Hydrolytic Dehalogenation Research Articles

New Hydroxylactones and Chloro-Hydroxylactones Obtained by Biotransformation of Bicyclic Halolactones and Their Antibacterial Activity.

The aim of this study was to obtain new halolactones with a gem-dimethyl group in the cyclohexane ring (at the C-3 or C-5 carbon) and a methyl group in the lactone ring and then subject them to biotransformations using filamentous fungi. Halolactones in the form of mixtures of two diasteroisomers were subjected to screening biotransformations, which showed that only compounds with a gem-dimethyl group located at the C-5 carbon were transformed. Strains from the genus Fusarium carried out hydrolytic dehalogenation, while strains from the genus Absidia carried out hydroxylation of the C-7 carbon. Both substrates and biotransformation products were then tested for antimicrobial activity against multidrug-resistant strains of both bacteria and yeast-like fungi. The highest antifungal activity against C. dubliniensis and C. albicans strains was obtained for compound 5b, while antimicrobial activity against S. aureus MRSA was obtained for compound 4a.

Catalytic role of histidine-114 in the hydrolytic dehalogenation of chlorothalonil by Pseudomonas sp. CTN-3.

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile; TPN) is an environmentally persistent fungicide that sees heavy use in the USA and is highly toxic to aquatic species and birds, as well as a probable human carcinogen. The chlorothalonil dehalogenase from Pseudomonas sp. CTN-3 (Chd, UniProtKB C9EBR5) degrades TPN to its less toxic 4-OH-TPN analog making it an exciting candidate for the development of a bioremediation process for TPN; however, little is currently known about its catalytic mechanism. Therefore, an active site residue histidine-114 (His114) which forms a hydrogen bond with the Zn(II)-bound water/hydroxide and has been suggested to be the active site acid/base, was substituted by an Ala residue. Surprisingly, ChdH114A exhibited catalytic activity with a kcat value of 1.07s-1, ~ 5% of wild-type (WT) Chd, and a KM of 32µM. Thus, His114 is catalytically important but not essential. The electronic and structural aspects of the WT Chd and ChdH114A active sites were examined using UV-Vis and EPR spectroscopy on the catalytically competent Co(II)-substituted enzyme as well as all-atomistic molecular dynamics (MD) simulations. Combination of these data suggest His114 can quickly and reversibly move nearly 2Å between one conformation that facilitates catalysis and another that enables product egress and active site recharge. In light of experimental and computational data on ChdH114A, Asn216 appears to play a role in substrate binding and preorganization of the transition-state while Asp116 likely facilitates the deprotonation of the Zn(II)-bound water in the absence of His114. Based on these data, an updated proposed catalytic mechanism for Chd is presented.

Community-integrated multi-omics facilitates the isolation of an organohalide dehalogenation microorganism

A variety of anthropogenic organohalide contaminants generated from industry are released into the environment and thus cause serious pollution that endangers human health. In the present study, we investigated the microbial community composition of industrial saponification wastewater using 16S rRNA sequencing, providing genomic insights of potential organohalide dehalogenation bacteria (OHDBs) by metagenomic sequencing. We also explored yet-to-culture OHDBs involved in the microbial community. Microbial diversity analysis reveals that Proteobacteria and Patescibacteria phyla dominate microbiome abundance of the wastewater. In addition, a total ofsix bacterial groups (Rhizobiales, Rhodobacteraceae, Rhodospirillales, Flavob a cteriales, Micrococcales, and Saccharimonadales) were found as biomarkers in the key organohalide removal module. Ninety-four metagenome-assembled genomes were reconstructed from the microbial community, and 105 hydrolytic dehalogenase genes within 42 metagenome-assembled genomes were identified, suggesting that the potential for organohalide hydrolytic dehalogenation is present in the microbial community. Subsequently, we characterized the organohalide dehalogenation of an isolated OHDB, Microbacterium sp. J1-1, which shows the dehalogenation activities of chloropropanol, dichloropropanol, and epichlorohydrin. This study provides a community-integrated multi-omics approach to gain functional OHDBs for industrial organohalide dehalogenation.

The potential for bacteria from carbon-limited deep terrestrial environments to participate in chlorine cycling.

