Dehalogenase Activity Research Articles

Mechanistic Dichotomy in Bacterial Trichloroethene Dechlorination Revealed by Carbon and Chlorine Isotope Effects.

Tetrachloroethene (PCE) and trichloroethene (TCE) are significant groundwater contaminants. Microbial reductive dehalogenation at contaminated sites can produce nontoxic ethene but often stops at toxic cis-1,2-dichloroethene ( cis-DCE) or vinyl chloride (VC). The magnitude of carbon relative to chlorine isotope effects (as expressed by ΛC/Cl, the slope of δ13C versus δ37Cl regressions) was recently recognized to reveal different reduction mechanisms with vitamin B12 as a model reactant for reductive dehalogenase activity. Large ΛC/Cl values for cis-DCE reflected cob(I)alamin addition followed by protonation, whereas smaller ΛC/Cl values for PCE evidenced cob(I)alamin addition followed by Cl- elimination. This study addressed dehalogenation in actual microorganisms and observed identical large ΛC/Cl values for cis-DCE (ΛC/Cl = 10.0 to 17.8) that contrasted with identical smaller ΛC/Cl for TCE and PCE (ΛC/Cl = 2.3 to 3.8). For TCE, the trend of small ΛC/Cl could even be reversed when mixed cultures were precultivated on VC or DCEs and subsequently confronted with TCE (ΛC/Cl = 9.0 to 18.2). This observation provides explicit evidence that substrate adaptation must have selected for reductive dehalogenases with different mechanistic motifs. The patterns of ΛC/Cl are consistent with practically all studies published to date, while the difference in reaction mechanisms offers a potential answer to the long-standing question of why bioremediation frequently stalls at cis-DCE.

Detection and Characterization of Bovine Rumen Microorganisms Resistant to Sodium Fluoroacetate

Background: Poisoning of animals due to toxic plants is found in Brazil and other countries. One of the known toxic plants in Brazil, with the active ingredient sodium fluoroacetate (SF), is Palicourea marcgravii. Dehalogenases that inactivate the fluor-carbon bonds are enzymes found in microorganisms and may prevent intoxication. This study evaluated the occurrence of rumen microorganisms naturally resistant to SF.Materials, Methods & Results: Two samples of rumen fluid of cattle from the Experimental Farm of Federal University of Mato Grosso fed with Brachiaria sp. were obtained via fistula in flasks. An aliquot of 2 mL was placed in a microtube and centrifuged at 9000 g for 1 min. Then, the sample was inoculated into 2 tubes, one containing 100 µL of clarified rumen fluid in 2 mL of modified liquid culture medium (0.1% ammonium sulfate, 0.1% potassium phosphate monobasic, 0.05% sodium phosphate dibasic, 0.01% magnesium sulfate, 0.01% yeast extract, pH 7.0) and 0.4% of SF and the other sample containing 2 mL of liquid culture medium and 100 µL of clarified rumen fluid. The 2 samples were incubated at 40°C for 24 h. Dilutions were performed under the same conditions every 24 h until the attainment of microorganisms resistant to SF, and the finaldilution containing 50 µL of each sample was plated in the middle containing SF (0.4%) and incubated at 40°C for 24 h for the isolation of bacteria. The bacterial colonies resistant to SF were identified by morphological methods, stained, and subjected to DNA extraction sequencing using the universal primers 27f and 1492r (16S rDNA) for the identification of the bacterial genus using Blast DNA identity analysis. These bacteria were cultured with and without SF (0.4%), and the presence of fluoride ions was detected by an ion-selective electrode (fluoride) during incubation for 0, 30, 60, 90, and 120 min. Two resistant microorganisms were isolated, one was a Gram-positive coccus and the other was a Gram-positive rod. DNA sequencing identified these organisms as Enterococcus faecalis (98% identity Genbank 1358689) and Bacillus sp. (89% identity Genbank 1358671). Fluoride ions were detected more at 60-min incubation time in both E. faecalis (0.0560 ppm) and Bacillus sp. (0.0488 ppm). Bioassay protection tests were performed in mice ofthe following four groups: negative control (NC) with saline administration, positive control (PC) with administration of plant containing SF, Bacillus group (BG) with administration of plant containing SF plus Bacillus sp., and coccus group (CG) with administration of SF and E. faecalis. Clinical signs were recorded, and statistical analyses were performed to confirm the differences in the groups. Bioassay protection tests showed clinical signs of intoxication in the PC group (83.3%), BG group (100%), and CG group (16.6%) but not in the NC group (0%), with a statistical difference between GC and PC groups (P < 0.05).Discussion: Several environmental bacteria possessing dehalogenase activity have been described, such as Pseudomonas sp., Moraxella sp., and Burkholderia sp. and Pigmentiphaga kullae and Ancylobacter dichloromethanicus isolated from the rumen. No previous study has yet reported an association between dehalogenase activity and E. faecalis, and the protection assay has been observed only in the E. faecalis group. Similar results were observed in experimental intoxication in goats that had previously consumed SF, with the microorganisms identified being Pigmentiphaga kullae and Ancylobacter dichloromethanicus. E. faecalis, isolated from the bovine rumen, exhibited a dehalogenase activity, which could help control animal poisoning by plants containing SF.

