High-Yield Expression, Purification, and Characterization of the Recombinant Diisopropylfluorophosphatase from Loligo vulgaris

Hartleib, Judith; Rüterjans, Heinz

doi:10.1006/prep.2000.1360

Cited by 68 publications

(51 citation statements)

References 30 publications

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“…Size exclusion chromatography analysis demonstrated that purified tag-free E3 was mainly a soluble monomer with smaller amounts of dimer and trimer (Figure 2-7), an observation consistent with the wild type huPONs recombinantly produced in mammalian cells 55 . Furthermore, CD spectra of purified tag-free E3 showed a broad minimum centered around 210 nm with a crossover point of 205 nm (Figure 2-8), in a good agreement with known β-fold proteins such as diisopropylfluorophosphatase 53,56 . And compared with wild type huPON1, E3 has 70 the negative peak shifted from 217 to 210 nm, indicating less composition of α-helices 57 .…”

Section: Generation Of Soluble Expressed Hupon2 Variantssupporting

confidence: 73%

“…Therefore, it is not necessary to localize therapeutic huPON2 on cell membranes or HDLs. Moreover, it is well accepted that extended hydrophobic patches on the protein surface promote coli 53 . On the basis of these considerations, we hypothesize that H1−H3 of huPON2 are not essential for its catalytic activity, and removal of these hydrophobic helices can substantially improve its solubility, a well-documented protein engineering method for solubility improvement 51, 54 .…”

Section: Generation Of Soluble Expressed Hupon2 Variantsmentioning

confidence: 99%

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Engineering Soluble Human Paraoxonase 2 for Quorum Quenching

Wang

Mulchandani

et al. 2016

ACS Chem. Biol.

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Many pathogenic bacteria utilize quorum sensing (QS) systems to regulate the expression of their virulence genes and promote the formation of biofilm, which renders pathogens with extreme resistance to conventional antibiotic treatments. As a novel approach for attenuating antibiotic resistance and in turn fighting chronic infections, enzymatic inactivation of QS signaling molecules, such as N-acyl homoserine lactones (AHLs), holds great promises. Instead of using bacterial lactonases that can evoke immune response when administered, we focus on the human paraoxonase 2 (huPON2). However, insolubility when heterologously overexpressed hinders its application as anti-infection therapeutics. In this study, huPON2 was engineered for soluble expression with minimal introduction of foreign sequences. On the basis of structure modeling, degenerate linkers were exploited for the removal of hydrophobic helices of huPON2 without disrupting its folding structure and thus retaining its enzymatic function. High soluble expression levels were achieved with a yield of 76 mg of fully human PON2 variants per liter of culture media. Particularly, two clones, D2 and E3, showed significant quorum quenching (QQ) bioactivities and effectively impeded Pseudomonas aeruginosa swimming and swarming motilities, signs of an early stage of biofilm formation. In addition, by correlating QQ with luminescence signal readouts, quantitative analysis of QQ toward natural or non-natural AHL-regulator combinations suggested that D2 and E3 exhibited strong lactone hydrolysis activities toward five AHLs of different side chain lengths and modifications widely utilized by a variety of biomedically important pathogens.

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Section: Generation Of Soluble Expressed Hupon2 Variantssupporting

confidence: 73%

Section: Generation Of Soluble Expressed Hupon2 Variantsmentioning

confidence: 99%

Engineering Soluble Human Paraoxonase 2 for Quorum Quenching

Wang

Mulchandani

et al. 2016

ACS Chem. Biol.

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“…The protein was prepared as reported previously (Hartleib & Rü terjans, 2001). Briefly, the overexpressed protein was initially purified by Ni-NTA affinity chromatography.…”

Section: Crystallization and Data Collectionmentioning

confidence: 99%

“…Diisopropyl fluorophosphatase (DFPase; EC 3.1.8.2; 314 amino acids; 35 kDa), a Ca 2+ -dependent phosphotriesterase, is capable of efficiently detoxifying a wide variety of organophosphorus nerve agents, such as sarin, cyclohexylsarin, soman and tabun (Hartleib & Rü terjans, 2001). As such, it is a prime candidate for the enzymatic decontamination of existing nerve-agent stocks.…”

Section: Introductionmentioning

confidence: 99%

Preliminary time-of-flight neutron diffraction study on diisopropyl fluorophosphatase (DFPase) fromLoligo vulgaris

et al. 2006

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The enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is capable of decontaminating a wide variety of toxic organophosphorus nerve agents. DFPase is structurally related to a number of enzymes, such as the medically important paraoxonase (PON). In order to investigate the reaction mechanism of this phosphotriesterase and to elucidate the protonation state of the active-site residues, large-sized crystals of DFPase have been prepared for neutron diffraction studies. Available H atoms have been exchanged through vapour diffusion against D 2 O-containing mother liquor in the capillary. A neutron data set has been collected to 2.2 Å resolution on a relatively small (0.43 mm 3 ) crystal at the spallation source in Los Alamos. The sample size and asymmetric unit requirements for the feasibility of neutron diffraction studies are summarized.

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“…Enzymatic degradation of OP compounds has received considerable attention because it provides the possibility of both environmentally friendly and in situ detoxification. Among the enzymes that hydrolyze OP compounds (10,15,24,28,33), the phosphotriesterase (PTE, EC 3.1.8.1) from the soil bacterium Brevundimonas (formerly Pseudomonas) diminuta (BdPTE) is most efficient and hydrolyzes a variety of neurotoxic OP compounds (including most pesticides and chemical warfare agents) (12,25). The best substrate identified to date is paraoxon, with a rate approaching the diffusion limit (k cat /K m Ն 4 ϫ 10 7 s Ϫ1 M Ϫ1 ) (12).…”

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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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High-Yield Expression, Purification, and Characterization of the Recombinant Diisopropylfluorophosphatase from Loligo vulgaris

Cited by 68 publications

References 30 publications

Engineering Soluble Human Paraoxonase 2 for Quorum Quenching

Engineering Soluble Human Paraoxonase 2 for Quorum Quenching

Preliminary time-of-flight neutron diffraction study on diisopropyl fluorophosphatase (DFPase) fromLoligo vulgaris

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

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