Rational engineering of a native hyperthermostable lactonase into a broad spectrum phosphotriesterase

Jacquet, Pauline; Hiblot, Julien; Daudé, David; Bergonzi, Céline; Gotthard, Guillaume; Armstrong, Nicholas; Chabrière, Eric; Elias, Mikael

doi:10.1038/s41598-017-16841-0

Download PDF

Article
Open access
Published: 01 December 2017

Rational engineering of a native hyperthermostable lactonase into a broad spectrum phosphotriesterase

Pauline Jacquet¹^na1,
Julien Hiblot¹^na1^nAff4,
David Daudé²^na1,
Céline Bergonzi^1,3,
Guillaume Gotthard¹,
Nicholas Armstrong ORCID: orcid.org/0000-0002-8365-2244¹,
Eric Chabrière¹ &
…
Mikael Elias³

Scientific Reports volume 7, Article number: 16745 (2017) Cite this article

2524 Accesses
37 Citations
1 Altmetric
Metrics details

Subjects

Abstract

The redesign of enzyme active sites to alter their function or specificity is a difficult yet appealing challenge. Here we used a structure-based design approach to engineer the lactonase SsoPox from Sulfolobus solfataricus into a phosphotriesterase. The five best variants were characterized and their structure was solved. The most active variant, αsD6 (V27A-Y97W-L228M-W263M) demonstrates a large increase in catalytic efficiencies over the wild-type enzyme, with increases of 2,210-fold, 163-fold, 58-fold, 16-fold against methyl-parathion, malathion, ethyl-paraoxon, and methyl-paraoxon, respectively. Interestingly, the best mutants are also capable of degrading fensulfothion, which is reported to be an inhibitor for the wild-type enzyme, as well as others that are not substrates of the starting template or previously reported W263 mutants. The broad specificity of these engineered variants makes them promising candidates for the bioremediation of organophosphorus compounds. Analysis of their structures reveals that the increase in activity mainly occurs through the destabilization of the active site loop involved in substrate binding, and it has been observed that the level of disorder correlates with the width of the enzyme specificity spectrum. This finding supports the idea that active site conformational flexibility is essential to the acquisition of broader substrate specificity.

In silico protein engineering shows that novel mutations affecting NAD+ binding sites may improve phosphite dehydrogenase stability and activity

Article Open access 01 February 2023

Soukayna Baammi, Rachid Daoud & Achraf El Allali

Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor

Article Open access 28 February 2024

Sumire Honda Malca, Nadine Duss, … Rebecca Buller

Enzymatic kinetic resolution of desmethylphosphinothricin indicates that phosphinic group is a bioisostere of carboxyl group

Article Open access 02 September 2020

Daniela De Biase, Francesca Cappadocio, … Alex Khomutov

Introduction

Enzymes are extraordinary molecules that can accelerate chemical reactions, including those that spontaneously occur extremely slowly, and can make them happen many times per second¹. Some of these reactions are of high biotechnological or industrial interest and enzymes are, therefore, the focus of numerous studies². However, enzymes are often not directly compatible with industrial processes for various reasons ranging from their sub-optimality^3,4 or lack of stability under certain conditions⁵. The properties of enzymes can be tuned by molecular engineering to fit these new requirements using high throughput methods such as directed evolution^5,6,7,8,9,10 and /or low throughput methods, reducing the size of the library by focusing on positions that can be saturated^11,12,13, or by using phylogenetically-inferred^14,15 or structure-based mutations to create combinatorial¹⁶, reduced libraries¹⁷.

Here, we focus on the molecular engineering of an enzymatic bio-decontaminant of organophosphates compounds (OPs), including nerve agents and insecticides. These compounds are neurotoxic because of their ability to irreversibly inhibit the acetylcholine esterase, a key enzyme in the nervous system¹⁸. Massively used in agriculture as pesticides¹⁹, they were modified before World War II to increase their toxicity and develop chemical warfare agents²⁰. OPs still represent major environmental, health and security concerns, as they cause significant pollution^21,22, and intoxication, and have been used in terrorist attacks^19,20. However, since current methods for removing them are cost prohibitive and lead to ecological concerns, enzymatic remediation is a promising way of developing efficient bio-decontamination methods^23,24,25.

