1,2,3-trichloropropane OR C009536 Dotaz Zobrazit nápovědu
Concise international chemical assessment document, ISSN 1020-6167 56
iv, 41 s. : tab. ; 30 cm
- MeSH
- hodnocení rizik MeSH
- propan škodlivé účinky toxicita MeSH
- vystavení vlivu životního prostředí MeSH
- zdraví MeSH
- Konspekt
- Veřejné zdraví a hygiena
- NLK Obory
- chemie, klinická chemie
- veřejné zdravotnictví
- environmentální vědy
- NLK Publikační typ
- publikace WHO
1,2,3-Trichloropropane (TCP) is a toxic compound that is recalcitrant to biodegradation in the environment. Attempts to isolate TCP-degrading organisms using enrichment cultivation have failed. A potential biodegradation pathway starts with hydrolytic dehalogenation to 2,3-dichloro-1-propanol (DCP), followed by oxidative metabolism. To obtain a practically applicable TCP-degrading organism, we introduced an engineered haloalkane dehalogenase with improved TCP degradation activity into the DCP-degrading bacterium Pseudomonas putida MC4. For this purpose, the dehalogenase gene (dhaA31) was cloned behind the constitutive dhlA promoter and was introduced into the genome of strain MC4 using a transposon delivery system. The transposon-located antibiotic resistance marker was subsequently removed using a resolvase step. Growth of the resulting engineered bacterium, P. putida MC4-5222, on TCP was indeed observed, and all organic chlorine was released as chloride. A packed-bed reactor with immobilized cells of strain MC4-5222 degraded >95% of influent TCP (0.33 mM) under continuous-flow conditions, with stoichiometric release of inorganic chloride. The results demonstrate the successful use of a laboratory-evolved dehalogenase and genetic engineering to produce an effective, plasmid-free, and stable whole-cell biocatalyst for the aerobic bioremediation of a recalcitrant chlorinated hydrocarbon.
- MeSH
- biodegradace MeSH
- biotransformace MeSH
- exprese genu MeSH
- hydrolasy genetika metabolismus MeSH
- látky znečišťující životní prostředí metabolismus MeSH
- metabolické inženýrství * MeSH
- metabolické sítě a dráhy genetika MeSH
- plazmidy MeSH
- propan analogy a deriváty metabolismus MeSH
- Pseudomonas putida genetika metabolismus MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- selekce (genetika) MeSH
- transpozibilní elementy DNA MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Haloalkane dehalogenases catalyze the hydrolytic cleavage of carbon-halogen bonds, which is a key step in the aerobic mineralization of many environmental pollutants. One important pollutant is the toxic and anthropogenic compound 1,2,3-trichloropropane (TCP). Rational design was combined with saturation mutagenesis to obtain the haloalkane dehalogenase variant DhaA31, which displays an increased catalytic activity towards TCP. Here, the 1.31 Å resolution crystal structure of substrate-free DhaA31, the 1.26 Å resolution structure of DhaA31 in complex with TCP and the 1.95 Å resolution structure of wild-type DhaA are reported. Crystals of the enzyme-substrate complex were successfully obtained by adding volatile TCP to the reservoir after crystallization at pH 6.5 and room temperature. Comparison of the substrate-free structure with that of the DhaA31 enzyme-substrate complex reveals that the nucleophilic Asp106 changes its conformation from an inactive to an active state during the catalytic cycle. The positions of three chloride ions found inside the active site of the enzyme indicate a possible pathway for halide release from the active site through the main tunnel. Comparison of the DhaA31 variant with wild-type DhaA revealed that the introduced substitutions reduce the volume and the solvent-accessibility of the active-site pocket.
