首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
Haloalkane dehalogenases catalyse environmentally important dehalogenation reactions. These microbial enzymes represent objects of interest for protein engineering studies, attempting to improve their catalytic efficiency or broaden their substrate specificity towards environmental pollutants. This paper presents the results of a comparative study of haloalkane dehalogenases originating from different organisms. Protein sequences and the models of tertiary structures of haloalkane dehalogenases were compared to investigate the protein fold, reaction mechanism and substrate specificity of these enzymes. Haloalkane dehalogenases contain the structural motifs of alpha/beta-hydrolases and epoxidases within their sequences. They contain a catalytic triad with two different topological arrangements. The presence of a structurally conserved oxyanion hole suggests the two-step reaction mechanism previously described for haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. The differences in substrate specificity of haloalkane dehalogenases originating from different species might be related to the size and geometry of an active site and its entrance and the efficiency of the transition state and halide ion stabilization by active site residues. Structurally conserved motifs identified within the sequences can be used for the design of specific primers for the experimental screening of haloalkane dehalogenases. Those amino acids which were predicted to be functionally important represent possible targets for future site-directed mutagenesis experiments.  相似文献   

2.
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 1-bromobutane, 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.  相似文献   

3.
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.  相似文献   

4.
Haloalkane dehalogenases are bacterial enzymes capable of carbon-halogen bond cleavage in halogenated compounds. To obtain insights into the mechanism of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB), we studied the steady-state and presteady-state kinetics of the conversion of the substrates 1-chlorohexane, chlorocyclohexane, and bromocyclohexane. The results lead to a proposal of a minimal kinetic mechanism consisting of three main steps: (i) substrate binding, (ii) cleavage of the carbon-halogen bond with simultaneous formation of an alkyl-enzyme intermediate, and (iii) hydrolysis of the alkyl-enzyme intermediate. Release of both products, halide and alcohol, is a fast process that was not included in the reaction mechanism as a distinct step. Comparison of the kinetic mechanism of LinB with that of haloalkane dehalogenase DhlA from Xantobacter autotrophicus GJ10 and the haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 shows that the overall mechanisms are similar. The main difference is in the rate-limiting step, which is hydrolysis of the alkylenzyme intermediate in LinB, halide release in DhlA, and liberation of an alcohol in DhaA. The occurrence of different rate-limiting steps for three enzymes that belong to the same protein family indicates that extrapolation of this important catalytic property from one enzyme to another can be misleading even for evolutionary closely related proteins. The differences in the rate-limiting step were related to: (i) number and size of the entrance tunnels, (ii) protein flexibility, and (iii) composition of the halide-stabilizing active site residues based on comparison of protein structures.  相似文献   

5.
Mechanistic insight into the biochemistry of carbon-halogen bond cleavage is rapidly growing because of recent structural, biochemical and computational studies that have provided further insight into how haloalkane dehalogenases achieve their impressive catalytic activity. An occluded water-free active-site cavity together with strong hydrogen bond donating groups reduce the transition state energy barrier compared with that of the non-enzymatic reaction in water. Even though all known haloalkane dehalogenases belong to the alpha/beta-hydrolase fold family, there are interesting differences in mechanistic and kinetic details, as shown by properties of mutant enzymes and transient-state kinetic studies. To improve enzymatic degradation of some environmentally important recalcitrant compounds, site-directed mutagenesis and directed-evolution studies are being done.  相似文献   

6.
Halohydrin dehalogenases, also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides. Three novel bacterial genes encoding halohydrin dehalogenases were cloned and expressed in Escherichia coli, and the enzymes were shown to display remarkable differences in substrate specificity. The halohydrin dehalogenase of Agrobacterium radiobacter strain AD1, designated HheC, was purified to homogeneity. The k(cat) and K(m) values of this 28-kDa protein with 1,3-dichloro-2-propanol were 37 s(-1) and 0.010 mM, respectively. A sequence homology search as well as secondary and tertiary structure predictions indicated that the halohydrin dehalogenases are structurally similar to proteins belonging to the family of short-chain dehydrogenases/reductases (SDRs). Moreover, catalytically important serine and tyrosine residues that are highly conserved in the SDR family are also present in HheC and other halohydrin dehalogenases. The third essential catalytic residue in the SDR family, a lysine, is replaced by an arginine in halohydrin dehalogenases. A site-directed mutagenesis study, with HheC as a model enzyme, supports a mechanism for halohydrin dehalogenases in which the conserved Tyr145 acts as a catalytic base and Ser132 is involved in substrate binding. The primary role of Arg149 may be lowering of the pK(a) of Tyr145, which abstracts a proton from the substrate hydroxyl group to increase its nucleophilicity for displacement of the neighboring halide. The proposed mechanism is fundamentally different from that of the well-studied hydrolytic dehalogenases, since it does not involve a covalent enzyme-substrate intermediate.  相似文献   