Development of Fluorescent Assay for Monitoring of Dehalogenase Activity.

The rapid accumulation of sequence data and powerful protein engineering techniques providing large mutant libraries have greatly heightened interest in efficient methods for biochemical characterization of proteins. Herein is reported a continuous assay for screening of enzymatic activity. The assay is developed and tested with the model enzymes haloalkane dehalogenases and relies upon a fluorescent change of a derivative of 8-hydroxypyrene-1,3,6-trisulphonic acid due to the pH drop associated with the dehalogenation reactions. The assay is performed in a microplate format using a purified enzyme, cell-free extract or intact cells, making the analysis quick and simple. The method exhibits high sensitivity with a limit of detection of 0.06 mM. The assay is successfully validated with gas chromatography and then applied for screening of 12 haloalkane dehalogenases with the environmental pollutant bis(2-chloroethyl) ether and chemical warfare agent sulfur mustard. Six enzymes exhibited detectable activity with both substrates. The within-day variability of the assay for five replicates (n = 5) was 21%.

Removal of gaseous dichloromethane using a solid–liquid partitioning bioreactor under gradual and stepped load increase

Silicone rubber was used as the solid non-aqueous phase of two-phase partitioning bioreactor (TPPB) for dichloromethane (DCM) removal. When the load gradually increased from 150 g m−3 h−1–485 g m−3 h−1 by increasing the gaseous DCM concentration, a steady elimination capacity (EC) of approximately 130 g m−3 h−1 was achieved in one-liquid-phase completely mixed stirred tank bioreactor (CSTB), whereas the EC of TPPB was enhanced from 155 g m−3 h−1–366 g m−3 h−1. This result indicated that DCM removal was controlled by the biological process in CSTB and by mass transfer in TPPB. Meanwhile, the biomass decreased from 534 mg l−1 to 381 mg l−1 in CSTB compared with that maintained at around 580 mg l−1 in TPPB due to the different resistance capabilities to DCM toxicity. Dehalogenase activity was maintained at a relatively high level under high DCM stress with the addition of silicon rubber. TPPB was shown to be more dependable in the overload from 120 g m−3 h−1–450 g m−3 h−1. This paper is the first to report that the crucial step in high-load pollutant removal can be converted from the biological process to mass transfer by adding solid non-aqueous phase.

Sensitive operation of enzyme-based biodevices by advanced signal processing.

Analytical devices that combine sensitive biological component with a physicochemical detector hold a great potential for various applications, e.g., environmental monitoring, food analysis or medical diagnostics. Continuous efforts to develop inexpensive sensitive biodevices for detecting target substances typically focus on the design of biorecognition elements and their physical implementation, while the methods for processing signals generated by such devices have received far less attention. Here, we present fundamental considerations related to signal processing in biosensor design and investigate how undemanding signal treatment facilitates calibration and operation of enzyme-based biodevices. Our signal treatment approach was thoroughly validated with two model systems: (i) a biodevice for detecting chemical warfare agents and environmental pollutants based on the activity of haloalkane dehalogenase, with the sensitive range for bis(2-chloroethyl) ether of 0.01–0.8 mM and (ii) a biodevice for detecting hazardous pesticides based on the activity of γ-hexachlorocyclohexane dehydrochlorinase with the sensitive range for γ-hexachlorocyclohexane of 0.01–0.3 mM. We demonstrate that the advanced signal processing based on curve fitting enables precise quantification of parameters important for sensitive operation of enzyme-based biodevices, including: (i) automated exclusion of signal regions with substantial noise, (ii) derivation of calibration curves with significantly reduced error, (iii) shortening of the detection time, and (iv) reliable extrapolation of the signal to the initial conditions. The presented simple signal curve fitting supports rational design of optimal system setup by explicit and flexible quantification of its properties and will find a broad use in the development of sensitive and robust biodevices.

