Biochemical and biophysical characterisation of haloalkane dehalogenases DmrA and DmrB in Mycobacterium strain JS60 and their role in growth on haloalkanes

Haloalkane dehalogenases (HLDs) catalyse the hydrolysis of haloalkanes to alcohols, offering a biological solution for toxic haloalkane industrial wastes. Hundreds of putative HLD genes have been identified in bacterial genomes, but relatively few enzymes have been characterised. We identified two novel HLDs in the genome of Mycobacterium rhodesiae strain JS60, an isolate from an organochlorine‐contaminated site: DmrA and DmrB. Both recombinant enzymes were active against C2–C6 haloalkanes, with a preference for brominated linear substrates. However, DmrA had higher activity against a wider range of substrates. The kinetic parameters of DmrA with 4‐bromobutyronitrile as a substrate were K_m = 1.9 ± 0.2 mM, k_cat = 3.1 ± 0.2 s^?1. DmrB showed the highest activity against 1‐bromohexane. DmrA is monomeric, whereas DmrB is tetrameric. We determined the crystal structure of selenomethionyl DmrA to 1.7 Å resolution. A spacious active site and alternate conformations of a methionine side‐chain in the slot access tunnel may contribute to the broad substrate activity of DmrA. We show that M. rhodesiae JS60 can utilise 1‐iodopropane, 1‐iodobutane and 1‐bromobutane as sole carbon and energy sources. This ability appears to be conferred predominantly through DmrA, which shows significantly higher levels of upregulation in response to haloalkanes than DmrB.     

DspA from Strongylocentrotus purpuratus: The first biochemically characterized haloalkane dehalogenase of non-microbial origin

Haloalkane dehalogenases are known as bacterial enzymes cleaving a carbon–halogen bond in halogenated compounds. Here we report the first biochemically characterized non-microbial haloalkane dehalogenase DspA from Strongylocentrotus purpuratus. The enzyme shows a preference for terminally brominated hydrocarbons and enantioselectivity towards β-brominated alkanes. Moreover, we identified other putative haloalkane dehalogenases of eukaryotic origin, representing targets for future experiments to discover dehalogenases with novel catalytic properties.     

Cloning,functional expression,biochemical characterization,and structural analysis of a haloalkane dehalogenase from <Emphasis Type="Italic">Plesiocystis pacifica</Emphasis> SIR-1

A haloalkane dehalogenase (DppA) from Plesiocystis pacifica SIR-1 was identified by sequence comparison in the NCBI database, cloned, functionally expressed in Escherichia coli, purified, and biochemically characterized. The three-dimensional (3D) structure was determined by X-ray crystallography and has been refined at 1.95 Å resolution to an R-factor of 21.93%. The enzyme is composed of an α/β-hydrolase fold and a cap domain and the overall fold is similar to other known haloalkane dehalogenases. Active site residues were identified as Asp123, His278, and Asp249 and Trp124 and Trp163 as halide-stabilizing residues. DppA, like DhlA from Xanthobacter autotrophicus GJ10, is a member of the haloalkane dehalogenase subfamily HLD-I. As a consequence, these enzymes have in common the relative position of their catalytic residues within the structure and also show some similarities in the substrate specificity. The enzyme shows high preference for 1-bromobutane and does not accept chlorinated alkanes, halo acids, or halo alcohols. It is a monomeric protein with a molecular mass of 32.6 kDa and exhibits maximum activity between 33 and 37°C with a pH optimum between pH 8 and 9. The K_m and k_cat values for 1-bromobutane were 24.0 mM and 8.08 s^?1. Furthermore, from the 3D-structure of DppA, it was found that the enzyme possesses a large and open active site pocket. Docking experiments were performed to explain the experimentally determined substrate preferences.     

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.     

Halogenated Metabolites Isolated from Penicillium citreonigrum

Three chromone analogs, 1 – 3 , a chlorinated alkaloid sclerotioramine ( 4 ), together with two 11‐noreremophilane‐type sesquiterpenes with a conjugated enolic OH group and a brominated one, 5 and 6 , respectively, were isolated from Penicillium citreonigrum (HQ738282). Compounds 1, 5 , and 6 were new. Biological tests revealed that 4 exhibited a significant activity (IC₅₀ 7.32 μg/ml), and 6 showed a moderate activity (IC₅₀ 16.31 μg/ml) in vitro against HepG2 cell line, and 4 also displayed an activity comparable to that of acarbose against α‐glucosidase.     

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.     