Phosphotriesterase-like Lactonases (PLLs) are appealing candidates, being natural lactonases endowed with promiscuous phosphotriesterase activity²⁶. They have long been related to the bacterial phosphotriesterases (PTEs), enzymes which are capable of hydrolyzing insecticides such as parathion with high catalytic efficiency²⁷. PTEs and PLLs belong to the amidohydrolase superfamily^28,29, and share the same (β/α)₈ barrel topology. Some PLL representatives are extremely thermostable^{30,31,32,33,34,35,36,37,38} and are particularly promising candidates for biotechnological use^39,40 by virtue of their high stability and resistance towards denaturing agents⁴¹. PLLs, however, exhibit much lower phosphotriesterase activity than PTEs. Engineering experiments with the aim of increasing PLLs’ phosphotriesterase activity were performed. In particular, because PTEs and PLLs’ active sites mainly differ in their loop length (7 and 8), sequences and conformations^16,26,42, loop grafting experiments were performed and proved to be difficult. This approach resulted in aggregation-prone or inactive enzymes^16,34, but was successful when using a stepwise approach⁴³. Recently, more classical engineering techniques were successful in increasing the phosphotriesterase activity of the PLL DrOPH⁴⁴.

In this study, we worked on the PLL SsoPox from the hyperthermophilic archaea Sulfolobus solfataricus ^32,45. SsoPox exhibits high acyl-homoserine lactones and oxo-lactones hydrolysis abilities¹¹, and promiscuous, low phosphotriesterase activity⁴⁶. Previous work showed that engineering can successfully improve SsoPox catalytic efficiency against phosphotriesters such as ethyl-paraoxon^11,47. These previous studies highlighted the role of a key active site loop 8 residue, W263, which modulates the active site loop conformational space¹¹. In this study, we aimed to further improve the phosphotriesterase activity of SsoPox, including against phosphothionoesters. SsoPox is a very appealing candidate for the bioremediation of phosphotriesters because of its unique thermal stability, and its ability to resist aging, solvent and protease treatments⁴⁸. On the other hand, SsoPox is a poor phosphothionoesterase, exhibiting a strong preference (100-fold) for methyl-paraoxon over methyl-parathion, a feature referred to as the thiono-effect⁴⁶. We used a rational strategy: taking advantage of the structural similarity between SsoPox and the bacterial PTE from the mesophilic Brevundimonas diminuta (BdPTE)²⁷, we designed and produced a structure-based combinatorial library, and screened this library for improved phosphotriesterase activity with the aim of obtaining heat stable, highly active variants with broad specificity. We obtained several improved variants, including against methyl-parathion, with a 2,210-fold improvement in activity. All these variants but variant αsB5 contained a mutation at position 263. Interestingly, the addition of the W263 mutation using site-saturation mutagenesis¹² on an αsB5 background revealed an incompatibility of these mutations, instead of the expected increase in phosphotrieseterase activity. Of these improved variants, we obtained a mutant that lost the thiono-effect against methyl-parathion, exhibiting a >2,000-fold (k_cat/K_M ~10⁴ M⁻¹.s⁻¹) as referred to the wild-type enzyme, and a methyl-paraoxon/methyl-parathion activity ratio of ~1. The X-ray structures of several improved variants reveal that the selected mutations increase the active site loop conformational flexibility and reshape the active site. The best variants were extensively characterized against eight commercially available insecticides, along with previously reported SsoPox monovariants harboring mutation at residue 263 and their specificity spectrum was determined.

Results and Discussion

The lactonase SsoPox was engineered for higher phosphotriesterase activity using structure-based combinatorial libraries. By comparing structures of enzymes with similar topology, it has been possible to redesign, using modelling tools, the active site cavity of SsoPox to mimic as closely as possible that of BdPTE (Fig. 1; Table S1). This method consisted of two main steps: (i) the identification of mutations corresponding to equivalent positions at the enzyme active sites using structural alignment and (ii) the rational selection of mutations, in a position with no equivalent residue, which mimicked the shape and chemical nature of the target enzyme cavity. A mutation dataset was thus obtained and used to develop a combinatorial library consisting of random combinations of our pre-selected mutations.

