Phosphotriesterase-like Lactonase Research Articles

Directed Evolution of a Thermostable Quorum-quenching Lactonase from the Amidohydrolase Superfamily

A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-L-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh β-strands. This E101N/R230I mutant had an increased value of k(cat)/K(m) of 72-fold toward 3-oxo-N-dodecanoyl-L-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-L-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the β-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis.

Improving the promiscuous nerve agent hydrolase activity of a thermostable archaeal lactonase

The thermostable Phosphotriesterase-Like Lactonase from Sulfolobus solfataricus (SsoPox) hydrolyzes lactones and, at a lower rate, neurotoxic organophosphorus compounds. The persistent demand of detoxification tools in the field of agricultural wastes and restoring of conditions after terrorist acts prompted us to exploit SsoPox as a "starter" to evolve its ancillary nerve agents hydrolytic capability. A directed evolution strategy yielded, among several variants, the single mutant W263F with k(cat) and specificity constant against paraoxon 16- and 6-fold enhanced, respectively, compared to the wild type. Furthermore, a phenomenon of enzyme activation by SDS has been observed, which allowed to increase those values 150- and 28-fold, respectively. The activity of SsoPox against the deadly nerve gas Cyclosarin has been reported for the first time and proved to be substantially unaffected for variant W263F. Finally, outperforming efficiency of W263F was demonstrated, under severe stressing conditions, with respect to the best known phosphotriesterase PTE from Brevundimonas diminuta.

Structural basis for thermostability revealed through the identification and characterization of a highly thermostable phosphotriesterase-like lactonase from Geobacillus stearothermophilus

A new enzyme homologous to phosphotriesterase was identified from the bacterium Geobacillus stearothermophilus (GsP). This enzyme belongs to the amidohydrolase family and possesses the ability to hydrolyze both lactone and organophosphate (OP) compounds, making it a phosphotriesterase-like lactonase (PLL). GsP possesses higher OP-degrading activity than recently characterized PLLs, and it is extremely thermostable. GsP is active up to 100 °C with an energy of activation of 8.0 kcal/mol towards ethyl paraoxon, and it can withstand an incubation temperature of 60 °C for two days. In an attempt to understand the thermostability of PLLs, the X-ray structure of GsP was determined and compared to those of existing PLLs. Based upon a comparative analysis, a new thermal advantage score and plot was developed and reveals that a number of different factors contribute to the thermostability of PLLs.

Enzymes with Phosphotriesterase and Lactonase Activities in Archaea

Organophosphates (OPs) are a class of synthetic compounds used both as pesticides and nerve agents. They are chemically phosphoesters and biologically potent irreversible inhibitors of serine esterases, particularly acetylcholinesterase (ACh), the key enzyme of the nervous system. Enzymes able to degrade these compounds are at the moment the focus of several research studies in the environmental decontamination/detoxification field. OP hydrolysing enzymes have been found in Bacteria, Archaea and Eukarya. The best known and characterised enzyme is phosphotriesterase from the soil bacterium Pseudomonas diminuta (PTE). It shows a high catalytic activity, close to the molecular diffusion limit, against some triester organophosphates. Its natural substrate and its main physiological function are still unknown. In the last few years, two phosphotriesterase activities have been found in the hyperthermoacidophilic archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius, which have been ascribed to two PTE-related genes (ssopox and sacpox). The corresponding proteins, SsoPox and SacPox, have recently been inserted in the Phosphotriesterase-Like-Lactonase (PLL) group. Their lactonase activity is around 100-fold higher than that of phosphotriesterase and it depends on the same active site. These enzymes are thermophilic and extremely thermostable. Stability in high temperatures and in other stressful conditions is an important feature for every biotechnological application in the industrial field. Finally, the lactonase activity would suggest a potential physiological role for SsoPox and SacPox in the quenching of signals involved in the pathways for cell-to-cell communication.