Bacteria capable of dehalogenation via reductive or hydrolytic pathways are ubiquitous. Little is known, however, about the prevalence of bacterial dechlorination in deep terrestrial environments with a limited carbon supply. In this study we analyzed published genomes from three deep terrestrial subsurface sites: a deep aquifer in Western Siberia, the Sanford Underground Research Facility in South Dakota, USA, and the Soudan Underground Iron Mine (SUIM) in Minnesota, USA to determine if there was evidence to suggest that microbial dehalogenation was possible in these environments. Diverse dehalogenase genes were present in all analyzed metagenomes, with reductive dehalogenase and haloalkane dehalogenase genes the most common. Taxonomic analysis of both hydrolytic and reductive dehalogenase genes was performed to explore their affiliation; this analysis indicated that at the SUIM site, hydrolytic dehalogenase genes were taxonomically affiliated with Marinobacter species. Because of this affiliation, experiments were also performed with Marinobacter subterrani strain JG233 ('JG233'), an organism containing three predicted hydrolytic dehalogenase genes and isolated from the SUIM site, to determine whether hydrolytic dehalogenation was an active process and involved in growth on a chlorocarboxylic acid. Presence of these genes in genome appears to be functional, as JG233 was capable of chloroacetate dechlorination with simultaneous chloride release. Stable isotope experiments combined with confocal Raman microspectroscopy demonstrated that JG233 incorporated carbon from 13C-chloroacetate into its biomass. These experiments suggest that organisms present in these extreme and often low-carbon environments are capable of reductive and hydrolytic dechlorination and, based on laboratory experiments, may use this capability as a competitive advantage by utilizing chlorinated organic compounds for growth, either directly or after dechlorination.

Microbial detoxification of 2,4,6-tribromophenol via a novel process with consecutive oxidative and hydrolytic debromination: Biochemical, genetic and evolutionary characterization

As a typical brominated flame retardants (BFRs), 2,4,6-tribromophenol (TBP) has serious hazard to the environmental health and its environmental fate has attracted considerable attention. Dehalogenation reaction plays key role in microbial TBP degradation and detoxification. So far, several halophenols-degrading enzymes have been reported to transform their substrate by oxidative dehalogenation; however, the molecular and biochemistry characterization of microbial hydrolytic dehalogenation is limited. In this study, Cupriavidus sp. CNP-8 with high TBP degradation activity was found to degrade TBP via an obviously differnet pathway as compared to other reported TBP-degraders. The transcription of hnp genes were significantly upregulated with TBP stimulation, indicating their involvment in TBP degradation. Enzymatic assays with 18O-labeling experiments showed that HnpAB, a two-component FAD-dependent monooxygenase, transformed TBP via consecutive oxidative and hydrolytic debromination reactions with the formation of 6-bromo-1,2,4-benzenetriol (BBT) as the ring-cleavage substrate. The function of the BBT ring-cleavage enzyme (HnpC) was also characterized both in vitro and in vivo. This finding provides new molecular mechanism of microbial detoxification of TBP and novel information of the environmental fate of this BFRs. Furthermore, to investigate the frequency of this novel dehalogenation mechanism in microbes, we also analyzed the distribution as well as the genetic structure of the hnpABC cluster by comparative genomics. Although hnpA homolog is distributed in several bacterial genera including Cupriavidus, Paraburkholderia, Variovorax and Streptomyces, the complete hnpABC cluster is only retrieved from Cupriavidus and strictly conservative in the genomes. This indicated that Cupriavidus have unique evolutionary pattern in acquiring the hnpABC to degrade TBP and its analogs, enhancing our understanding of the microbial adaptive evolution in halophenols-contaminated environment.

The Operon Encoding Hydrolytic Dehalogenation of 4-Chlorobenzoate Is Transcriptionally Regulated by the TetR-Type Repressor FcbR and Its Ligand 4-Chlorobenzoyl Coenzyme A.

