Biochemical and structural characterisation of a haloalkane dehalogenase from a marine Rhodobacteraceae

A putative haloalkane dehalogenase has been identified in a marine Rhodobacteraceae and subsequently cloned and over-expressed in Escherichia coli. The enzyme has highest activity towards the substrates 1,6-dichlorohexane, 1-bromooctane, 1,3-dibromopropane and 1-bromohexane. The crystal structures of the enzyme in the native and product bound forms reveal a large hydrophobic active site cavity. A deeper substrate binding pocket defines the enzyme preference towards substrates with longer carbon chains. Arg136 at the bottom of the substrate pocket is positioned to bind the distal halogen group of extended di-halogenated substrates.     

一种来源于红螺菌科细菌新型卤醇脱卤酶的克隆表达及其酶学性质鉴定

作为一类多功能生物催化剂,卤醇脱卤酶在手性β-取代醇和环氧化合物合成应用方面备受关注.目前催化功能较为清楚的卤醇脱卤酶不足40种,且绝大部分催化性能并不能满足科学研究和实际应用的要求,因此挖掘并鉴定更多的卤醇脱卤酶具有重要意义.本文克隆表达了来源于红螺菌科细菌Rhodospirillaceae bacterium中一个...     

Biochemical characteristics of the novel haloalkane dehalogenase DatA, isolated from the plant pathogen Agrobacterium tumefaciens C58

We report the biochemical characterization of a novel haloalkane dehalogenase, DatA, isolated from the plant pathogen Agrobacterium tumefaciens C58. DatA possesses a peculiar pair of halide-stabilizing residues, Asn-Tyr, which have not been reported to play this role in other known haloalkane dehalogenases. DatA has a number of other unique characteristics, including substrate-dependent and cooperative kinetics, a dimeric structure, and excellent enantioselectivity toward racemic mixtures of chiral brominated alkanes and esters.     

Degradation of beta-hexachlorocyclohexane by haloalkane dehalogenase LinB from gamma-hexachlorocyclohexane-utilizing bacterium Sphingobium sp. MI1205

The technical formulation of hexachlorocyclohexane (HCH) mainly consists of the insecticidal γ-isomer and noninsecticidal α-, β-, and δ-isomers, among which β-HCH is the most recalcitrant and has caused serious environmental problems. A γ-HCH-utilizing bacterial strain, Sphingobium sp. MI1205, was isolated from soil which had been contaminated with HCH isomers. This strain degraded β-HCH more rapidly than the well-characterized γ-HCH-utilizing strain Sphingobium japonicum UT26. In MI1205, β-HCH was converted to 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL) via 2,3,4,5,6-pentachlorocyclohexanol (PCHL). A haloalkane dehalogenase LinB (LinB_MI) that is 98% identical (seven amino-acid differences among 296 amino acids) to LinB from UT26 (LinB_UT) was identified as an enzyme responsible for the two-step conversion of β-HCH to TCDL. This property of LinB_MI contrasted with that of LinB_UT, which catalyzed only the first step conversion of β-HCH to PCHL. Site-directed mutagenesis and computer modeling suggested that two of the seven different amino acid residues (V134 and H247) forming a catalytic pocket of LinB are important for the binding of PCHL in an orientation suitable for the reaction in LinB_MI. However, mutagenesis also indicated the involvement of other residues for the activity unique to LinB_MI. Sequence analysis revealed that MI1205 possesses the IS6100-flanked cluster that contains two copies of the linB _MI gene. This cluster is identical to the one located on the exogenously isolated plasmid pLB1, suggesting that MI1205 had recruited the linB genes by a horizontal transfer event.     

Purification and characterization of hydrolytic haloalkane dehalogenase from Xanthobacter autotrophicus GJ10.