Structure-based design produced mutants with improved phosphotriesterase activity

The structure-based design considerably reshaped the active site cavity of SsoPox, theoretically bringing it closer to that of BdPTE, both in shape and nature (Figure S1). A total of 14 key positions within the active site were identified, and were mutated to specific residues, or degenerated into several possible amino acids (see Methods). The paraoxonase activity of 184 randomly selected clones was screened and the 14 best were sequenced. Most of these 14 were found to possess the same mutations (e.g. W263F/L/M/A; 73% of cases) and (Figure S2A; Table S3). Of them, the most active variants, αsA1 (C258L-I261F-W263A), αsA6 (F46L-C258A-W263M-I280T), αsB5 (V27A-I76T-Y97W-Y99F-L130P-L226V), αsC6 (L72I-Y99F-I122L-L228M-F229S-W263L), and αsD6 (V27A-Y97W-L228M-W263M) were subject to kinetic and structural characterization. It should be noted that αsA1 was obtained in a previous engineering effort⁴⁷, and that four of the five selected variants contained a substitution of the key residue W263. We previously highlighted the role of this residue in substrate binding and modulation of the active site loop 8 conformation, resulting in an increase in activity against promiscuous substrates¹¹. Variant αsB5 does not harbor any substitution at position W263. We therefore decided to test the W263 substitution on an αsB5 background, using saturation mutagenesis strategy.

The five variants (αsA1, αsA6, αsB5, αsC6 & αsD6) were characterized against several phosphotriesters, including ethyl-paraoxon (I), ethyl-parathion (II), methyl-paraoxon (III), methyl-parathion (IV) and malathion (V) (Fig. 2). Overall, all variant exhibits improved catalytic efficiencies against all tested substrates, with the exception of αsA1 with malathion (~2-fold decrease) (Fig. 3A; Table 2). The best improved variant, αsD6, exhibits a 2,210-fold increase in catalytic efficiency against methyl-parathion (Table 2). The largest improvements were observed for ethyl/methyl-parathion, two bad substrates for wild-type SsoPox. Interestingly, while the wt enzyme shows a clear preference for small substituents⁴⁶, most selected variants lost this preference, possibly indicating an enlargement of the active site cavity. These improvements were in the range of what was previously obtained but with higher intensive mutation protocols and for only one OP substrate⁴⁹. Finally, it was noted that two variants (αsA6 & αsC6) presented a substrate inhibition for some thiono-phosphotriesters.

Table 2 Kinetic characterization of SsoPox variants.

Full size table

The five selected variants were further evaluated for their lactonase activity (Table 2). Interestingly, variants αsA6 and αsC6 exhibit enhanced lactonase activity against γ- and δ-lactones, while αsA1, αsB5 and αsD6 show reduced lactonase catalytic efficiencies. This emphasizes the fact that the improvement of the phosphotriesterase activity does not necessarily compromise the cognate, lactonase activity of the enzyme. These mutations therefore dramatically increased a new activity (phosphotriesterase) without the complete loss of the native/original function (lactonase)⁴. These mutants are different from those generated on another PLLs, through loop insertion. While the stepwise insertion of residues in loop 7 can significantly increase the ability of PLLs to degrade phosphotriesters, it also drastically decreases their lactonase activity⁴³. Insertion in loop 7 has been previously described as a key evolutionary event in the transition from lactonase to phosphotriesterase⁵⁰.

Structural analysis of improved variants reveals increased mobility of the active site loop 8