The bacterial hydrolytic dehalogenation of 4-chlorobenzoate (4CBA) is a coenzyme A (CoA)-activation-type catabolic pathway that is usually a common part of the microbial mineralization of chlorinated aromatic compounds. Previous studies have shown that the transport and dehalogenation genes for 4CBA are typically clustered as an fcbBAT1T2T3C operon and inducibly expressed in response to 4CBA. However, the associated molecular mechanism remains unknown. In this study, a gene (fcbR) adjacent to the fcb operon was predicted to encode a TetR-type transcriptional regulator in Comamonas sediminis strain CD-2. The fcbR knockout strain exhibited constitutive expression of the fcb cluster. In the host Escherichia coli, the expression of the Pfcb -fused green fluorescent protein (gfp) reporter was repressed by the introduction of the fcbR gene, and genetic studies combining various catabolic genes suggest that the ligand for FcbR may be an intermediate metabolite. Purified FcbR could bind to the Pfcb DNA probe in vitro, and the metabolite 4-chlorobenzyl-CoA (4CBA-CoA) prevented FcbR binding to the P fcb DNA probe. Isothermal titration calorimetry (ITC) measurements showed that 4CBA-CoA could bind to FcbR at a 1:1 molar ratio. DNase I footprinting showed that FcbR protected a 42-bp DNA motif (5'-GGAAATCAATAGGTCCATAGAAAATCTATTGACTAATCGAAT-3') that consists of two sequence repeats containing four pseudopalindromic sequences (5'-TCNATNGA-3'). This binding motif overlaps with the -35 box of Pfcb and was proposed to prevent the binding of RNA polymerase. This study characterizes a transcriptional repressor of the fcb operon, together with its ligand, thus identifying halogenated benzoyl-CoA as belonging to the class of ligands of transcriptional regulators.IMPORTANCE The bacterial hydrolytic dehalogenation of 4CBA is a special CoA-activation-type catabolic pathway that plays an important role in the biodegradation of polychlorinated biphenyls and some herbicides. With genetic and biochemical approaches, the present study identified the transcriptional repressor and its cognate effector of a 4CBA hydrolytic dehalogenation operon. This work extends halogenated benzoyl-CoA as a new member of CoA-derived effector compounds that mediate allosteric regulation of transcriptional regulators.

Biotechnological Approach for the Production of Enantiomeric Hydroxylactones Derived from Benzaldehyde and Evaluation of Their Cytotoxic Activity

The β-aryl-δ-halo-γ-lactones are known for their antiproliferative activity towards numerous cancer cell lines. The aim of this study was to obtain in the biotransformation process new β-aryl-δ-hydroxy-γ-lactones and compare their activity with the antiproliferative activity of parent compounds. The racemic cis-5-(1-iodoethyl)-4-phenyldihydrofuran-2-one as well as separate enantiomers were transformed in fungal cultures. Among ten tested biocatalysts, three (Absidia cylindrospora AM336, Absidia glauca AM254, and Fusarium culmorum AM10) were able to catalyze the hydrolytic dehalogenation process. The biotransformations processes were highly stereoselective and enantiomerically pure hydroxylactones were obtained (ee ≥ 99%). The iodo- and hydroxylactone enantiomers were subjected to cytotoxic activity evaluation on canine leukemia and lymphoma cell lines. The iodolactones exhibited higher biological potential towards tested cell lines than hydroxylactones. Higher cytotoxic potential was also characteristic for (+)-(4S,5S,6R)-enantiomer of iodolactone compared to its antipode.

Structural basis for the hydrolytic dehalogenation of the fungicide chlorothalonil

Cleavage of aromatic carbon-chlorine bonds is critical for the degradation of toxic industrial compounds. Here, we solved the X-ray crystal structure of chlorothalonil dehalogenase (Chd) from Pseudomonas sp. CTN-3, with 15 of its N-terminal residues truncated (ChdT), using single-wavelength anomalous dispersion refined to 1.96 Å resolution. Chd has low sequence identity (<15%) compared with all other proteins whose structures are currently available, and to the best of our knowledge, we present the first structure of a Zn(II)-dependent aromatic dehalogenase that does not require a coenzyme. ChdT forms a "head-to-tail" homodimer, formed between two α-helices from each monomer, with three Zn(II)-binding sites, two of which occupy the active sites, whereas the third anchors a structural site at the homodimer interface. The catalytic Zn(II) ions are solvent-accessible via a large hydrophobic (8.5 × 17.8 Å) opening to bulk solvent and two hydrophilic branched channels. Each active-site Zn(II) ion resides in a distorted trigonal bipyramid geometry with His117, His257, Asp116, Asn216, and a water/hydroxide as ligands. A conserved His residue, His114, is hydrogen-bonded to the Zn(II)-bound water/hydroxide and likely functions as the general acid-base. We examined substrate binding by docking chlorothalonil (2,4,5,6-tetrachloroisophtalonitrile, TPN) into the hydrophobic channel and observed that the most energetically favorable pose includes a TPN orientation that coordinates to the active-site Zn(II) ions via a CN and that maximizes a π-π interaction with Trp227 On the basis of these results, along with previously reported kinetics data, we propose a refined catalytic mechanism for Chd-mediated TPN dehalogenation.

