe solute bands in the 450-cm−1 region. The far-infrared spectroelectrochemistry of [MoFe2S4Cl4]2− and its tungsten analogue was investigated. The disappearance of the oxidation bands and the appearance of bands due to the reduced product could be clearly observed. The origin of the vibrational bands could be clearly identified using 34S-substituted complexes. In addition to the far-infrared bands, the resonance Raman spectroelectrochemistry of the oxidized and reduced complex, along with the 34S-substituted complexes was obtained. Far-infrared and resonance Raman spectroelectrochemistry can be combined to understand the electrochemical mechanism of transition metal complexes. The far-infrared spectroelectrochemistry of [MoFe2S4Cl4]2− and its tungsten analogue was investigated. The disappearance of the initial bands and the appearance of bands due to the reduced product could be clearly observed. Resonance Raman spectroscopy and the use of 34S-substituted complexes were used for characterization of the reactant and products

Diverse reductive dehalogenases are associated with Clostridiales-enriched microcosms dechlorinating 1,2-dichloroethane

The achievement of successful biostimulation of active microbiomes for the cleanup of a polluted site is strictly dependent on the knowledge of the key microorganisms equipped with the relevant catabolic genes responsible for the degradation process. In this work, we present the characterization of the bacterial community developed in anaerobic microcosms after biostimulation with the electron donor lactate of groundwater polluted with 1,2-dichloroethane (1,2-DCA). Through a multilevel analysis, we have assessed (i) the structural analysis of the bacterial community; (ii) the identification of putative dehalorespiring bacteria; (iii) the characterization of functional genes encoding for putative 1,2-DCA reductive dehalogenases (RDs). Following the biostimulation treatment, the structure of the bacterial community underwent a notable change of the main phylotypes, with the enrichment of representatives of the order Clostridiales. Through PCR targeting conserved regions within known RD genes, four novel variants of RDs previously associated with the reductive dechlorination of 1,2-DCA were identified in the metagenome of the Clostridiales-dominated bacterial community

Mechanistic analysis of an asymmetric palladium-catalyzed conjugate addition of arylboronic acids to β-substituted cyclic enones.

An asymmetric palladium-catalyzed conjugate addition reaction of arylboronic acids to enone substrates was investigated mechanistically. Desorption electrospray ionization coupled to mass spectrometry was used to identify intermediates of the catalytic cycle and delineate differences in substrate reactivity. Our findings provide evidence for the catalytic cycle proceeding through formation of an arylpalladium(II) cation, subsequent formation of an arylpalladium-enone complex, and, ultimately, formation of the new C-C bond. Reaction monitoring in both positive and negative ion modes revealed that 4-iodophenylboronic acid formed a relatively stable trimeric species under the reaction conditions

Filling of mater-BI with nanoclays to enhance the biofilm rigidity

We investigated the efficacy of several nanoclays (halloysite, sepiolite and laponite) as nanofillers for Mater-Bi, which is a commercial bioplastic extensively used within food packaging applications. The preparation of Mater-Bi/nanoclay nanocomposite films was easily achieved by means of the solvent casting method from dichloroethane. The prepared bio-nanocomposites were characterized by dynamic mechanical analysis (DMA) in order to explore the effect of the addition of the nanoclays on the mechanical behavior of the Mater-Bi-based films. Tensile tests found that filling Mater-Bi with halloysite induced the most significant improvement of the mechanical performances under traction force, while DMA measurements under the oscillatory regime showed that the polymer glass transition was not affected by the addition of the nanoclay. The tensile properties of the Mater-Bi/halloysite nanotube (HNT) films were competitive compared to those of traditional petroleum plastics in terms of the elastic modulus and stress at the breaking point. Both the mechanical response to the temperature and the tensile properties make the bio-nanocomposites appropriate for food packaging and smart coating purposes. Here, we report a preliminary study of the development of sustainable hybrid materials that could be employed in numerous industrial and technological applications within materials science and pharmaceutics

Theoretical calculations of an osmium molecular switch

Indexación: Web of Science; Scielo.ABSTRACT We have investigated the molecular, electronic and optical properties of the [Os(tpy-py)2]2+ complex (tpy-py = 4'-(4-pyridyl)-2,2':6',2"-terpyridine) and its protonated derivative [Os(tpy-pyH)2]4+ through Density Functional Relativistic calculations including Scalar and Spin Orbit corrections. The molecular geometry of the parent complex is not strongly modified by the protonation at the basic nitrogen atoms of the pyridine moieties of the terpyridine ligands in the complex. On the other hand, the optical properties of these complexes can be controlled by a change in the chemical acid-base environment, converting them into suitable materials to act as molecular switches or pH sensor devices.http://ref.scielo.org/54hzc