The wild type bacterial Co2+/Co2+-phosphotriesterase shows a middle-range thermostability

Rochu, Daniel; Beaufet, Nadège; Renault, Frédérique; Viguié, Nathalie; Masson, Patrick

doi:10.1016/s0005-2728(01)00224-9

Cited by 24 publications

(28 citation statements)

References 40 publications

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“…Using optimized conditions, as shown in Fig. 10, it was possible to determine a T m = 587C, for the unfolding of Co-OPH [109]. Provided by the diode array detector, the UV-absorbance spectra of enzyme before and after the transition showed differences in conformation between native and unfolded protein states populated by the heat-treatment.…”

Section: Metal and Ph-dependence Of Stability Of Oph As Discriminatedmentioning

confidence: 99%

Multiple advantages of capillary zone electrophoresis for exploring protein conformational stability

Rochu

Masson

2002

Electrophoresis

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This review summarizes the work of our laboratory to explore the use of capillary zone electrophoretic (CZE) methods for the investigation of protein conformational stability. Early CZE works on protein denaturation as well as fundamental and theoretical considerations are discussed. Instrumental aspects of the CE-based approach including general and particular CE requirements are documented. Several aspects dealing with estimation of stability of enzymes (cholinesterases and organophosphate-hydrolyzing enzymes) interacting with organophosphates profusely illustrate the multiple advantages of CZE. The discrimination of parameters controlling the "good compromise" stability/plasticity for allowing functional efficiency of these enzymes is exemplified. Thermal stability, susceptibility to high electric field, alteration of stability by bound ligands and the role of associated cations in metalloenzymes have been successfully investigated.

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Section: Metal and Ph-dependence Of Stability Of Oph As Discriminatedmentioning

confidence: 99%

Multiple advantages of capillary zone electrophoresis for exploring protein conformational stability

Rochu

Masson

2002

Electrophoresis

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“…The results revealed that the GkaP variants were still remarkably thermostable. For example, the least thermostable variant, 26A8C, had a T 50 15 of 65°C, while the BdPTE had a T 50 15 of 48°C (38). Obviously, the GkaP variants described here retain a high level of thermostability, and the enhancements in OP-degrading activity have increased their potential for practical applications.…”

Section: Discussionmentioning

confidence: 88%

“…The phosphotriesterase represents an ideal candidate for OP detoxification. However, a weakness of thermostability has limited the use of mesophilic PTEs in practical applications (38).…”

mentioning

confidence: 99%

Enhancing the Promiscuous Phosphotriesterase Activity of a Thermostable Lactonase (GkaP) for the Efficient Degradation of Organophosphate Pesticides

Zhang

et al. 2012

Appl Environ Microbiol

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ABSTRACTThe phosphotriesterase-like lactonase (PLL) enzymes in the amidohydrolase superfamily hydrolyze various lactones and exhibit latent phosphotriesterase activities. These enzymes serve as attractive templates forin vitroevolution of neurotoxic organophosphates (OPs) with hydrolytic capabilities that can be used as bioremediation tools. Here, a thermostable PLL fromGeobacillus kaustophilusHTA426 (GkaP) was targeted for joint laboratory evolution with the aim of enhancing its catalytic efficiency against OP pesticides. By a combination of site saturation mutagenesis and whole-gene error-prone PCR approaches, several improved variants were isolated. The most active variant, 26A8C, accumulated eight amino acid substitutions and demonstrated a 232-fold improvement over the wild-type enzyme in reactivity (kcat/Km) for the OP pesticideethyl-paraoxon. Concomitantly, this variant showed a 767-fold decrease in lactonase activity with δ-decanolactone, imparting a specificity switch of 1.8 × 105-fold. 26A8C also exhibited high hydrolytic activities (19- to 497-fold) for several OP pesticides, including parathion, diazinon, and chlorpyrifos. Analysis of the mutagenesis sites on the GkaP structure revealed that most mutations are located in loop 8, which determines substrate specificity in the amidohydrolase superfamily. Molecular dynamics simulation shed light on why 26A8C lost its native lactonase activity and improved the promiscuous phosphotriesterase activity. These results permit us to obtain further insights into the divergent evolution of promiscuous enzymes and suggest that laboratory evolution of GkaP may lead to potential biological solutions for the efficient decontamination of neurotoxic OP compounds.

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“…The wide application of green fluorescent protein as fusion partner opened new capabilities in the field of proteomics [68,69]. The importance of affinity-tag technology will increase because of its necessity for the peptide/protein chip design, high-throughput purification, peptide/protein libraries, drug delivery systems, and strategies of large-scale production.…”

Section: Hybrid Proteinsmentioning

confidence: 99%

Postgenomic chemistry: New problems and challenges

Varfolomeyev

Алиев

Ефременко

2004

Pure and Applied Chemistry

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New trends in chemistry induced by genetic engineering and genomic information are analyzed. Genomic information and bioinformatics make the identification of all molecular participants of biological processes possible. The methods of identification of enzymeactive sites from sequence data were demonstrated. The problems of "chemical proteomics" were formulated and analyzed. New biocatalytic processes based on a new generation of enzymes, including CO 2 reduction and enzyme fuel cell construction, are demonstrated. It was shown that in many reactions the enzymes are substantially more efficient in comparison with classical chemical catalysts. The creation of recombinant proteins from unnatural amino acids is demonstrated, and organisms containing such proteins are described. The new trends of proteomics and bioanalytical chemistry in the postgenomic era are discussed.

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The wild type bacterial Co2+/Co2+-phosphotriesterase shows a middle-range thermostability

Cited by 24 publications

References 40 publications

Multiple advantages of capillary zone electrophoresis for exploring protein conformational stability

Multiple advantages of capillary zone electrophoresis for exploring protein conformational stability

Enhancing the Promiscuous Phosphotriesterase Activity of a Thermostable Lactonase (GkaP) for the Efficient Degradation of Organophosphate Pesticides

Postgenomic chemistry: New problems and challenges

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