Recent progress in 2-haloacid dehalogenases

2-Haloacid dehalogenases (EC 3.8.1.X) catalyze the hydrolytic dehalogenation of 2-haloacids, releasing halogen ions and producing corresponding 2-hydroxyacids. The enzymes not only degrade xenobiotic halogenated pollutants, but also show wide substrate profile and astonishing efficiency for enantiomer resolution, making them valuable in environmental protection and the green synthesis of optically pure chiral compounds. A variety of 2-haloacid dehalogenases have been biochemically characterized so far. Further studies have been made in protein crystal structures and catalytic mechanisms. Here, we review the recent progresses of 2-haloacid dehalogenases in their source, protein structures, reaction mechanisms, catalytic properties and application. We also suggest further research directions for 2-haloacid dehalogenase.

Multi-elemental C-Br-Cl isotope analysis for characterizing biotic and abiotic transformations of 1-bromo-2-chloroethane (BCE)

Multi-elemental C-Br-Cl compound-specific isotope analysis was applied for characterizing abiotic and biotic degradation of the environmental pollutant 1-bromo-2-chloroethane (BCE). Isotope effects were determined in the model processes following hydrolytic dehalogenation and dihaloelimination pathways as well as in a microcosm experiment by the microbial culture from the contaminated site. Hydrolytic dehalogenation of BCE under alkaline conditions and by DhaA enzyme resulted in similar dual isotope slopes (ɅC/Br 21.9 ± 4.7 and 19.4 ± 1.8, respectively, and ɅC/Cl ~ ∞). BCE transformation by cyanocobalamin (B12) and by Sulfurospirillum multivorans followed dihaloelimination and was accompanied by identical, within the uncertainty range, dual isotope slopes (ɅC/Br 8.4 ± 1.7 and 7.9 ± 4.2, respectively, and ɅC/Cl 2.4 ± 0.3 and 1.5 ± 0.6, respectively). Changes over time in the isotope composition of BCE from the contaminated groundwater showed only a slight variation in δ13C values and were not sufficient for the elucidation of the BCE degradation pathway in situ. However, an anaerobic microcosm experiment with the enrichment cultures from the contaminated groundwater presented dual isotope slopes similar to the hydrolytic pathway, suggesting that the potential for BCE degradation in situ by the hydrolytic dehalogenation pathway exists in the contaminated site.

Optical enzymatic biosensor membrane for rapid in situ detection of organohalide in water samples

An optical biosensor employing immobilized haloalkane dehalogenase (HLD), the halide degrading enzyme for the detection of halogenated organic in environmental water and drinking water samples was developed. The enzymatic biosensor was fabricated by incorporating H+ ion selective chromoionophore ETH5294 and HLD enzyme in a stacked chitosan films system on a glass slide. Hydrolytic dehalogenation of dichloroethane (DCA) by the carbon-halide degrading HLD enzyme resulting in the release of a halogen, a proton and a primary alcohol. The halocarbon concentration was optically transduced by the pH transducer layer as a result of protonation reaction of the chromoionophore pH indicator dye embedded in the underneath layer. The resulting colour change of the protonated chromoionophore was measured by fiber optic reflectance spectrophotometry method. Under optimized conditions the detection limit of the proposed reflectance-based enzymatic biosensor membrane was estimated to be 1 mg L−1 with a wide dynamic linear concentration range of 5–60 mg L−1 DCA (R2 = 0.9792) and satisfactory reproducibility within the relative standard deviation (RSD) range of 3.4–4.3%. Validation test demonstrated that the optical halocarbon biosensor could be a promising tool for rapid (6 min) in situ and direct evaluation of organohalide in river water, tap water and bottled water samples without any sample pre-treatment or extraction steps.