Exploration of Enzyme Diversity by Integrating Bioinformatics with Expression Analysis and Biochemical Characterization

Millions of protein sequences are being discovered at an incredible pace, representing an inexhaustible source of biocatalysts. Here, we describe an integrated system for automated in silico screening and systematic characterization of diverse family members. The workflow consists of (i) identification and computational characterization of relevant genes by sequence/structural bioinformatics, (ii) expression analysis and activity screening of selected proteins, and (iii) complete biochemical/biophysical characterization and was validated against the haloalkane dehalogenase family. The sequence-based search identified 658 potential dehalogenases. The subsequent structural bioinformatics prioritized and selected 20 candidates for exploration of protein functional diversity. Out of these 20, the expression analysis and the robotic screening of enzymatic activity provided 8 soluble proteins with dehalogenase activity. The enzymes discovered originated from genetically unrelated Bacteria, Eukaryota, and also A...

Cloning and expression of haloacid dehalogenase gene from Bacillus cereus IndB1

Organohalogen compounds, widely used as pesticides in agriculture and solvents in the industrial sector, cause environmental pollution and health problems due to their toxicity and persistence. Numerous studies have been conducted on the biodegradation of organohalogen compounds, with many focusing on the use of dehalogenase from bacteria. Haloacid dehalogenase is a group of enzymes that cleaves the carbon-halogen bond in halogenated aliphatic acids. In a previous study, the bcfd1 gene encoded haloacid dehalogenase from Bacillus cereus IndB1 was successfully isolated and characterized. This research aimed to create an expression system of the bcfd1 gene by subcloning this gene into pET expression vector and to overexpress the gene in Escherichia coli BL21 (DE3). In addition, the recombinant protein was characterized to gain a better understanding of the catalytic action of this enzyme. A high expression of bcfd1 was obtained by inducing the culture at OD550 0.8–1.0 using 0.01 mM IPTG as determined by SDS-PAGE. Zymogram analysis proved that the recombinant protein possessed dehalogenase activity. Bcfd1 activity toward monochloroacetic acid (MCA) showed specific activity of 37 U/mg at 30°C, pH 9. The predicted tertiary structure of Bcfd1 was estimated has conserved α/ß hydrolase folding motif for haloacid dehalogenase superfamily.

Expression of haloacid dehalogenase gene and its molecular protein characterization from Klebsiella pneumoniae ITB1

Organohalogen compounds are widely used industrially and agriculturally, as well as in households as flame retardants and refrigerants. However, these compounds can become significant pollutants through their accidental or deliberate release into the environment in large quantities. Dehalogenase is an enzyme with the potential to be used in the removal of organohalogen contaminants. A previous study successfully subcloned a 690 bp of haloacid dehalogenase gene (hakp1) from Klebsiella pneumoniae ITB1 into a pET-30a(+) expression system to achieve high enzyme productivity. IPTG was used as an inducer to express a pET-hakp1 recombinant clone in Escherichia coli BL21 (DE3). The molecular mass of the haloacid dehalogenase Hakp1 protein was 30 kDa as determined by SDS-PAGE. Zymogram analysis showed that this recombinant protein has dehalogenase activity as shown by the formation of AgCl white precipitate. A quantitative assay of haloacid dehalogenase Hakp1 gave a specific activity of 84.29 U/mg with the optimum temperature of 40°C at pH 9. Predicted three-dimensional structure of Hakp1 showed α/β motif folding which comprised of cap and core domain. The predicted active sites of Hakp1 were Asp8, Glu10, Leu22, Phe23, Trp90, Ser125, Ser126, Lys159, and Asp184 with Asp8, Glu10, Ser126, and Lys159 act as binding residue. This recombinant haloacid dehalogenase clone provides an alternative agent for effective bioremediation of organohalogen pollutants.