A new enzyme, haloalkane dehalogenase, was isolated from the 1,2-dichloroethane-utilizing bacterium Xanthobacter autotrophicus GJ10. The purified enzyme catalyzed the hydrolytic dehalogenation of n-halogenated C1 to C4 alkanes, including chlorinated, brominated, and iodinated compounds. The highest activity was found with 1,2-dichloroethane, 1,3-dichloropropane, and 1,2-dibromoethane. The enzyme followed Michaelis-Menten kinetics, and the Km for 1,2-dichloroethane was 1.1 mM. Maximum activity was found at pH 8.2 and 37 degrees C. Thiol reagents such as p-chloromercuribenzoate and iodoacetamide rapidly inhibited the enzyme. The protein consists of a single polypeptide chain of a molecular weight of 36,000, and its amino acid composition and N-terminal sequence are given.     

Construction and characterization of histidine-tagged haloalkane dehalogenase (LinB) of a new substrate class from a gamma-hexachlorocyclohexane-degrading bacterium, Sphingomonas paucimobilis UT26.

The linB gene product (LinB), which is involved in the degradation of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis UT26, is a member of haloalkane dehalogenases with a broad range of substrate specificity. Elucidation of the factors determining its substrate specificity is of interest. Aiming to facilitate purification of recombinant LinB protein for site-directed mutagenesis analysis, a 6-histidyl tail was added to the C-terminus of LinB. The His-tagged LinB was specifically bound with Ni-NTA resin in the buffer containing 10 mM imidazole. After elution with 500 mM imidazole, quantitative recovery of protein occurred. The steady-state kinetic parameters of the His-tagged LinB for four substrates were in good agreement with that of wild-type recombinant LinB. Although the His-tagged LinB expressed in an average of 80% of the activity of the wild type LinB for 10 different substrates, the decrease was very similar for different substrates with the standard deviation of 5.5%. The small activity reduction is independent of the substrate shape, size, or number of substituents, indicating that the His-tagged LinB can be used for further mutagenesis studies. To confirm the suitability of this system for mutagenesis studies, two mutant proteins with substitution in putative halide binding residues (W109 and F151) were constructed, purified, and tested for activity. As expected, complete loss in activity of W109L and sustained activity of F151W were observed.     

Crystallization of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10

Haloalkane dehalogenases are enzymes that release chloride or bromide from n-halogenated alkanes. X-ray quality crystals of haloalkane dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 have been grown at room temperature from 64% saturated ammonium sulfate solutions (pH 6.2 to 6.4). The crystals diffract in the X-ray beam to at least 2.4 A resolution (1 A = 0.1 nm). Their space group is P2(1)2(1)2, with cell dimensions a = 94.1 A, b = 72.8 A, c = 41.4 A and alpha = beta = gamma = 90 degrees. There is one monomer (molecular weight 36,000) per asymmetric unit.     

Generating segmental mutations in haloalkane dehalogenase: a novel part in the directed evolution toolbox

Directed evolution techniques allow us to genuinely mimic molecular evolution in vitro. To enhance this imitation of natural evolutionary processes on a laboratory scale in even more detail, we developed an in vitro method for the generation of random deletions and repeats. The pairwise fusion of two fragments of the same gene that are truncated by exonuclease BAL-31 either at the 3′ or 5′ side results in a deletion or a repeat at the fusion point. Although in principle the method randomly covers the whole gene, it can also be limited to a predefined area in the sequence by controlling the level of the initial truncation. To test the procedure and to illustrate its potential, we used haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) as a model enzyme, since the adaptation of this enzyme towards new substrates is known to occur via the generation of this type of mutation. The results show that the mutagenesis method presented here is an effective tool for accessing formerly unexplorable sequence space and can contribute to the success of future directed evolution experiments.     

In vivo screening of haloalkane dehalogenase mutants

Haloalkane dehalogenase (Dh1A) from Xanthobacter autotrophicus GJ10 catalyzes the dehalogenation of short chain primary alkyl halides. Due to the high Km and low turnover, wild type Dh1A is not optimal for applications in bioremediation. We have developed an in vivo screen, based on a colorimetric pH indicator, to identify Dh1A mutant with improved catalytic activity. After screening 50,000 colonies, we identified a Dh1A mutant with a lower pH optimum. Sequence analysis of the mutant revealed a single substitution, alanine 149 to threonine, which is located close to the active site of Dh1A. Replacement of alanine 149 via site-directed mutagenesis with threonine, serine or cysteine retained the mutant phenotype. Other substitutions at position 149 show little or no activity.     