Detection of halogenated hydrocarbon pollutants using enzymatic reflectance biosensor

A reflectometric biosensor for enzymatic determination of halogenated organic pollutant in environmental water sample was developed. Polyacrylate microspheres modified with succinimide functional groups were employed as the substrate matrix for covalent immobilization of haloalkane dehalogenase (DhlA) enzyme, and ETH5294 Nile Blue chromoionophore (NBC) pH dye was physically immobilized on the acrylic micromatrix to function as an optical proton sensor. The hydrolytic dehalogenation of halocarbon catalyzed by the immobilized DhlA resulting in the release of a halogen and a proton, which protonated the immobilized NBC proton indicator, and a colour change of the biosensor from violet to blue was measured with a reflectance spectrophotometer for indirect quantification of the halogenated hydrocarbon concentration. The enzymatic halocarbon reflectance biosensor exhibited a dynamic linear response range of 1–30 mg L−1 1,2-dichloroethane (DCA) (R2 = 0.9762) with a detection limit of 0.3 mg L−1. The proposed enzymatic biosensor gave rapid response, within 2 min, towards the detection of 50 mg L−1 DCA at 662 nm, and demonstrated stable response up to 6 days of storage period at 4 °C and pH 8.0. Validation between the developed biosensor with standard gas chromatography–electron capture detector (GC-ECD) showed that both methods have a comparable accuracy in detecting halocarbon species.

Salt mine microorganisms used for the biotransformation of chlorolactones.

The aim of the project was to find new catalysts capable of chlorolactone biotransformation. Three bicyclic chlorolactones with structures possessing one or two methyl groups in their cyclohexane ring were subjected to screening biotransformation using seven bacterial strains and one fungal strain from a salt mine. Three strains of bacteria (Micrococcus luteus Pb10, Micrococcus luteus WSP45, Gordonia alkanivorans Pd25) and one fungal strain (Aspergillus sydowii KGJ10) were able to catalyse hydrolytic dehalogenation of one substrate. The classification of the strains that were effective biocatalysts was confirmed by 16S rDNA analysis. The best result (76%) was obtained using Aspergillus sydowii KGJ10. All strains catalysed hydrolytic dehalogenation without changing the conformation. The equatorial position of the chlorine atom in the substrate turned out to be warrant of the positive result of the biotransformation process.

Insights into the molecular mechanism of dehalogenation catalyzed by D-2-haloacid dehalogenase from crystal structures

D-2-haloacid dehalogenases (D-DEXs) catalyse the hydrolytic dehalogenation of D-2-haloacids, releasing halide ions and producing the corresponding 2-hydroxyacids. A structure-guided elucidation of the catalytic mechanism of this dehalogenation reaction has not been reported yet. Here, we report the catalytic mechanism of a D-DEX, HadD AJ1 from Pseudomonas putida AJ1/23, which was elucidated by X-ray crystallographic analysis and the H218O incorporation experiment. HadD AJ1 is an α-helical hydrolase that forms a homotetramer with its monomer including two structurally axisymmetric repeats. The product-bound complex structure was trapped with L-lactic acid in the active site, which is framed by the structurally related helices between two repeats. Site-directed mutagenesis confirmed the importance of the residues lining the binding pocket in stabilizing the enzyme-substrate complex. Asp205 acts as a key catalytic residue and is responsible for activating a water molecule along with Asn131. Then, the hydroxyl group of the water molecule directly attacks the C2 atom of the substrate to release the halogen ion instead of forming an enzyme-substrate ester intermediate as observed in L-2-haloacid dehalogenases. The newly revealed structural and mechanistic information on D-DEX may inspire structure-based mutagenesis to engineer highly efficient haloacid dehalogenases.

Hydrogen Isotope Fractionation during the Biodegradation of 1,2-Dichloroethane: Potential for Pathway Identification Using a Multi-element (C, Cl, and H) Isotope Approach.

Even though multi-element isotope fractionation patterns provide crucial information with which to identify contaminant degradation pathways in the field, those involving hydrogen are still lacking for many halogenated groundwater contaminants and degradation pathways. This study investigates for the first time hydrogen isotope fractionation during both aerobic and anaerobic biodegradation of 1,2-dichloroethane (1,2-DCA) using five microbial cultures. Transformation-associated isotope fractionation values (εbulkH) were -115 ± 18‰ (aerobic C-H bond oxidation), -34 ± 4‰ and -38 ± 4‰ (aerobic C-Cl bond cleavage via hydrolytic dehalogenation), and -57 ± 3‰ and -77 ± 9‰ (anaerobic C-Cl bond cleavage via reductive dihaloelimination). The dual-element C-H isotope approach (ΛC-H = Δδ2H/Δδ13C ≈ εbulkH/εbulkC, where Δδ2H and Δδ13C are changes in isotope ratios during degradation) resulted in clearly different ΛC-H values: 28 ± 4 (oxidation), 0.7 ± 0.1 and 0.9 ± 0.1 (hydrolytic dehalogenation), and 1.76 ± 0.05 and 3.5 ± 0.1 (dihaloelimination). This result highlights the potential of this approach to identify 1,2-DCA degradation pathways in the field. In addition, distinct trends were also observed in a multi- (i.e., Δδ2H versus Δδ37Cl versus Δδ13C) isotope plot, which opens further possibilities for pathway identification in future field studies. This is crucial information to understand the mechanisms controlling natural attenuation of 1,2-DCA and to design appropriate strategies to enhance biodegradation.