Residues His172 and Lys238 are Essential for the Catalytic Activity of the Maleylacetate Reductase from Sphingobium chlorophenolicum Strain L-1

Maleylacetate reductase (PcpE), the last enzyme in the pentachlorophenol biodegradation pathway in Sphingobium chlorophenolicum L-1, catalyzes two consecutive reductive reactions, reductive dehalogenation of 2-chloromaleylacetate (2-CMA) to maleylacetate (MA) and subsequent reduction of MA to 3-oxoadipate (3-OXO). In each reaction, one molecule of NADH is consumed. To better understand its catalytic function, we undertook a structural model-based site-directed mutagenesis and steady-state kinetics study of PcpE. Our results showed that the putative catalytic site of PcpE is located in a positively charged solvent channel at the interface of the two domains and the binding of 2-CMA/MA involves seven basic amino acids, His172, His236, His237, His241 and His251, Lys140 and Lys238. Mutagenesis studies showed that His172 and Lys238 are essential for the catalytic activity of PcpE. However, the mutation of His236 to an alanine can increase the catalytic efficiency (kcat/Km) of PcpE by more than 2-fold, implying that PcpE is still in an early stage of molecular evolution. Similar to tetrachlorobenzoquinone reductase (PcpD), PcpE is also inhibited by pentachlorophenol in a concentration-dependent manner. Furthermore, our studies showed that PcpE exhibits an extremely low but detectable level of alcohol dehalogenase activity toward ethanol and supports the notion that it is evolved from an iron-containing alcohol dehydrogenase.

Kinetic and structural characterization of a cis-3-Chloroacrylic acid dehalogenase homologue in Pseudomonas sp. UW4: A potential step between subgroups in the tautomerase superfamily

A Pseudomonas sp. UW4 protein (UniProt K9NIA5) of unknown function was identified as similar to 4-oxalocrotonate tautomerase (4-OT)-like and cis-3-chloroacrylic acid dehalogenase (cis-CaaD)-like subgroups of the tautomerase superfamily (TSF). This protein lacks only Tyr-103 of the amino acids critical for cis-CaaD activity (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, Glu-114). As it may represent an important variant of these enzymes, its kinetic and structural properties have been determined. The protein shows tautomerase activity with phenylenolpyruvate, but lacks native 4-OT activity and dehalogenase activity with the isomers of 3-chloroacrylic acid. It shows mostly low-level hydratase activity at pH 7.0, converting 2-oxo-3-pentynoate to acetopyruvate, consistent with cis-CaaD-like behavior. At pH 9.0, this compound results primarily in covalent modification of Pro-1, which is consistent with 4-OT-like behavior. These observations could reflect a pKa for Pro-1 that is closer to that of cis-CaaD (∼9.2) than to 4-OT (∼6.4). A structure of the native enzyme, at 2.6 Å resolution, highlights differences at the active site from those of 4-OT and cis-CaaD that add to our understanding of how contemporary TSF reactions and mechanisms may have diverged from a common 4-OT-like ancestor.

Ylehd, an epoxide hydrolase with promiscuous haloalkane dehalogenase activity from tropical marine yeast Yarrowia lipolytica is induced upon xenobiotic stress

Recalcitrant environmental pollutants, like bromoorganics and epoxides are hydrolysed with limited substrate specificities by microbial oxygenases, reductases, hydrolases and dehalogenases. Here, we report the identification and characterisation of a protein (XP_504164) from the tropical marine yeast Yarrowia lipolytica NCIM 3589, known to degrade bromoorganics and epoxides. Multiple sequence alignment suggests it belongs to α/β superfamily with conservation of catalytic triad and oxyanion hole motifs. The corresponding gene cloned and protein (Ylehd) expressed in E. coli BL21AI exhibited epoxide hydrolase activity (24 ± 0.7 nmol s−1 mg−1 protein) at pH 8.0 and promiscuous haloalkane dehalogenase (1.5 ± 0.2 nmol s−1 mg−1 protein) at pH 4.5. Recombinant Ylehd catalyses structurally diverse epoxides and bromoorganics with maximum catalytic efficiency (kcat/Km) of 96.56 and 10.1 mM−1 s−1 towards 1,2-Epoxyoctane (EO) and 1-Bromodecane (BD). The expression of Ylehd was highly induced in presence of BD and EO but not in glucose grown cells as studied by immunoblot analyses, q-PCR and activity levels. Immunoelectron microscopy confirmed higher expression in presence of xenobiotics and located it to cytosol. Such inducible nature of Ylehd suggests its physiological role in xenobiotic stress mitigation. This study represents the first functional characterisation of a bifunctional EH/HLD in eukaryotic microbes with broad substrate specificity making it a potential biocatalyst for bioremediation/biosensing of mixed pollutants.

