Sandbox 7654

Phosphotriesterase-Like Lactonase (PLL)

Overview

Phosphotriesterase-Like Lactonase (PLL) family includes a group of enzymes that have main lactonase activity on lactones and acyl-homoserin lactones (AHLs) and, in addition, low promiscuous phosphotriesterase activity towards organophosphates compound (OPs). At the beginning most of them has been identified as putative phosphotriesterases and were called "Paraoxonases" (Pox) because able to degrade pesticides such as paraoxon. However, further structural, phylogenetic, and biochemical studies have revealed that these enzymes have a proficient lactonase activity, beside the weak phosphotriesterase activity.

SsoPox

Sso Pox is a protein of 314 aa deriving from the hyperthermophilic archaeon Sulfolobus solfataricus and it is the first protein with phosphotriesterase activities to be identified in Archaea. It has an exceptional thermal stability with denaturation half-life of 4h and 90 min at 95 °C and 100 °C, respectively. Its activity depends on the presence of metal ions, with cobalt significantly enhancing catalysis. SsoPox have been reported to catalyse the hydrolysis of different N-acyl homoserine lactones AHLs; suggesting a physiological role as a quorum quencher lactonase. Infact the AHLs are natural molecules involved in the cell–cell communication process known as quorum sensing (QS) and any bacterial species may produce different AHLs, which vary in the length and substitution of the acyl chain. The anti-QS mechanisms of the enzyme works by the hydrolysis of the lactone bond of these AHLs.

Structural features

SsoPox is homodimeric, and the monomer is roughly globular. The SsoPox structure could be described as a distorted . The SsoPox active site consists of a cavity containing a , located at the C terminus of the β -barrel. These two metal cations are bridged by a putative catalytic , and by . As for the metal cation coordination, are also involved, as well as and a . The most deeply buried metal cation (called α) adopts a trigonal bipyramidal geometry, being bound by coordination bonds with . The most solvent exposed (called β ) has a distorted trigonal bipyramidal geometry, and is bound to and the second water molecule. So SsoPox results in a protein with heterobinuclear centre constituted by . The 3D structure of SsoPox has been solved in the apo form and in complex with a quorum sensing lactone mimics N-decanoyl-Lhomocysteine thiolactone (C10-HTL). The structure reveals a unique hydrophobic channel that perfectly accommodates the acyl chain of C10-HTL. Results suggest that the high thermal stability of SsoPox resides in the larger number of surface salt bridges, which are involved in surface networks, and in the optimization of the interactions at the interface between the two monomers, which stabilize the dimeric structure of SsoPox. The crystal structure of SsoPox shows that the charged residues are principally located in solvent accessible regions, on the protein surface. Half of these surface-charged residues are involved in salt bridges; in particular, SsoPox contains 25 salt bridges per monomer. In each monomer of SsoPox, the two chain termini are linked by a salt bridge between the terminal carboxylic group of Ser314 and the guanidinium group of Arg 2; moreover, the residues Pro4 and Pro309 confer rigidity to the chain termini in the SsoPox structure, two large hydrophobic clusters are formed, each constituted by the side chains of Phe104 and Leu107 of one subunit, and Tyr97, Tyr99 and Trp263 of the other subunit, contributing to the anchoring of the two monomers.

Biotechnological applications

Ssopox belongs to the PLL family of enzyme which has the peculiar characteristic to have promiscuous activites toward lactones and organophosphates compounds. Owing to these promiscuous activities, the thermostability and rare properties, SsoPox is considered an excellent starting point for biotechnological applications directed towards the achievement of efficient bioscavengers for organophosphorus compounds and against certain pathogens. In vitro directed evolution experiments demonstrated the possibility that, if there is an immediate selective advantage, promiscuous activities of enzymes can diverge quickly in new functions by means of a limited number of mutations. By enhancing the stability and, in particular, the thermostability of some hydrolytic enzymes the protein gains a structural stability; a convenient prerequisite for every in vitro evolutive approach. The protein’s tolerance to substitutions (its neutrality) allows the analysis of kinetically more mutated forms, allowing a wider screening and a complete overview of the amino acids’ position roles [83]. For these reasons SsoPox and other enzymes of the same family appear good candidates for engineering approaches aimed at biotechnological applications in the industrial field. One of the promising applications is the use of PLLs (Sso Pox) for enzymatic detoxification of Ops. This has become the subject of many studies because alternative methods of removing them, such as bleach treatments and incineration are impractical due to high costs or environmental concerns. OPs are toxic compounds for all vertebrates because they irreversibly inhibit acetylcholinesterase, a key enzyme of the nervous system. They have been distributed globally since the end of WorldWar II and their toxic properties have also been exploited for the development of chemical warfare agents such as sarin, soman and VX as well as for the production of agricultural insecticides. For this application, enzymes that catalyse the hydrolysis of phosphoester bonds in OPs represent an excellent bio-based solution.

Function

Disease

Relevance

Structural highlights

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