Reconstruction of mycobacterial dehalogenase Rv2579 by cumulative mutagenesis of haloalkane dehalogenase LinB

The homology model of protein Rv2579 from Mycobacterium tuberculosis H37Rv was compared with the crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26, and this analysis revealed that 6 of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. To characterize the effect of replacement of these six amino acid residues, mutations were introduced cumulatively into the six amino acid residues of LinB. The sixfold mutant, which was supposed to have the active site of Rv2579, exhibited haloalkane dehalogenase activity with the haloalkanes tested, confirming that Rv2579 is a member of the haloalkane dehalogenase protein family.     

Crystal structure of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26

The haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) is the enzyme involved in the degradation of the important environmental pollutant gamma-hexachlorocyclohexane. The enzyme hydrolyzes a broad range of halogenated cyclic and aliphatic compounds. Here, we present the 1.58 A crystal structure of LinB and the 2.0 A structure of LinB with 1,3-propanediol, a product of debromination of 1,3-dibromopropane, in the active site of the enzyme. The enzyme belongs to the alpha/beta hydrolase family and contains a catalytic triad (Asp108, His272, and Glu132) in the lipase-like topological arrangement previously proposed from mutagenesis experiments. The LinB structure was compared with the structures of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 and from Rhodococcus sp. and the structural features involved in the adaptation toward xenobiotic substrates were identified. The arrangement and composition of the alpha-helices in the cap domain results in the differences in the size and shape of the active-site cavity and the entrance tunnel. This is the major determinant of the substrate specificity of this haloalkane dehalogenase.     

Structure and activity of DmmA, a marine haloalkane dehalogenase

DmmA is a haloalkane dehalogenase (HLD) identified and characterized from the metagenomic DNA of a marine microbial consortium. Dehalogenase activity was detected with 1,3-dibromopropane as substrate, with steady-state kinetic parameters typical of HLDs (K_m = 0.24 ± 0.05 mM, k_cat = 2.4 ± 0.1 s⁻¹). The 2.2-Å crystal structure of DmmA revealed a fold and active site similar to other HLDs, but with a substantially larger active site binding pocket, suggestive of an ability to act on bulky substrates. This enhanced cavity was shown to accept a range of linear and cyclic substrates, suggesting that DmmA will contribute to the expanding industrial applications of HLDs.     

Characterization of 1,2-dibromoethane-degrading haloalkane dehalogenase from Bradyrhizobium japonicum USDA110

Haloalkane dehalogenases (DHAs, E.C. 3.8.1.5) are very promising biocatalytic tools for the bioremediation of environmental pollutants which consists of haloalkanes. In the present work, we investigated the DHA from Bradyrhizobium japonicum USDA110 (BjDHA). The dehalogenase activity of B. japonicum USDA110 and RT-PCR analysis revealed that the BjDHA gene expression is induced by 1,2-dibromoethane (1,2-DBE) during the early exponential phase. The BjDHA gene was cloned, expressed in Escherichia coli BL21 (DE3) and characterized. The enzyme catalyzes the irreversible hydrolysis of a variety of haloalkanes to the corresponding alcohol, halide, and a hydrogen ion. The catalytic properties of the recombinant enzyme were investigated and the kinetic parameters (K_m, k_cat) for a number of substrates were determined. The results showed that the BjDHA displays wide substrate specificity towards haloalkanes and particular high activity towards 1,2-DBE. The enzyme has a different catalytic triad topology compared to the Xanthobacter haloalkane dehalogenase and is more similar to the Rhodococcus enzyme. In addition, consistent with its broad specificity, the BjDHA has a substantially larger and more polar active site cavity compared to the Xanthobacter and Rhodococcus enzymes and as a consequence, BjDHA is able to dehalogenate longer and polar compounds. These properties make this enzyme very promising bioremediation tool for environmental applications.     

