Mn2+ modulates the kinetic properties of an archaeal member of the PLL family

Porzio, Elena; Gennaro, Spartaco Di; Palma, Achille; Manco, Giuseppe

doi:10.1016/j.cbi.2012.11.003

Cited by 14 publications

(16 citation statements)

References 22 publications

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“…This is in contrast with the previous result in which SsoPox was shown to possess heterobinuclear metal center constituted by an iron at α-site and a cobalt cation at β-site after purification in the presence of Co 2+ ion in the buffer [34]. However, in a previous work we demonstrated the possibility to change the metal content in PLL members with sometimes significant changes in enzyme properties [21]. The details of active sites of 3Mut ( Figure 6; blue) and 4Mut ( Figure 6; red) were shown with metal cofactors (Co 2+ ; red spheres) detected.…”

Section: D Structure Of 4mutcontrasting

confidence: 99%

“…Several reports suggest that catalytic activity of PLL enzymes as well as PTE depends on the presence of metal ions in the active site. In addition, the species of metals also modulate the activity [18,20,21,[24][25][26]. In order to examine the metal ions for the enhancement of paraoxonase activity, new mutant V82L/C258L/I261F/W263A (4Mut) was expressed in E. coli BL21(DE3) under the presence of different metal ions including Co 2+ , Mn 2+ , Zn 2+ , Ni 2+ or Cd 2+ .…”

Section: Optimal Metal Ionmentioning

confidence: 99%

“…In this context, our group has focused on the studies of an enzyme family of lactonases with promiscuous phosphotriesterase activity, dubbed Phosphotriesterase-Like-Lactonases (PLLs) [17][18][19][20][21][22]. PLL family enzymes are closely related to mesophilic bdPTEs, being classified in the same amidohydrolase superfamily.…”

Section: Introductionmentioning

confidence: 99%

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Structural and Functional Characterization of New SsoPox Variant Points to the Dimer Interface as a Driver for the Increase in Promiscuous Paraoxonase Activity

Suzumoto¹,

Dim

Roviello

et al. 2020

IJMS

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Increasing attention is more and more directed toward the thermostable Phosphotriesterase-Like-Lactonase (PLL) family of enzymes, for the efficient and reliable decontamination of toxic nerve agents. In the present study, the DNA Staggered Extension Process (StEP) technique was utilized to obtain new variants of PLL enzymes. Divergent homologous genes encoding PLL enzymes were utilized as templates for gene recombination and yielded a new variant of SsoPox from Saccharolobus solfataricus. The new mutant, V82L/C258L/I261F/W263A (4Mut) exhibited catalytic efficiency of 1.6 × 10 5 M −1 s −1 against paraoxon hydrolysis at 70 • C, which is more than 3.5-fold and 42-fold improved in comparison with C258L/I261F/W263A (3Mut) and wild type SsoPox, respectively. 4Mut was also tested with chemical warfare nerve agents including tabun, sarin, soman, cyclosarin and VX. In particular, 4Mut showed about 10-fold enhancement in the hydrolysis of tabun and soman with respect to 3Mut. The crystal structure of 4Mut has been solved at the resolution of 2.8 Å. We propose that, reorganization of dimer conformation that led to increased central groove volume and dimer flexibility could be the major determinant for the improvement in hydrolytic activity in the 4Mut.

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Section: D Structure Of 4mutcontrasting

confidence: 99%

Section: Optimal Metal Ionmentioning

confidence: 99%

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Structural and Functional Characterization of New SsoPox Variant Points to the Dimer Interface as a Driver for the Increase in Promiscuous Paraoxonase Activity

Suzumoto¹,

Dim

Roviello

et al. 2020

IJMS

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“…Metal cations are coordinated by four histidines, an aspartic acid and a carboxylated lysine residue [9]. While the nature of the bimetallic center can vary depending on the enzyme nature and the purification procedure [3,5,13,14], the catalytic mechanism is presumed to be identical. The bimetallic center activates a water molecule into a hydroxide ion which performs a nucleophilic attack onto the electrophilic center [9,15].…”

Section: Introductionmentioning

confidence: 99%

“…Being an enzyme from a hyperthermophile, Sac Pox is however less stable than Sso Pox (half-life of 5 min at 90°C [4] and of 4 h at 95°C [3,46], respectively). The kinetic characterizations performed on Sac Pox revealed that it hydrolyzes OP, ester and lactone molecules at high temperature [4,13]. However, only few substrates have been tested, and no natural lactones were assayed as substrate.…”

Section: Introductionmentioning

confidence: 99%

Sac Pox from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius is a proficient lactonase

et al. 2014

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BackgroundSacPox, an enzyme from the extremophilic crenarchaeal Sulfolobus acidocaldarius (Sac), was isolated by virtue of its phosphotriesterase (or paraoxonase; Pox) activity, i.e. its ability to hydrolyze the neurotoxic organophosphorus insecticides. Later on, SacPox was shown to belong to the Phosphotriesterase-Like Lactonase family that comprises natural lactonases, possibly involved in quorum sensing, and endowed with promiscuous, phosphotriesterase activity.ResultsHere, we present a comprehensive and broad enzymatic characterization of the natural lactonase and promiscuous organophosphorus hydrolase activities of SacPox, as well as a structural analysis using a model.ConclusionKinetic experiments show that SacPox is a proficient lactonase, including at room temperature. Moreover, we discuss the observed differences in substrate specificity between SacPox and its closest homologues SsoPox and SisLac together with the possible structural causes for these observations.

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An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination

Giudice

Coppolecchia

Merone

et al. 2015

Biotech & Bioengineering

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In vitro evolution of enzymes represents a powerful device to evolve new or to improve weak enzymatic functions. In the present work a semi-rational engineering approach has been used to design an efficient and thermostable organophosphate hydrolase, starting from a lactonase scaffold (SsoPox from Sulfolobus solfataricus). In particular, by in vitro evolution of the SsoPox ancillary promiscuous activity, the triple mutant C258L/I261F/W263A has been obtained which, retaining its inherent stability, showed an enhancement of its hydrolytic activity on paraoxon up to 300-fold, achieving absolute values of catalytic efficiency up to 10(5) M(-1) s(-1). The kinetics and structural determinants of this enhanced activity were thoroughly investigated and, in order to evaluate its potential biotechnological applications, the mutant was tested in formulations of different solvents (methanol or ethanol) or detergents (SDS or a commercial soap) for the cleaning of pesticide-contaminated surfaces.

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Mn2+ modulates the kinetic properties of an archaeal member of the PLL family

Cited by 14 publications

References 22 publications

Structural and Functional Characterization of New SsoPox Variant Points to the Dimer Interface as a Driver for the Increase in Promiscuous Paraoxonase Activity

Structural and Functional Characterization of New SsoPox Variant Points to the Dimer Interface as a Driver for the Increase in Promiscuous Paraoxonase Activity

Sac Pox from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius is a proficient lactonase

An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination

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