Acyl-homoserine lactones (AHLs) are produced by a number of bacterial species and are used by them to regulate the expression of virulence genes in a process known as quorum-sensing. Each bacterial cell has a basal level of AHL and, once the population density reaches a critical level, it triggers AHL-signalling which, in turn, initiates the expression of particular virulence genes . Plants or animals capable of degrading AHLs would have a therapeutic advantage in avoiding bacterial infection as they could prevent AHL-signalling and the expression of virulence genes in quorum-sensing bacteria . N-(3-Oxohexanoyl)-L-homoserine lactone, N-(3-oxododecanoyl)-L-homoserine lactone, N-butanoyl-L-homoserine lactone and N-(3-oxooctanoyl)-L-homoserine lactone can act as substrates .
The taxonomic range for the selected organisms is: Saccharolobus solfataricus The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
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SYSTEMATIC NAME
IUBMB Comments
N-acyl-L-homoserine-lactone lactonohydrolase
Acyl-homoserine lactones (AHLs) are produced by a number of bacterial species and are used by them to regulate the expression of virulence genes in a process known as quorum-sensing. Each bacterial cell has a basal level of AHL and, once the population density reaches a critical level, it triggers AHL-signalling which, in turn, initiates the expression of particular virulence genes [5]. Plants or animals capable of degrading AHLs would have a therapeutic advantage in avoiding bacterial infection as they could prevent AHL-signalling and the expression of virulence genes in quorum-sensing bacteria [5]. N-(3-Oxohexanoyl)-L-homoserine lactone, N-(3-oxododecanoyl)-L-homoserine lactone, N-butanoyl-L-homoserine lactone and N-(3-oxooctanoyl)-L-homoserine lactone can act as substrates [5].
no activity with coumaphos, fensulfothion, fenitrothion, diazinon, chlorpyrifos, and ethyl parathion. Low activity with malathion, methyl parathion methyl paraoxon, and ethyl paraoxon
the enzyme has activity on acyl-homoserine lactones and 5-thiobutyl butyrolactone (TBBL). Substrate docking analysis. The C258 residue in the active site is involved in an interaction with the oxygen atom from the amide in the lactone analogue. C258 residue might have a key role in the lactone hydrolysis mechanism
the enzyme has lactonase activity with a preference toward N-acyl homoserine lactones with 8-10 carbon aliphatic chains and oxo-lactones with shorter chains
the enzyme has a binuclear metal-centre. The activity depends on the presence of divalent metal cations, the highest activity is observed with Co2+. The binuclear centre is used to activate a bridging water molecule to a hydroxide ion and the substrate for nucleophilic attack by polarizing the phosphoryl-oxygen bond. The nucleophilic bridging hydroxide ion attacks the electrophilic centre (phosphorus or carbon) via a SN2 mechanism, forming transition states that bridge the two metals
enzyme SsoPox belongs to the phosphotriesterase-like lactonase (PLL) family of enzymes, to the PLL-A subfamily, a group of lactonases showing a preference for acyl-homoserine lactones. SsoPox shares only about 30% sequence identity with phosphotriesterases (PTEs) but all amino acids coordinating the binuclear metal-centre are conserved. The coexistence of lactonase and phosphotriesterase activities has been already reported for many members of PLL family
SsoPox is a thermostable phosphotriesterase-like lactonase (PLL) that hydrolyses lactones (primary activity) and, at a lower rate, neurotoxic organophosphorus compounds (promiscuous activity)
the enzyme also exhibits promiscuous phosphotriesterase activity for the degradation of organophosphorous chemicals including insecticides and chemical warfare agents
the mutant demonstrates a large increase in catalytic efficiencies over the wild-type enzyme, with increases of 2210fold, 163fold, 58fold, 16fold against methyl parathion, malathion, ethyl paraoxon, and methyl paraoxon, respectively
site-directed mutagenesis, the W263 residue is previously demonstrated to be involved in the formation of an hydrophobic channel for the substrate leaving group, the mutant enzyme shows decreased lactonase activity compared to the wild-type
evolution of a lactonase into a phosphotriesterase, semi-rational engineering approach is used to design an efficient and thermostable organophosphate hydrolase, starting from enzyme SsoPox from Sulfolobus solfataricus as a lactonase scaffold. In particular, by in vitro evolution of the SsoPox ancillary promiscuous activity, the triple mutant C258L/I261F/W263A is obtained which, retaining its inherent stability, shows an enhancement of its hydrolytic activity on paraoxon up to 300fold. The mutant is tested in formulations of different solvents (methanol or ethanol) or detergents (SDS or a commercial soap) for the cleaning of pesticide-contaminated surfaces. Construction of a chimeric gene ssopox-pte by insertion of 16 conserved residues of pte gene in the ssopox sequence. Recombination by DNA StEP between ssopox-pte chimera and ssopox gene
site-directed mutagenesis, the mutant is not able to hydrolyze C8-HSL or C10-HSL and its paraoxonase activity is 3fold higher than the lactonase activity on 5-thiobutyl butyrolactone, as opposed to the wild-type paraoxonase activity which is 760fold lower than the lactonase activity. The combination of C258L, I261F, and W263A mutations in the SsoPox triple mutant improves the hydrolytic specific activity in terms of kcat/KM toward paraoxon 12fold, the kcat 294fold compared to wild-type SsoPox, while the KM value increases
106°C is the melting temperature. The temperatures required to lose one half of enzymatic activity in 5 minutes are 92°C and 130°C in liquid and solid states respectively. More than 30% activity remains after autoclaving the enzyme powder at 121°C for 15 min
the enzyme activity is not affected by acetone, acetonitrile, butanone, butyl acetate, chloroform, dichloromethane, diethyl ether, ethanol, ethyl acetate, isopropanol, methanol, and methoxypropanol
gene ssopox and gene ssopox-pte, sequence comparisons, recombinant expression of wild-type, point mutation, and chimeric mutant enzymes in Escherichia coli strains TOP10 and BL21(DE3)
lactonase SsoPox-W263I is significantly more effective than the tested quorum sensing inhibitors at their respective concentration optimum in inhibition of the virulence of 51 clinical Pseudomonas aeruginosa isolates from diabetic foot ulcers. SsoPox may be incorporated into medical devices such as functionalised catheters and antivirulence dressings