OleB from Bacterial Hydrocarbon Biosynthesis Is a β-Lactone Decarboxylase That Shares Key Features with Haloalkane Dehalogenases.

OleB is an α/β-hydrolase found in bacteria that biosynthesize long-chain olefinic hydrocarbons, but its function has remained obscure. We report that OleB from the Gram-negative bacterium Xanthomonas campestris performs an unprecedented β-lactone decarboxylation reaction, to complete cis-olefin biosynthesis. OleB reactions monitored by 1H nuclear magnetic resonance spectroscopy revealed a selectivity for decarboxylating cis-β-lactones and no discernible activity with trans-β-lactones, consistent with the known configuration of pathway intermediates. Protein sequence analyses showed OleB proteins were most related to haloalkane dehalogenases (HLDs) and retained the canonical Asp-His-Asp catalytic triad of HLDs. Unexpectedly, it was determined that an understudied subfamily, denoted as HLD-III, is comprised mostly of OleB proteins encoded within oleABCD gene clusters, suggesting a misannotation. OleB from X. campestris showed very low dehalogenase activity only against haloalkane substrates with long alkyl chains. A haloalkane substrate mimic alkylated wild-type X. campestris OleB but not OleBD114A, implicating this residue as the active site nucleophile as in HLDs. A sequence-divergent OleB, found as part of a natural OleBC fusion and classified as an HLD-III, from the Gram-positive bacterium Micrococcus luteus was demonstrated to have the same activity, stereochemical preference, and dependence on the proposed Asp nucleophile. H218O studies with M. luteus OleBC suggested that the canonical alkyl-enzyme intermediate of HLDs is hydrolyzed differently by OleB enzymes, as 18O is not incorporated into the nucleophilic aspartic acid. This work defines a previously unrecognized reaction in nature, functionally identifies some HLD-III enzymes as β-lactone decarboxylases, and posits an enzymatic mechanism of β-lactone decarboxylation.

Phenol Is the Initial Product Formed during Growth and Degradation of Bromobenzene by Tropical Marine Yeast, Yarrowia lipolytica NCIM 3589 via an Early Dehalogenation Step.

Bromobenzene (BrB), a hydrophobic, recalcitrant organic compound, is listed by the environmental protection agencies as an environmental and marine pollutant having hepatotoxic, mutagenic, teratogenic, and carcinogenic effects. The tropical marine yeast Yarrowia lipolytica 3589 was seen to grow aerobically on BrB and displayed a maximum growth rate (μmax) of 0.04 h-1. Furthermore, we also observed an increase in cell size and sedimentation velocity for the cells grown on BrB as compared to the glucose grown cells. The cells attached to the hydrophobic bromobenzene droplets through its hydrophobic and acid–base interactions. The BrB (0.5%, 47.6 mM) was utilized by the cells with the release of a corresponding amount of bromide (12.87 mM) and yielded a cell mass of 1.86 g/L after showing 34% degradation in 96 h. Maximum dehalogenase activity of 16.16 U/mL was seen in the cell free supernatant after 24 h of growth. Identification of metabolites formed as a result of BrB degradation, namely, phenol, catechol, cis, cis muconic acid, and carbon dioxide were determined by LC–MS and GC–MS. The initial attack on bromobenzene by Y. lipolytica cells lead to the transient accumulation of phenol as an early intermediate which is being reported for the first time. Degradation of phenol led to catechol which was degraded by the ortho- cleavage pathway forming cis, cis muconic acid and then to Krebs cycle intermediates eventually leading to CO2 production. The study shows that dehalogenation via an extracellular dehalogenase occurs prior to ring cleavage with phenol as the preliminary degradative compound being produced. The yeast was also able to grow on the degradative products, i.e., phenol and catechol, to varying degrees which would be of potential relevance in the degradation and remediation of xenobiotic environmental bromoaromatic pollutants such as bromobenzene.

Concurrent Haloalkanoate Degradation and Chlorate Reduction by Pseudomonas chloritidismutans AW-1T.