7.
We propose Cys-X scanning as a semisynthetic approach to engineer the functional properties of recombinant proteins. As in the case of Ala scanning, key residues in the primary structure are identified, and one of them is replaced by Cys via site-directed mutagenesis. The thiol of the residue introduced is subsequently modified by alternative chemical reagents to yield diverse Cys-X mutants of the protein. This chemical approach is orthogonal to Ala or Cys scanning and allows the expansion of the repertoire of amino acid side chains far beyond those present in natural proteins. In its present application, we have introduced Cys-X residues in human glutathione transferase (GST) M2-2, replacing Met-212 in the substrate-binding site. To achieve selectivity of the modifications, the Cys residues in the wild-type enzyme were replaced by Ala. A suite of simple substitutions resulted in a set of homologous Met derivatives ranging from normethionine to S-heptyl-cysteine. The chemical modifications were validated by HPLC and mass spectrometry. The derivatized mutant enzymes were assayed with alternative GST substrates representing diverse chemical reactions: aromatic substitution, epoxide opening, transnitrosylation, and addition to an ortho-quinone. The Cys substitutions had different effects on the alternative substrates and differentially enhanced or suppressed catalytic activities depending on both the Cys-X substitution and the substrate assayed. As a consequence, the enzyme specificity profile could be changed among the alternative substrates. The procedure lends itself to large-scale production of Cys-X modified protein variants.  相似文献   

8.
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.  相似文献   

9.
Two putative haloalkane dehalogenases (HLDs) of the HLD‐I subfamily, DccA from Caulobacter crescentus and DsaA from Saccharomonospora azurea, have been identified based on sequence comparisons with functionally characterized HLD enzymes. The two genes were synthesized, functionally expressed in E. coli and shown to have activity toward a panel of haloalkane substrates. DsaA has a moderate activity level and a preference for long (greater than 3 carbons) brominated substrates, but little activity toward chlorinated alkanes. DccA shows high activity with both long brominated and chlorinated alkanes. The structure of DccA was determined by X‐ray crystallography and was refined to 1.5 Å resolution. The enzyme has a large and open binding pocket with two well‐defined access tunnels. A structural alignment of HLD‐I subfamily members suggests a possible basis for substrate specificity is due to access tunnel size.  相似文献   

10.
The role of a polypeptide loop in tyrosine hydroxylase (TyrH) whose homolog in phenylalanine hydroxylase (PheH) takes on a different conformation when substrates are bound has been studied using site-directed mutagenesis. The loop spans positions 177 to 191; alanine was introduced into those positions, introducing one alanine substitution per TyrH variant. Mutagenesis of residues in the center of the loop resulted in alterations in the KM values for substrates, the Vmax value for dihydroxyphenylalanine (DOPA) synthesis, and the coupling of tetrahydropterin oxidation to tyrosine hydroxylation. The variant with the most altered KM value for 6-methyltetrahydropterin was TyrH F184A. The variants with the most affected K(tyr) values were those with substitutions in the center of the loop, TyrH K183A, F184A, D185A, P186A and D187A. These five variants also had the most reduced Vmax values for DOPA synthesis. Alanine substitution in positions 182-186 resulted in lowered ratios of tyrosine hydroxylation to tetrahydropterin oxidation. TyrH F184Y and PheH Y138F, variants with the residue at the center of the loop substituted with the residue present at the homologous position in the other hydroxylase, were also studied. The V/K(tyr) to V/K(phe) ratios for these variants were altered significantly, but the results did not suggest that F184 of TyrH or Y138 of PheH plays a dominant role in determining amino acid substrate specificity.  相似文献   

11.
We attempted to expand the range of chlorinated solvents degraded by Xanthobacter autotrophicus GJ10 to include trichloroethylene by the rational modification of the enzyme haloalkane dehalogenase. The amino acids Phe164, Asp170, Phe172 and Trp175 were individually replaced with alanine by site-directed mutagenesis. All substitutions produced enzymes with lower than wild type activity with 1,2-dichloroethane. The Phe164Ala and Asp170Ala mutants were 3 and 2 times more active than was the wild type enzyme in dechlorinating 1,6-dichlorohexane. The Asp170Ala mutant resembled the wild type enzyme in its relative activity against longer chain substrates. No mutant was active with trichloroethylene.  相似文献   