PEGylation with the thiosuccinimido butylamine linker significantly increases the stability of haloalkane dehalogenase DhaA

Haloalkane dehalogenase (HLD) can catalyze the hydrolytic dehalogenation of halogenated compounds. However, HLD suffers from the poor stability to resist the environmental stress. PEGylation is an effective approach to enhance the stability of enzymes. The linker is an important stabilization factor of PEGylation. Thus, the linkers of the PEGylated HLD were optimized to improve the stability of HLD in the present study. The PEGylated haloalkane dehalogenase DhaAs with methylamine (Ml), carbamate (Cm) and thiosuccinimido butylamine (Tb) linkers were prepared, respectively. The effects of the Ml, Cm and Tb linkers on the stability of the PEGylated DhaAs were investigated under different environmental stresses. Among the three linkers, the Tb linker showed the highest efficacy to improve the stability of the PEGylated DhaA. The Tb linker significantly increased the thermal stability of the PEGylated DhaA by slowing its structural unfolding, and the pH stability of the PEGylated DhaA by slowing the protonation process. In addition, the PEGylated DhaA with the Tb linker showed the maximum resistance to high ionic strength (1M NaCl) and organic solvent (40% DMSO). PEGylation with the Tb linker is of general interest to effectively improve the stability of proteins, particularly the protein with poor stability.

Biotransformation of hexabromocyclododecanes with hexachlorocyclohexane-transforming Sphingobium chinhatense strain IP26

Bacterial evolution has resulted in the appearance of several Sphingomonadacea strains that gained the ability to metabolize hexachlorocyclohexanes (HCHs). HCHs have been widely used as pesticides but were banned under the Stockholm Convention on persistent organic pollutants (POPs) in 2009. Here we present evidence for bacterial transformation reactions of hexabromocyclododecanes (HBCDs), which are structurally related to HCHs. HBCDs were used as flame retardants. They are now also considered as POPs and their production and use is restricted since 2013. Racemic α-, β-, and γ-HBCDs and their mixture were exposed to Sphingobium chinhatense IP26 in resting cell assays in parallel to β-HCH. All HBCD stereoisomers were converted with (−)β-HBCD being the best and both α-HBCD enantiomers the poorest substrates. HBCD conversion rates were 27–430 times slower than that of β-HCH. Three generations of hydroxylated transformation products were observed, 7 pentabromocyclododecanol isomers (PeBCD-ols), 11 tetrabromocyclododecadiols (TeBCD-diols) and 3 tribromocyclododecatriols (TrBCD-triols). The conversion of (+)α-, (−)β- and (−)γ-HBCD was faster than those of their enantiomers. Therefore the respective enantiomeric excess increased to 3 ± 1%, 36 ± 1% and 6 ± 2% during 48 h of bacterial exposure. PeBCD-ols appeared first, followed by TeBCD-diols and TrBCD-triols indicating stepwise hydrolytic dehalogenation reactions. In conclusion, severe HCH pollution at geographically distinct dumpsites triggered bacterial evolution to express enzymes transforming such compounds. We used S. chinhatense IP26 bacteria to transform structurally related HBCDs, also regulated under the Stockholm Convention. Such bacteria might be useful for bioremediation but the toxicity of the numerous transformation products observed must be assessed in advance.