- MeSH
- bakteriální proteiny chemie metabolismus MeSH
- biodegradace MeSH
- hydrolasy chemie metabolismus MeSH
- hydrolýza MeSH
- katalytická doména MeSH
- krystalografie rentgenová MeSH
- látky znečišťující životní prostředí chemie metabolismus MeSH
- molekulární modely MeSH
- mutageneze MeSH
- propan analogy a deriváty chemie metabolismus MeSH
- Rhodococcus chemie enzymologie MeSH
- sekundární struktura proteinů MeSH
- terciární struktura proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The anthropogenic compound 1,2,3-trichloropropane (TCP) has recently drawn attention as an emerging groundwater contaminant. No living organism, natural or engineered, is capable of the efficient aerobic utilization of this toxic industrial waste product. We describe a novel biotechnology for transforming TCP based on an immobilized synthetic pathway. The pathway is composed of three enzymes from two different microorganisms: engineered haloalkane dehalogenase from Rhodococcus rhodochrous NCIMB 13064, and haloalcohol dehalogenase and epoxide hydrolase from Agrobacterium radiobacter AD1. Together, they catalyze consecutive reactions converting toxic TCP to harmless glycerol. The pathway was immobilized in the form of purified enzymes or cell-free extracts, and its performance was tested in batch and continuous systems. Using a packed bed reactor filled with the immobilized biocatalysts, 52.6 mmol of TCP was continuously converted into glycerol within 2.5 months of operation. The efficiency of the TCP conversion to the intermediates was 97%, and the efficiency of conversion to the final product glycerol was 78% during the operational period. Immobilized biocatalysts are suitable for removing TCP from contaminated water up to a 10 mM solubility limit, which is an order of magnitude higher than the concentration tolerated by living microorganisms.
- MeSH
- Agrobacterium enzymologie MeSH
- biodegradace účinky léků MeSH
- biokatalýza účinky léků MeSH
- bioreaktory mikrobiologie MeSH
- biotransformace účinky léků MeSH
- časové faktory MeSH
- chemické látky znečišťující vodu metabolismus toxicita MeSH
- enzymy imobilizované metabolismus MeSH
- hydrolasy metabolismus MeSH
- metabolické sítě a dráhy * účinky léků MeSH
- propan analogy a deriváty chemie metabolismus toxicita MeSH
- Rhodococcus enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
1,2,3-Trichloropropane (TCP) is a highly toxic and recalcitrant compound. Haloalkane dehalogenases are bacterial enzymes that catalyze the cleavage of a carbon-halogen bond in a wide range of organic halogenated compounds. Haloalkane dehalogenase LinB from Sphingobium japonicum UT26 has, for a long time, been considered inactive with TCP, since the reaction cannot be easily detected by conventional analytical methods. Here we demonstrate detection of the weak activity (k(cat) = 0.005 s(-1)) of LinB with TCP using X-ray crystallography and microcalorimetry. This observation makes LinB a useful starting material for the development of a new biocatalyst toward TCP by protein engineering. Microcalorimetry is proposed to be a universal method for the detection of weak enzymatic activities. Detection of these activities is becoming increasingly important for engineering novel biocatalysts using the scaffolds of proteins with promiscuous activities.
Haloalkane dehalogenases hydrolyze carbon-halogen bonds in a wide range of halogenated aliphatic compounds. The potential use of haloalkane dehalogenases in bioremediation applications has stimulated intensive investigation of these enzymes and their engineering. The mutant DhaA31 was constructed to degrade the anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. This strategy enhances activity towards TCP by decreasing the accessibility of the active site to water molecules, thereby promoting formation of the activated complex. The structure of DhaA31 will help in understanding the structure-function relationships involved in the improved dehalogenation of TCP. The mutant protein DhaA31 was crystallized by the sitting-drop vapour-diffusion technique and crystals of DhaA31 in complex with TCP were obtained using soaking experiments. Both crystals belonged to the triclinic space group P1. Diffraction data were collected to high resolution: to 1.31 Å for DhaA31 and to 1.26 Å for DhaA31 complexed with TCP.
- MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- difrakce rentgenového záření MeSH
- hydrolasy chemie genetika metabolismus MeSH
- krystalizace MeSH
- molekulární sekvence - údaje MeSH
- propan analogy a deriváty chemie metabolismus MeSH
- Rhodococcus enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The anthropogenic toxic compound 1,2,3-trichloropropane is poorly degradable by natural enzymes. We have previously constructed the haloalkane dehalogenase DhaA31 by focused directed evolution ( Pavlova, M. et al. Nat. Chem. Biol. 2009 , 5 , 727 - 733 ), which is 32 times more active than the wild-type enzyme and is currently the most active variant known against that substrate. Recent evidence has shown that the structural basis responsible for the higher activity of DhaA31 was poorly understood. Here we have undertaken a comprehensive computational study of the main steps involved in the biocatalytic hydrolysis of 1,2,3-trichloropropane to decipher the structural basis for such enhancements. Using molecular dynamics and quantum mechanics approaches we have surveyed (i) the substrate binding, (ii) the formation of the reactive complex, (iii) the chemical step, and (iv) the release of the products. We showed that the binding of the substrate and its transport through the molecular tunnel to the active site is a relatively fast process. The cleavage of the carbon-halogen bond was previously identified as the rate-limiting step in the wild-type. Here we demonstrate that this step was enhanced in DhaA31 due to a significantly higher number of reactive configurations of the substrate and a decrease of the energy barrier to the SN2 reaction. C176Y and V245F were identified as the key mutations responsible for most of those improvements. The release of the alcohol product was found to be the rate-limiting step in DhaA31 primarily due to the C176Y mutation. Mutational dissection of DhaA31 and kinetic analysis of the intermediate mutants confirmed the theoretical observations. Overall, our comprehensive computational approach has unveiled mechanistic details of the catalytic cycle which will enable a balanced design of more efficient enzymes. This approach is applicable to deepen the biochemical knowledge of a large number of other systems and may contribute to robust strategies in the development of new biocatalysts.
- MeSH
- biokatalýza * MeSH
- hydrolasy chemie genetika metabolismus MeSH
- katalytická doména MeSH
- kinetika MeSH
- mutace MeSH
- počítačová simulace * MeSH
- Rhodococcus enzymologie MeSH
- simulace molekulární dynamiky MeSH
- simulace molekulového dockingu MeSH
- termodynamika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The enzyme DhaA from Rhodococcus rhodochrous NCIMB 13064 belongs to the haloalkane dehalogenases, which catalyze the hydrolysis of haloalkanes to the corresponding alcohols. The haloalkane dehalogenase DhaA and its variants can be used to detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Three mutants named DhaA04, DhaA14 and DhaA15 were constructed in order to study the importance of tunnels connecting the buried active site with the surrounding solvent to the enzymatic activity. All protein mutants were crystallized using the sitting-drop vapour-diffusion method. The crystals of DhaA04 belonged to the orthorhombic space group P2(1)2(1)2(1), while the crystals of the other two mutants DhaA14 and DhaA15 belonged to the triclinic space group P1. Native data sets were collected for the DhaA04, DhaA14 and DhaA15 mutants at beamline X11 of EMBL, DESY, Hamburg to the high resolutions of 1.30, 0.95 and 1.15 A, respectively.
In our previous work, we designed and implemented a synthetic metabolic pathway for 1,2,3-trichloropropane (TCP) biodegradation in Escherichia coli. Significant effects of metabolic burden and toxicity exacerbation were observed on single cell and population levels. Deeper understanding of mechanisms underlying these effects is extremely important for metabolic engineering of efficient microbial cell factories for biotechnological processes. In this paper, we present a novel mathematical model of the pathway. The model addresses for the first time the combined effects of toxicity exacerbation and metabolic burden in the context of bacterial population growth. The model is calibrated with respect to the real data obtained with our original synthetically modified E. coli strain. Using the model, we explore the dynamics of the population growth along with the outcome of the TCP biodegradation pathway considering the toxicity exacerbation and metabolic burden. On the methodological side, we introduce a unique computational workflow utilising algorithmic methods of computer science for the particular modelling problem.
- Publikační typ
- časopisecké články MeSH