Haloalkanoates are environmental pollutants that can be degraded aerobically by microorganisms producing hydrolytic dehalogenases. However, there is a lack of information about the anaerobic degradation of haloalkanoates. Genome analysis of Pseudomonas chloritidismutans AW-1T, a facultative anaerobic chlorate-reducing bacterium, showed the presence of two putative haloacid dehalogenase genes, the l-DEX gene and dehI, encoding an l-2-haloacid dehalogenase (l-DEX) and a halocarboxylic acid dehydrogenase (DehI), respectively. Hence, we studied the concurrent degradation of haloalkanoates and chlorate as a yet-unexplored trait of strain AW-1T The deduced amino acid sequences of l-DEX and DehI revealed 33 to 37% and 26 to 86% identities with biochemically/structurally characterized l-DEX and the d- and dl-2-haloacid dehalogenase enzymes, respectively. Physiological experiments confirmed that strain AW-1T can grow on chloroacetate, bromoacetate, and both l- and d-α-halogenated propionates with chlorate as an electron acceptor. Interestingly, growth and haloalkanoate degradation were generally faster with chlorate as an electron acceptor than with oxygen as an electron acceptor. In line with this, analyses of l-DEX and DehI dehalogenase activities using cell-free extract (CFE) of strain AW-1T grown on dl-2-chloropropionate under chlorate-reducing conditions showed up to 3.5-fold higher dehalogenase activity than the CFE obtained from AW-1T cells grown on dl-2-chloropropionate under aerobic conditions. Reverse transcription-quantitative PCR showed that the l-DEX gene was expressed constitutively independently of the electron donor (haloalkanoates or acetate) or acceptor (chlorate or oxygen), whereas the expression of dehI was induced by haloalkanoates. Concurrent degradation of organic and inorganic halogenated compounds by strain AW-1T represents a unique metabolic capacity in a single bacterium, providing a new piece of the puzzle of the microbial halogen cycle.IMPORTANCE Halogenated organic and inorganic compounds are important environmental pollutants that have carcinogenic and genotoxic effects on both animals and humans. Previous research studied the degradation of organic and inorganic halogenated compounds separately but not concurrently. This study shows concurrent degradation of halogenated alkanoates and chlorate as an electron donor and acceptor, respectively, coupled to growth in a single bacterium, Pseudomonas chloritidismutans AW-1T Hence, besides biogenesis of molecular oxygen from chlorate reduction enabling a distinctive placement of strain AW-1T between aerobic and anaerobic microorganisms, we can now add another unique metabolic potential of this bacterium to the roster. The degradation of different halogenated compounds under anoxic conditions by a single bacterium is also of interest for the natural halogen cycle in different aquatic and terrestrial ecosystems where ample natural production of halogenated compounds has been documented.

Genetic programming of catalytic Pseudomonas putida biofilms for boosting biodegradation of haloalkanes

Bacterial biofilms outperform planktonic counterparts in whole-cell biocatalysis. The transition between planktonic and biofilm lifestyles of the platform strain Pseudomonas putida KT2440 is ruled by a regulatory network controlling the levels of the trigger signal cyclic di-GMP (c-di-GMP). This circumstance was exploited for designing a genetic device that over-runs the synthesis or degradation of c-di-GMP – thus making P. putida to form biofilms at user׳s will. For this purpose, the transcription of either yedQ (diguanylate cyclase) or yhjH (c-di-GMP phoshodiesterase) from Escherichia coli was artificially placed under the tight control of a cyclohexanone-responsive expression system. The resulting strain was subsequently endowed with a synthetic operon and tested for 1-chlorobutane biodegradation. Upon addition of cyclohexanone to the culture medium, the thereby designed P. putida cells formed biofilms displaying high dehalogenase activity. These results show that the morphologies and physical forms of whole-cell biocatalysts can be genetically programmed while purposely designing their biochemical activity.

A novel putative non-ligninolytic dehalogenase activity for 3-chloropropionic acid (3CP) utilization by Trichoderma asperellum strain SD1

A novel putative non-ligninolytic dehalogenase activity for 3-chloropropionic acid (3CP) utilization by Trichoderma asperellum strain SD1

Improvement of the thermostability and activity of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by engineering C-terminal amino acids

In the current study, a three-tiered mutagenesis strategy was employed to simultaneously improve the thermostability and activity of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) by engineering the last ten amino acids (Met245∼Glu254) of its C-terminal region. Initially, truncated mutagenesis results displayed that C-terminal deletions decreased the thermostability and/or activity of HheC. Then ten residues were subjected to single-site saturation mutagenesis, resulting in 20 beneficial single-point variants related to the thermostability or activity of HheC. The results clearly indicated that residues Met252∼Glu254 and Trp249 are crucial for regulating enzyme thermostability and activity, respectively. Finally, the beneficial substitutions were combined using efficient multi-site combinatorial mutagenesis approaches, leading to an outstanding variant PX14 (Trp249Pro/Met252Leu/Pro253Asp), which had a 17.8-fold higher half-life and a 4.0-fold higher kcat value than that of wild-type HheC. These results indicated that the C-terminal residues play an important role in modulating both the thermostability and activity of HheC.