Complete Reductive Dehalogenation of Brominated Biphenyls by Anaerobic Microorganisms in Sediment

We sought to determine whether microorganisms from the polychlorinated biphenyl (PCB)-contaminated sediment in Woods Pond (Lenox, Mass.) could dehalogenate brominated biphenyls. The PCB dechlorination specificities for the microorganisms in this sediment have been well characterized. This allowed us to compare the dehalogenation specificities for brominated biphenyls and chlorinated biphenyls within a single sediment. Anaerobic sediment microcosms were incubated separately at 25°C with 16 different mono- to tetrabrominated biphenyls (350 μM) and disodium malate (10 mM). Samples were extracted and analyzed by gas chromatography with an electron capture detector and a mass spectrometer detector at various times for up to 54 weeks. All of the tested brominated biphenyls were dehalogenated. For most congeners, including 2,6-dibromobiphenyl (26-BB) and 24-25-BB, the dehalogenation began within 1 to 2 weeks. However, for 246-BB and 2-2-BB, debromination was first observed at 7 and 14 weeks, respectively. Most intermediate products did not persist, but when 2-2-BB was produced as a dehalogenation product, it persisted for at least 15 weeks before it was dehalogenated to 2-BB and then to biphenyl. The dehalogenation specificities for brominated and chlorinated biphenyls were similar: meta and para substituents were generally removed first, and ortho substituents were more recalcitrant. However, the brominated biphenyls were better dehalogenation substrates than the chlorinated biphenyls. All of the tested bromobiphenyls, including those with ortho and unflanked meta and para substituents, were ultimately dehalogenated to biphenyl, whereas their chlorinated counterparts either were not dehalogenation substrates or were only partially dehalogenated. Our data suggest that PCB-dechlorinating microorganisms may be able to dehalogenate brominated biphenyls and may exhibit a relaxed specificity for these substrates.     

Identification of a novel Baeyer‐Villiger monooxygenase from Acinetobacter radioresistens: close relationship to the Mycobacterium tuberculosis prodrug activator EtaA

This work demonstrates that Acinetobacter radioresistens strain S13 during the growth on medium supplemented with long‐chain alkanes as the sole energy source expresses almA gene coding for a Baeyer‐Villiger monooxygenase (BVMO) involved in alkanes subterminal oxidation. Phylogenetic analysis placed the sequence of this novel BVMO in the same clade of the prodrug activator ethionamide monooxygenase (EtaA) and it bears only a distant relation to the other known class I BVMO proteins. In silico analysis of the 3D model of the S13 BVMO generated by homology modelling also supports the similarities with EtaA by binding ethionamide to the active site. In vitro experiments carried out with the purified enzyme confirm that this novel BVMO is indeed capable of typical Baeyer‐Villiger reactions as well as oxidation of the prodrug ethionamide.     

CYP116B5: a new class VII catalytically self‐sufficient cytochrome P450 from Acinetobacter radioresistens that enables growth on alkanes

A gene coding for a class VII cytochrome P450 monooxygenase (CYP116B5) was identified from Acinetobacter radioresistens S13 growing on media with medium (C14, C16) and long (C24, C36) chain alkanes as the sole energy source. Phylogenetic analysis of its N‐ and C‐terminal domains suggests an evolutionary model involving a plasmid‐mediated horizontal gene transfer from the donor Rhodococcus jostii RHA1 to the receiving A. radioresistens S13. This event was followed by fusion and integration of the new gene in A. radioresistens chromosome. Heterologous expression of CYP116B5 in Escherichia coli BL21, together with the A. radioresistens Baeyer–Villiger monooxygenase, allowed the recombinant bacteria to grow on long‐ and medium‐chain alkanes, showing that CYP116B5 is involved in the first step of terminal oxidation of medium‐chain alkanes overlapping AlkB and in the first step of sub‐terminal oxidation of long‐chain alkanes. It was also demonstrated that CYP116B5 is a self‐sufficient cytochrome P450 consisting of a heme domain (aa 1–392) involved in the oxidation step of n‐alkanes degradation, and its reductase domain (aa 444–758) comprising the NADPH‐, FMN‐ and [2Fe2S]‐binding sites. To our knowledge, CYP116B5 is the first member of this class to have its natural substrate and function identified.     

