Directed Evolution of a Ring-cleaving Dioxygenase for Polychlorinated Biphenyl Degradation

Fortin, Pascal D.; Macpherson, Iain; Neau, David; Bolin, Jeffrey T.; Eltis, Lindsay D.

doi:10.1074/jbc.m510456200

Cited by 22 publications

(12 citation statements)

References 29 publications

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“…The last two enzymes have been target of early applications of SSM [107,108], as well as refinement of previous successful directed evolution approaches [109]. Further work, more focused on developing enzyme catalysts for bioremediation, has been developed on dioxygenases containing a single iron atom such as ring-cleaving dioxygenases acting on polychlorinated biphenyls [110], aniline [111,112], dinitrotoluene [113] and chlorinated catechols [114]. The engineering of the extradiol dioxygenase (DoxG) that displays a low activity in 3,4-dihydroxybiphenyls ring cleavage was achieved by a combination of error-prone PCR, SSM at hot spots and DNA shuffling applied in sequence.…”

Section: Recent Successful Applicationsmentioning

confidence: 99%

Improvement of Biocatalysts for Industrial and Environmental Purposes by Saturation Mutagenesis

Valetti

Gilardi

2013

Biomolecules

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Laboratory evolution techniques are becoming increasingly widespread among protein engineers for the development of novel and designed biocatalysts. The palette of different approaches ranges from complete randomized strategies to rational and structure-guided mutagenesis, with a wide variety of costs, impacts, drawbacks and relevance to biotechnology. A technique that convincingly compromises the extremes of fully randomized vs. rational mutagenesis, with a high benefit/cost ratio, is saturation mutagenesis. Here we will present and discuss this approach in its many facets, also tackling the issue of randomization, statistical evaluation of library completeness and throughput efficiency of screening methods. Successful recent applications covering different classes of enzymes will be presented referring to the literature and to research lines pursued in our group. The focus is put on saturation mutagenesis as a tool for designing novel biocatalysts specifically relevant to production of fine chemicals for improving bulk enzymes for industry and engineering technical enzymes involved in treatment of waste, detoxification and production of clean energy from renewable sources.

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Section: Recent Successful Applicationsmentioning

confidence: 99%

Improvement of Biocatalysts for Industrial and Environmental Purposes by Saturation Mutagenesis

Valetti

Gilardi

2013

Biomolecules

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“…3-Chlorocatechol is a metabolic intermediate of 3-chlorobiphenyl degradation, which severely affect the PCB metabolism [Sondossi et al, 1992]. Efforts were directed towards the possible optimization of extradiol dioxygenases through directed evolution [Fortin et al, 2005b]. However, although variants with increased resistance to 3-chlorocatechol could be obtained [Ohnishi et al, 2004], this increase was only twofold and not comparable to natural variants of catechol 2,3-dioxygenases previously observed in chlorobenzene degrading Pseudomonas strains which are highly resistant to 3-chlorocatechol mediated inactivation [Göbel et al, 2004;Kaschabek et al, 1998].…”

Section: The Biphenyl Upper (Bph) Pathwaymentioning

confidence: 99%

Bacterial Metabolism of Polychlorinated Biphenyls

2008

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Microbial metabolism is responsible for the removal of persistent organic pollutants including PCBs from the environment. Anaerobic dehalogenation of highly chlorinated congeners in aquatic sediments is an important process, and recent evidence has indicated that Dehalococcoides and related organisms are predominantly responsible for this process. Such anaerobic dehalogenation generates lower chlorinated congeners which are easily degraded aerobically by enzymes of the biphenyl upper pathway (bph). Initial biphenyl 2,3-dioxygenases are generally considered the key enzymes of this pathway which determine substrate range and extent of PCB degradation. These enzymes have been subject to different protein evolution strategies, and subsequent enzymes have been considered as crucial for metabolism. Significant advances have been made regarding the mechanistic understanding of these enzymes, which has also included elucidation of the function of BphK glutathione transferase. So far, the genomes of two important PCB-metabolizing organisms, namely Burkholderia xenovorans strain LB400 and Rhodococcus sp. strain RHA1, have been sequenced, with the rational to better understand their overall physiology and evolution. Genomic and proteomic analysis also allowed a better evaluation of PCB toxicity. Like all bph gene clusters which have been characterized in detail, particularly in strains LB400 and RHA1, these genes were localized on mobile genetic elements endowing single strains and microbial communities with a high flexibility and adaptability. However, studies show that our knowledge on enzymes and genes involved in PCB metabolism is still rather fragmentary and that the diversity of bacterial strategies is highly underestimated. Overall, metabolism of biphenyl and PCBs should not be regarded as a simple linear pathway, but as a complex interplay between different catabolic gene modules.

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“…The extradiol aromatic ring-cleaving dioxygenases (E.C.1.13.11) catalyze the addition of both atoms of molecular oxygen into catechol and its chloro-, methyl-, and ethyl-substituted derivatives resulting in cleavage of the bond between the carbon atoms at positions 2 and 3 (proximal extradiol cleavage) or the bond between these at positions 1 and 6 (distal extradiol cleavage) [Bugg and Ramaswamy, 2008;Fortin et al, 2005;Okuta et al, 2004;Palaniandavar and Mayilmurugan, 2007]. Based on their amino acid sequence and structural folds, similar extradiol dioxygenases can be divided into three types [Eltis and Bolin, 1996;Vaillancourt et al, 2006].…”

Section: Introductionmentioning

confidence: 99%

Cloning and Mutagenesis of Catechol 2,3-Dioxygenase Gene from the Gram-Positive <b><i>Planococcus</i></b> sp. Strain S5

Hupert-Kocurek¹,

Stawicka²,

Wojcieszyńska³

2013

Microb Physiol

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In this study, the catechol 2,3-dioxygenase gene that encodes a 307- amino-acid protein was cloned from Planococcus sp. S5. The protein was identified to be a member of the superfamily I, subfamily 2A of extradiol dioxygenases. In order to study residues and regions affecting the enzyme's catalytic parameters, the c23o gene was randomly mutated by error-prone PCR. The wild-type enzyme and mutants containing substitutions within either the C-terminal or both domains were functionally produced in Escherichia coli and their activity towards catechol was characterized. The C23OB65 mutant with R296Q substitution showed significant tolerance to acidic pH with an optimum at pH 5.0. In addition, it showed activity more than 1.5 as high as that of the wild type enzyme and its K_m was 2.5 times lower. It also showed altered sensitivity to substrate inhibition. The results indicate that residue at position 296 plays a role in determining pH dependence of the enzyme and its activity. Lower activity toward catechol was shown for mutants C23OB58 and C23OB81. Despite lower activity, these mutants showed higher affinity to catechol and were more sensitive to substrate concentration than nonmutated enzyme.

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Directed Evolution of a Ring-cleaving Dioxygenase for Polychlorinated Biphenyl Degradation

Cited by 22 publications

References 29 publications

Improvement of Biocatalysts for Industrial and Environmental Purposes by Saturation Mutagenesis

Improvement of Biocatalysts for Industrial and Environmental Purposes by Saturation Mutagenesis

Bacterial Metabolism of Polychlorinated Biphenyls

Cloning and Mutagenesis of Catechol 2,3-Dioxygenase Gene from the Gram-Positive <b><i>Planococcus</i></b> sp. Strain S5

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