Resolution of the uncertainty in the kinetic mechanism for the trans-3-Chloroacrylic acid dehalogenase-catalyzed reaction

trans- and cis-3-Chloroacrylic acid dehalogenase (CaaD and cis-CaaD, respectively) catalyze the hydrolytic dehalogenation of their respective isomers and represent key steps in the bacterial conversion of 1,3-dichloropropene to acetaldehyde. In prior work, a kinetic mechanism for the CaaD-catalyzed reaction could not be unequivocally determined because (1) the order of product release could not be determined and (2) the fluorescence factor for the enzyme species, E*PQ (where P = bromide and Q = malonate semialdehyde, the two products of the reaction) could not be assigned. The ambiguities in the model have now been resolved by stopped-flow experiments following the reaction using an active site fluorescent probe, αY60W-CaaD and 3-bromopropiolate, previously shown to be a mechanism-based inhibitor of CaaD, coupled with the rate of bromide release in the course of CaaD inactivation. A global fit of the combined datasets provides a complete minimal model for the reaction of αY60W-CaaD and 3-bromoacrylate. In addition, the global fit produces kinetic constants for CaaD inactivation by 3-bromopropiolate and implicates the acyl bromide as the inactivating species. Finally, a comparison of the model with that for cis-CaaD shows that for both enzymes turnover is limited by product release and not chemistry.

Degradation of three monochlorobenzoate isomers by different bacteria isolated from a contaminated soil

Three monochlorobenzoate isomer-utilizing bacterial strains, Pseudomonas sp. 2-CBA, Pseudomonas sp. 3-CBA, and Hydrogenophaga sp. 4-CBA, were isolated from a haloaromatic-contaminated soil in a disused chemical factory in Nanjing, China. Compared to strain 3-CBA and strain 4-CBA, which could only utilize its corresponding monochlorobenzoate isomer (3-chlorobenzoate and 4-chlorobenzoate) as the sole carbon source for growth, strain 2-CBA could utilize both 2-chlorobenzoate and 3-chlorobenzoate for growth. The three monochlorobenzoate isomers were catabolized via three different dechlorination pathways in the three isolates, respectively. 3-Chlorobenzoate was initially catabolized by the benzoate 1,2-dioxygenase (BenABCD) in strain 3-CBA, whereas 2-chlorobenzoate was catabolized by the hybrid 2-halobenzoate 1,2-dioxygenase system in strain 2-CBA, which comprised of the terminal oxygenase component of 2-halobenzoate dioxygenase system (CbdAB) and the reductase component of benzoate 1,2-dioxygenase system (BenC). For 4-chlorobenzoate, it was catabolized via the classic hydrolytic dehalogenation pathway in strain 4-CBA. While, strain 4-CBA seemed to have a more compact gene cluster of fcbABC compared to previously reported strains. This study provides new insights into the catabolic diversity of structurally similar isomers in a contaminated niche.

Biotransformation of Lactones with Methylcyclohexane Ring and Their Biological Activity

The aim of the study was to obtain biological active compounds during biotransformation. Three bicyclic halolactones with methylcyclohexane ring (2-chloro-4-methyl-9-oxabicyclo-[4.3.0]nonan-8-one, 2-bromo-4-methyl-9-oxabicyclo[4.3.0]nona- -8-one and 2-iodo-4-methyl-9-oxabicyclo[4.3.0]nonan-8-one) obtained from the corresponding γ,δ-unsaturated acid were subjected to a screening biotransformation using 22 fungal strains. Two of these strains (Cunninghamella japonica AM472 and Fusarium culmorum AM10) were able to transform halolactones into 2-hydroxy-4-methyl-9-oxabicyclo[4.3.0]nonan-8-one by hydrolytic dehalogenation with good yield. The biotransformation product was structurally different from its synthetically prepared analog. All halolactones and hydroxylactones were tested for their biological activity. The chlorolactone inhibited growth of Staphylococcus aureus (max ΔOD = 0), Escherichia coli (max ΔOD = 0.3) and Candida albicans (max ΔOD = 0) strains. Bromolactone caused inhibition of growth of Staphylococcus aureus (max ΔOD = 0) and Fusarium linii (max ΔOD = 0) strains. Iodolactone limited growth of Staphylococcus aureus (max ΔOD = 0), Escherichia coli (max ΔOD = 0.25), Candida albicans (max ΔOD = 0.45) and Pseudomonas fluorescens (max ΔOD = 0.42) strains. Hydroxylactone caused inhibition of growth of Staphylococcus aureus (max ΔOD = 0.36) and Pseudomonas fluorescens (max ΔOD = 0.39) strains only. The test performed on aphids Myzus persicae (Sulz.) showed that chloro- and bromolactone exhibited deterrent activity after 24 h (ID = 0.5 and 0.4, respectively), while hydroxylactone was a weak attractant (ID = −0.3).