Biodegradation of C7 and C8 iso‐alkanes under methanogenic conditions

Iso‐alkanes comprise a substantial proportion of petroleum and refined products that impact the environment, but their fate is cryptic under methanogenic conditions. We investigated methanogenic biodegradation of C₇ and C₈ iso‐alkanes found in naphtha, specifically 2‐methylhexane, 3‐methylhexane, 2‐methylheptane, 4‐methylheptane and 3‐ethylhexane. These were incubated as a mixture or individually with enrichment cultures derived from oil sands tailings ponds that generate methane from naphtha components; substrate depletion and methane production were monitored for up to 663 days. 3‐Methylhexane and 4‐methylheptane were degraded both singly and in the mixture, whereas 2‐methylhexane and 2‐methylheptane resisted degradation as single substrates but were depleted in the iso‐alkane mixture, suggesting co‐metabolism. 3‐Ethylhexane was degraded neither singly nor with co‐substrates. Putative metabolites consistent with succinylated C₇ and C₈ were detected, suggesting activation by addition of iso‐alkanes to fumarate and corresponding to detection of alkylsuccinate synthase‐like genes. 454 pyrotag sequencing, cloning and terminal restriction fragment length polymorphism of 16S rRNA genes revealed predominance of a novel member of the family Peptococcaceae (order Clostridiales) and Archaea affiliated with Methanoregula and Methanosaeta. We report here isomer‐specific metabolism of C₇‐C₈ iso‐alkanes under methanogenic conditions and propose their activation by a novel Peptococcaceae via addition to fumarate.     

Essential‐Oil Constituents and Alkanes of Cephalaria ambrosioides Roem. & Schult. (Family Caprifoliaceae,Subfamily Dipsacaceae) and (Chemo)taxonomic Discernment of the Subfamilies Dipsacaceae and Morinaceae

Herein, the results of the first study of the volatile and alkane profiles of Cephalaria ambrosioides Roem. & Schult . (Caprifoliaceae, subfamily Dipsacaceae) were reported. The GC‐FID and GC/MS analyses of the essential oils hydrodistilled from leaves and stems (CA1) and flowers (CA2) of C. ambrosioides allowed the identification of 284 different components. The main compounds of the studied oil samples were palmitic acid (24.3 and 32.5% for CA1 and CA2, resp.), hexahydrofarnesyl acetone (1.4 and 10.8% for CA1 and CA2, resp.), (Z)‐hex‐3‐en‐1‐ol (7.0 and <0.1% for CA1 and CA2, resp.), and linoleic acid (1.9 and 6.5% for CA1 and CA2, resp.). Essential‐oil compositional data of selected plant species belonging to the Dipsacaceae (15) and Morinaceae (2) subfamilies were used to resolve taxonomical ambiguities regarding the genus Cephalaria and its infrageneric relations, especially concerning the subfamily Morinaceae (formerly a genus within Dipsacaceae). The results of multivariate statistical analyses (25 different essential‐oil samples) supported the exclusion of Morina species from the Dipsacaceae subfamily. The relative abundances of alkanes from n‐, iso‐, and anteiso‐series followed a (distorted) Gaussian‐like distribution and suggested that the biosyntheses of n‐ and branched alkanes in C. ambrosioides are possibly not controlled by the same elongase. Also, the obtained results suggested that there was a difference in the biosynthesis/accumulation of alkanes in the vegetative and reproductive parts of C. ambrosioides.     

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.     

Crystal structure and enhanced activity of a cutinase‐like enzyme from Cryptococcus sp. strain S‐2

The structural and enzymatic characteristics of a cutinase‐like enzyme (CLE) from Cryptococcus sp. strain S‐2, which exhibits remote homology to a lipolytic enzyme and a cutinase from the fungus Fusarium solani (FS cutinase), were compared to investigate the unique substrate specificity of CLE. The crystal structure of CLE was solved to a 1.05 Å resolution. Moreover, hydrolysis assays demonstrated the broad specificity of CLE for short and long‐chain substrates, as well as the preferred specificity of FS cutinase for short‐chain substrates. In addition, site‐directed mutagenesis was performed to increase the hydrolysis activity on long‐chain substrates, indicating that the hydrophobic aromatic residues are important for the specificity to the long‐chain substrate. These results indicate that hydrophobic residues, especially the aromatic ones exposed to solvent, are important for retaining lipase activity. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Dechlorination of Pesticides by a Specific Bacterial Glutathione S‐transferase,BphKLB400: Potential for Bioremediation

The bphK gene located in the bph operon of Burkholderia xenovorans LB400 encodes a protein, BphK^LB400, with significant sequence similarity to glutathione‐S‐transferases (GSTs). GSTs are a superfamily of enzymes involved in the detoxification of many endobiotic and xenobiotic substances. Recently, BphK^LB400 was shown to catalyze the dechlorination of a number of toxic chlorinated organic compounds. Comparison of the amino acid sequence of BphK^LB400 with GSTs from other bacteria that degrade polychlorinated biphenyls identified a number of highly conserved amino acids in the C‐terminal region of the protein thought to be associated with substrate specificity. Mutating the conserved amino acid at position 180 of BphK^LB400 from an alanine to a proline residue resulted in an increase in GST activity of bacterial cell extracts towards a number of chlorinated organic substrates tested including commonly used pesticides. Laboratory scale plant protection experiments suggested that E. coli expressing BphK^LB400 [wildtype and mutant (Ala180Pro)] could protect pea plants from the effects of chloromequat chloride. Therefore, BphK^LB400, identified as having dechlorination activity towards toxic chlorinated organic compounds used in the environment, could have potential in bioremediation.     