12.
Haloalkane dehalogenases are enzymes that catalyze the cleavage of the carbon-halogen bond by a hydrolytic mechanism. Genomes of Mycobacterium tuberculosis and M. bovis contain at least two open reading frames coding for the polypeptides showing a high sequence similarity with biochemically characterized haloalkane dehalogenases. We describe here the cloning of the haloalkane dehalogenase genes dmbA and dmbB from M. bovis 5033/66 and demonstrate the dehalogenase activity of their translation products. Both of these genes are widely distributed among species of the M. tuberculosis complex, including M. bovis, M. bovis BCG, M. africanum, M. caprae, M. microti, and M. pinnipedii, as shown by the PCR screening of 48 isolates from various hosts. DmbA and DmbB proteins were heterologously expressed in Escherichia coli and purified to homogeneity. The DmbB protein had to be expressed in a fusion with thioredoxin to obtain a soluble protein sample. The temperature optimum of DmbA and DmbB proteins determined with 1,2-dibromoethane is 45 degrees C. The melting temperature assessed by circular dichroism spectroscopy of DmbA is 47 degrees C and DmbB is 57 degrees C. The pH optimum of DmbA depends on composition of a buffer with maximal activity at 9.0. DmbB had a single pH optimum at pH 6.5. Mycobacteria are currently the only genus known to carry more than one haloalkane dehalogenase gene, although putative haloalkane dehalogenases can be inferred in more then 20 different bacterial species by comparative genomics. The evolution and distribution of haloalkane dehalogenases among mycobacteria is discussed.  相似文献   

13.
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.  相似文献   

14.
Microbial dehalogenation   总被引:5,自引:0,他引:5  
Novel dehalogenases have been identified recently in various bacteria that utilise halogenated substrates. X-ray studies and sequence analysis have revealed insight into the molecular mechanisms of hydrolytic dehalogenases. Furthermore, genetic and biochemical studies have indicated that reductive dehalogenases are extra-cytoplasmic corrinoid-containing iron-sulphur proteins. Sequence analysis and mutagenesis studies indicate that several dehalogenases are homologous to enzymes that carry out transformations on non-halogenated substrates.  相似文献   

15.
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.  相似文献   

16.
Haloalkane dehalogenases are key enzymes for the degradation of halogenated aliphatic pollutants. Two rhizobial strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, have open reading frames (ORFs), mlr5434 and blr1087, respectively, that encode putative haloalkane dehalogenase homologues. The crude extracts of Escherichia coli strains expressing mlr5434 and blr1087 showed the ability to dehalogenate 18 halogenated compounds, indicating that these ORFs indeed encode haloalkane dehalogenases. Therefore, these ORFs were referred to as dmlA (dehalogenase from Mesorhizobium loti) and dbjA (dehalogenase from Bradyrhizobium japonicum), respectively. The principal component analysis of the substrate specificities of various haloalkane dehalogenases clearly showed that DbjA and DmlA constitute a novel substrate specificity class with extraordinarily high activity towards beta-methylated compounds. Comparison of the circular dichroism spectra of DbjA and other dehalogenases strongly suggested that DbjA contains more alpha-helices than the other dehalogenases. The dehalogenase activity of resting cells and Northern blot analyses both revealed that the dmlA and dbjA genes were expressed under normal culture conditions in MAFF303099 and USDA110 strain cells, respectively.  相似文献   

17.
18.
Tryptophan residues may play several roles in integral membrane proteins including direct interaction with substrates. In this work we studied the contribution of tryptophan residues to substrate binding in EmrE, a small multidrug transporter of Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons. Each of the four tryptophan residues was replaced by site-directed mutagenesis. The only single substitutions that affected the protein's activity were those in position 63. While cysteine and tyrosine replacements yielded a completely inactive protein, the replacement of Trp63 with phenylalanine brought about a protein that, although it could not confer any resistance against the toxicants tested, could bind substrate with an affinity 2 orders of magnitude lower than that of the wild-type protein. Double or multiple cysteine replacements at the other positions generate proteins that are inactive in vivo but regain their activity upon solubilization and reconstitution. The findings suggest a possible role of the tryptophan residues in folding and/or insertion. Substrate binding to the wild-type protein and to a mutant with a single tryptophan residue in position 63 induced a very substantial fluorescence quenching that is not observed in inactive mutants or chemically modified protein. The reaction is dependent on the concentration of the substrate and saturates at a concentration of 2.57 microM with the protein concentration of 5 microM supporting the contention that the functional unit is a dimer. These findings strongly suggest the existence of an interaction between Trp63 and substrate, and the nature of this interaction can now be studied in more detail with the tools developed in this work.  相似文献   

19.
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.  相似文献   

20.
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.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号