A selection assay for haloalkane dehalogenase activity based on toxic substrates.

Based on natural selection and the survival of the fittest by evolutionary adaption, a smart high-throughput system was developed to select active haloalkane dehalogenase variants from a large mutant library. Only active enzyme variants can hydrolyse toxic halogenated alkanes to promote growth, whereas inactive mutants starve or die due to the toxic compound. With this powerful tool, huge enzyme mutant libraries can be screened within a few days. The selection is done without any artificial substrates that are hard to synthesize and they also resemble typical ones for haloalkane dehalogenases. Three saturation libraries, with a size of more than 10(6) cells, based on inactive variants of the haloalkane dehalogenases DhaA or DhlA were successfully screened to retrieve active enzymes. The enrichment of the active wild-type enzyme in contrast to the inactive variants was about 340-fold. In addition, this selection approach can be applied for continuous directed evolution experiments for the enrichment of cells expressing adapted haloalkane dehalogenases.

MDC-Analyzer-facilitated combinatorial strategy for improving the activity and stability of halohydrin dehalogenase from Agrobacterium radiobacter AD1

Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) displays a broad substrate range with high regio- and enantioselectivity of both ring-closure and ring-opening reactions, making the enzyme a useful catalyst for the production of optically pure epoxides and β-substituted alcohols. In this study, we report a novel method using an MDC-Analyzer-facilitated combinatorial strategy to improve the activity and stability of HheC by simultaneously randomizing multiple contiguous residues. Six contiguous active-site residues, which are the hotspots for improving the activity of HheC, were simultaneously selected and randomized using the MDC-Analyzer-facilitated combinatorial strategy, resulting in a high-quality mutagenesis library. After screening a total of 1152 clones, three positive mutants were obtained, which exhibited approximately 3.5–5.9-fold higher kcat values than the wild-type HheC toward 1,3-dichloro-2-propanol (1,3-DCP). However, the inactivation half-life of the best mutant (DG9) at 55°C decreased 9-fold compared with that of the wild-type HheC. To improve the stability of mutant DG9, seven contiguous potential surface amino acids were revealed by using the B-FITTER tool. Two charged amino acids, Glu and Lys, which are more abundant in thermophilic proteins than in their mesophilic counterparts, were selected to substitute those seven amino acids and were combined together via an MDC-Analyzer-facilitated combinatorial strategy. Two mutants displaying 1.6- and 2.3-fold higher half-life τ1/2 (55°C) values than their DG9 template were obtained after screening only 384 clones. The results indicated that an MDC-Analyzer-facilitated combinatorial strategy represents an efficient tool for the directed evolution of functional enzymes with multiple contiguous targeting sites.

Functional Characterization of Structural Genomics Proteins in the Crotonase Superfamily

There are over 13,000 Structural Genomics (SG) proteins deposited in the PDB. However, most of these SG proteins are of unknown or uncertain biochemical function. Although many SG proteins have putative functional assignments, they are often incorrect. The Crotonase Superfamily consists of five diverse functional subgroups that are well characterized structurally and functionally, representing different types of reactivity: hydratase, isomerase, and dehalogenase activities. This superfamily also contains at least 70 SG proteins, so it is ideal to test predictions of protein function. Our approach is based on local structure matching at the computationally predicted active site. First, Partial Order Optimum Likelihood (POOL) is used to predict the functionally important residues of each SG protein and of the proteins of known function in the superfamily. Next, Structurally Aligned Local Site of Activity (SALSA) is used to align the predicted catalytic residues of the well characterized members in the superfamily. From this analysis we generate chemical signatures for each functional subgroup and compare them to the sets of catalytic residues predicted for the SG proteins. We demonstrate based on these computational methods that the majority of the putative annotations in this superfamily are likely incorrect. Currently, biochemical assays are being used to test these predictions. Preliminary biochemical results show that one SG protein classified as a probable enoyl-CoA hydratase possesses hydrolase activity as predicted by our methods. The outcome of this project will be to successfully classify the biochemical functions of SG proteins based on their local structure at the predicted active sites. This work is supported by NSF-CHE-1305655.