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.     

Membrane topology and identification of key residues of EaDAcT,a plant MBOAT with unusual substrate specificity

Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) catalyzes the transfer of an acetyl group from acetyl‐CoA to the sn‐3 position of diacylglycerol to form 3‐acetyl‐1,2‐diacyl‐sn‐glycerol (acetyl‐TAG). EaDAcT belongs to a small, plant‐specific subfamily of the membrane bound O‐acyltransferases (MBOAT) that acylate different lipid substrates. Sucrose gradient density centrifugation revealed that EaDAcT colocalizes to the same fractions as an endoplasmic reticulum (ER)‐specific marker. By mapping the membrane topology of EaDAcT, we obtained an experimentally determined topology model for a plant MBOAT. The EaDAcT model contains four transmembrane domains (TMDs), with both the N‐ and C‐termini orientated toward the lumen of the ER. In addition, there is a large cytoplasmic loop between the first and second TMDs, with the MBOAT signature region of the protein embedded in the third TMD close to the interface between the membrane and the cytoplasm. During topology mapping, we discovered two cysteine residues (C187 and C293) located on opposite sides of the membrane that are important for enzyme activity. In order to identify additional amino acid residues important for acetyltransferase activity, we isolated and characterized acetyltransferases from other acetyl‐TAG‐producing plants. Among them, the acetyltransferase from Euonymus fortunei possessed the highest activity in vivo and in vitro. Mutagenesis of conserved amino acids revealed that S253, H257, D258 and V263 are essential for EaDAcT activity. Alteration of residues unique to the acetyltransferases did not alter the unique acyl donor specificity of EaDAcT, suggesting that multiple amino acids are important for substrate recognition.     

Reductive dehalogenation of brominated ethenes by Sulfurospirillum multivorans and Desulfitobacterium hafniense PCE‐S

Sulfurospirillum multivorans and Desulfitobacterium hafniense PCE‐S are anaerobes that can utilize tetrachloroethene (PCE) as an electron acceptor in their energy metabolism. The end‐product of PCE reduction for both organisms is cis‐1,2‐dichloroethene, which is formed via trichloroethene as the intermediate. The bacteria were able to dehalogenate cis‐ and trans‐1,2‐dibromoethene (cDBE and tDBE) in growing cultures and cell extracts. Dibromoethene supported growth of both organisms. The organisms debrominated cDBE and tDBE to vinyl bromide (VB); D. hafniense PCE‐S also produced ethene in addition to VB. The PCE reductive dehalogenases (PCE dehalogenases) of S. multivorans and D. hafniense PCE‐S mediated the debromination of tribromoethene (TBE) and both isomers of 1,2‐DBE, indicating that this enzyme was responsible for the reductive dehalogenation of brominated ethenes. cDBE, tDBE, 1,1‐DBE and VB were formed upon TBE debromination; VB was the major end‐product. The PCE dehalogenase of D. hafniense PCE‐S also formed ethene. With the purified enzymes from both organisms the kinetic properties of dehalogenation of brominated alkenes were studied and compared with those of their chlorinated analogues.     

Substrate specificity of haloalkane dehalogenases

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.     

Discovery of Novel Haloalkane Dehalogenase Inhibitors

Haloalkane dehalogenases (HLDs) have recently been discovered in a number of bacteria, including symbionts and pathogens of both plants and humans. However, the biological roles of HLDs in these organisms are unclear. The development of efficient HLD inhibitors serving as molecular probes to explore their function would represent an important step toward a better understanding of these interesting enzymes. Here we report the identification of inhibitors for this enzyme family using two different approaches. The first builds on the structures of the enzymes'' known substrates and led to the discovery of less potent nonspecific HLD inhibitors. The second approach involved the virtual screening of 150,000 potential inhibitors against the crystal structure of an HLD from the human pathogen Mycobacterium tuberculosis H37Rv. The best inhibitor exhibited high specificity for the target structure, with an inhibition constant of 3 μM and a molecular architecture that clearly differs from those of all known HLD substrates. The new inhibitors will be used to study the natural functions of HLDs in bacteria, to probe their mechanisms, and to achieve their stabilization.