Crystal structure of the novel haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 reveals a special halide-stabilizing pair and enantioselectivity mechanism

A novel haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 belongs to the HLD-II subfamily and hydrolyzes brominated and iodinated compounds, leading to the generation of the corresponding alcohol, a halide ion, and a proton. Because DatA possesses a unique Asn-Tyr pair instead of the Asn-Trp pair conserved among the subfamily members, which was proposed to keep the released halide ion stable, the structural basis for its reaction mechanism should be elucidated. Here, we determined the crystal structures of DatA and its Y109W mutant at 1.70 and 1.95 Å, respectively, and confirmed the location of the active site by using its novel competitive inhibitor. The structural information from these two crystal structures and the docking simulation suggested that (i) the replacement of the Asn-Tyr pair with the Asn-Trp pair increases the binding affinity for some halogenated compounds, such as 1,3-dibromopropane, mainly due to the electrostatic interaction between Trp109 and halogenated compounds and the change of substrate-binding mode caused by the interaction and (ii) the primary halide-stabilizing residue is only Asn43 in the wild-type DatA, while Tyr109 is a secondary halide-stabilizing residue. Furthermore, docking simulation using the crystal structures of DatA indicated that its enantioselectivity is determined by the large and small spaces around the halogen-binding site.     

Expression of glycosylated haloalkane dehalogenase LinB in Pichia pastoris

Heterologous expression of the bacterial enzyme haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 in methylotrophic yeast Pichia pastoris is reported. The haloalkane dehalogenase gene linB was subcloned into the pPICZalphaA vector and integrated into the genome of P. pastoris. The recombinant LinB secreted from the yeast was purified to homogeneity and biochemically characterized. The deglycosylation experiment and mass spectrometry measurements showed that the recombinant LinB expressed in P. pastoris is glycosylated with a 2.8 kDa size of high mannose core. The specific activity of the glycosylated LinB was 15.6 +/- 3.7 micromol/min/mg of protein with 1,2-dibromoethane and 1.86 +/- 0.36 micromol/min/mg of protein with 1-chlorobutane. Activity and solution structure of the protein produced in P. pastoris is comparable with that of recombinant LinB expressed in Escherichia coli. The melting temperature determined by the circular dichroism (41.7+/-0.3 degrees C for LinB expressed in P. pastoris and 41.8 +/- 0.3 degrees C expressed in E. coli) and thermal stability measured by specific activity to 1-chlorobutane were also similar for two enzymes. Our results show that LinB can be extracellularly expressed in eukaryotic cell and glycosylation had no effect on activity, protein fold and thermal stability of LinB.     

Enzymatic dehalogenation of gas phase substrates with haloalkane dehalogenase

Haloalkane dehalogenase is an enzyme capable of catalyzing the conversion of short-chained (C(2)-C(8)) aliphatic halogenated hydrocarbons to a corresponding primary alcohol. Because of its broad substrate specificity for mono-, di-, and trisubstituted halogenated hydrocarbons and cofactor independence, haloalkane dehalogenases are attractive biocatalysts for gas-phase bioremediation of pollutant halogenated vapor emissions. A solid preparation of haloalkane dehalogenase from Rhodococcus rhodochrous was used to catalyze the dehalogenation reaction of 1-chlorobutane or 1,3-dichloropropane delivered in the gas phase. For optimal gas-phase dehalogenase activity, a relative humidity of 100%, a(w) = 1, was desired. With a 50% reduction in the vapor-phase hydration level, an 80% decrease in enzymatic activity was observed. The enzyme kinetics for the gas-phase substrates obeyed an Arrhenius-"like" behavior and the solid haloalkane dehalogenase preparation was more thermally stable than its water-soluble equivalent. Triethylamine was added to the gaseous reaction environment in efforts to increase the rate of reaction. A tenfold increase in the dehalogenase activity for the vapor-phase substrates was observed with the addition of triethylamine. Triethylamine altered the electrostatic environment of haloalkane dehalogenase via a basic shift in local pH, thereby minimizing the effect of the pH-reducing reaction product on enzyme activity. Both organic phase and solid-state buffers were used to confirm the activating role of the altered ionization state.     

Biochemical characterization of thermophilic dextranase from a thermophilic bacterium, Thermoanaerobacter pseudethanolicus

TPDex, a putative dextranase from Thermoanaerobacter pseudethanolicus, was purified as a single 70 kDa band of 7.37 U/mg. Its optimum pH was 5.2 and the enzyme was stable between pH 3.1 and 8.5 at 70 degrees C. A half-life comparison showed that TPDex was stable for 7.4 h at 70 degrees C, whereas Chaetominum dextranase (CEDex), currently used as a dextranase for sugar milling, was stable at 55 degrees C. TPDex showed broad dextranase activity regardless of dextran types, including dextran T2000, 742CB dextran, and alternan. TPDex showed the highest thermostability among the characterized dextranases, and may be a suitable enzyme for use in sugar manufacture without decreased temperature.     

Cloning and expression of the haloalkane dehalogenase gene dhmA from Mycobacterium avium N85 and preliminary characterization of DhmA

Haloalkane dehalogenases are microbial enzymes that catalyze cleavage of the carbon-halogen bond by a hydrolytic mechanism. Until recently, these enzymes have been isolated only from bacteria living in contaminated environments. In this report we describe cloning of the dehalogenase gene dhmA from Mycobacterium avium subsp. avium N85 isolated from swine mesenteric lymph nodes. The dhmA gene has a G+C content of 68.21% and codes for a polypeptide that is 301 amino acids long and has a calculated molecular mass of 34.7 kDa. The molecular masses of DhmA determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography are 34.0 and 35.4 kDa, respectively. Many residues essential for the dehalogenation reaction are conserved in DhmA; the putative catalytic triad consists of Asp123, His279, and Asp250, and the putative oxyanion hole consists of Glu55 and Trp124. Trp124 should be involved in substrate binding and product (halide) stabilization, while the second halide-stabilizing residue cannot be identified from a comparison of the DhmA sequence with the sequences of three dehalogenases with known tertiary structures. The haloalkane dehalogenase DhmA shows broad substrate specificity and good activity with the priority pollutant 1,2-dichloroethane. DhmA is significantly less stable than other currently known haloalkane dehalogenases. This study confirms that a hydrolytic dehalogenase is present in the facultative pathogen M. avium. The presence of dehalogenase-like genes in the genomes of other mycobacteria, including the obligate pathogens Mycobacterium tuberculosis and Mycobacterium bovis, as well as in other bacterial species, including Mesorhizobium loti, Xylella fastidiosa, Photobacterium profundum, and Caulobacter crescentus, led us to speculate that haloalkane dehalogenases have some other function besides catalysis of hydrolytic dehalogenation of halogenated substances.     

X-ray crystal structure of Mycobacterium tuberculosis haloalkane dehalogenase Rv2579

Haloalkane dehalogenases are enzymes well known to be important in bioremediation; the organisms from which they are produced are able to clean up toxic organohalides from polluted environments. However, besides being found in such contaminated environments, these enzymes have also been found in root or tissue-colonizing bacterial species. The haloalkane dehalogenase Rv2579 from Mycobacterium tuberculosis H37Rv has been cloned, expressed, purified and its crystal structure determined at high resolution (1.2A). In addition, the crystal structure of the enzyme has been determined in complex with the product from the reaction with 1,3-dibromopropane, i.e. 1,3-propanediol and in complex with the classical substrate of haloalkane dehalogenases, 1,2-dichloroethane. The enzyme is a two-domain protein having a catalytic domain of an alpha/beta hydrolase fold and a cap domain. The active site residues and the halide-stabilizing residues have been identified as Asp109, Glu133, His273, Asn39 and Trp110. Its overall structure is similar to those of other known haloalkane dehalogenases. Its mechanism of action involves an SN2 nucleophilic displacement.