The crystal structures of variants αsA1, αsA6, αsB5, αsC6 and αsD6 were solved (Table 1). While most structures were solved at high or medium resolutions (1.4–2.55 Å), mutant asD6 could only be solved at low resolution (2.95 Å). Given this resolution, we limited our interpretations and retained this data for the consistency of the study as it exhibits structural features (loop 8 disorder) that are consistent with all the other mutants. Overall, the mutants’ structures are similar to that of wt-SsoPox. However, the active site cavities are larger for the mutants: most selected mutations replace residues with smaller ones: W263 is mutated into M/L/I, V27 into A, I76 into T, L130 into P, Y99 into F. Only two selected substitutions relate to bulkier side chains: Y97W and L228M. This enlargement is difficult to quantify even with the structure of these mutants because of the extremely mobile nature of loop 8 in the variants. Enlarging the active site was a main objective of the active site redesign of SsoPox, to enable the bulkier phosphotriesters to bind within the natural lactonase enzyme. Additionally, the loop 8 conformations differ in mutants, as compared to the wt-enzyme. Because part of loop 8, including position 263, is located at the enzyme dimer interface, altered loop 8 conformations modulate the relative orientation of both monomers. In the case of the mutants characterized in this study, the dimer reorientation yields significant displacement up to 5.2 Å (e.g., αsA1, between equivalent carbon α positions), as compared to wt-SsoPox (Figure S5). Similar reorientations were previously observed upon substrate binding or mutations of W263^11,42.

Table 1 Data collection and refinement statistics of SsoPox variants structures.

Full size table

Although the active sites of the improved variant structures superpose well onto the wt-structure, all selected mutants display altered loop 8 conformations. Changes are subtle for some of the variants (e.g. αsA1), but are large for others (e.g. αsC6) (Fig. 4). Notably, mutant αsA6 was crystallized in two different conformations: one in which loop 8 adopts a wt-like conformation, referred to as closed conformation (CC), and another one where loop 8 is unfolded, referred to as open conformation (OC) (Figure S6). In the two other mutants, αsD6 and αsB5, loop 8 could not be modeled due to the lack of electronic density, which was likely to be due to the high level of motion of this enzyme region. Analysis of the normalized thermal motion B-factor supports this hypothesis. It also confirms that loop 8 is highly mobile in all selected mutants, as compared to wt-SsoPox (Fig. 5). The higher mobility of loop 8 may also partly explain the improved ability of the variants to hydrolyze phosphotriesters with large substituents.

Improved mutant αsD6 lost the thiono-effect

SsoPox presents a marked preference for oxono-OP substrates as compared to thiono-ones (>100-fold in catalytic efficiency)⁴⁶. Conversely, PTEs do not exhibit such a drastic preference, paraoxon being a slightly better substrate than parathion for the enzyme^27,51. Interestingly, some selected variants retained a strong preference for oxono-phosphotriesters (~10-fold), whereas others such as αsD6 lost the thiono-effect (Figure S7). The wild-type enzyme exhibits a low K_M value with methyl-parathion (121 µM), but also a very low k_cat value (1.10 × 10⁻³ s⁻¹) (Table 2). This may indicate a stronger interaction between the thiono moiety and the metal cations (low K_M value), which would subsequently impair catalysis (low k_cat values)⁵². Interestingly, the improved variants do not exhibit significant difference in K_M values for thiono-phosphotriesters, but show a dramatic increase in k_cat values, as compared to wild-type enzymes (e.g. k_cat value of 6.89 s⁻¹ and 1.10 × 10⁻³ s⁻¹, as compared to methyl-parathion for the αsD6 variant and wild-type enzymes, respectively). The two selected variants, namely αsB5 and αsD6, which lost the thiono-effect have only two mutations in common: V27A and Y97W. Both residues are located relatively close to the bi-metallic active site, yet do not interact directly with the metals (V27-Fe: 6.7 Å, Y97-Co: 4.3 Å; Fig. 6). Because other PLLs have been shown to exhibit high charge coupling between the β metal and the conserved tyrosine residue^33,53, Y97 is a likely candidate for the thiono-effect, and will be examined in future studies.

Mutations of W263 and αsB5 are incompatible

αsB5 (V27A-I76T-Y97W-Y99F-L130P-L226V) is the only selected mutant that does not contain a substitution of position W263. Because W263 substitutions were previously shown to be key for increasing SsoPox’s phosphotriesterase activity, we used saturation mutagenesis to introduce W263 mutations to the background of αsB5¹². The best selected mutants against paraoxon (Figure S2C) were found to be αsB5-W263I/L/M. Surprisingly, none of these mutants demonstrated an increase in phosphotriesterase activity in comparison with αsB5, but rather a decrease in activity (Table 2). This is intriguing because, taken individually, these mutations have been shown to greatly improve phosphotriesterase activity. For example, with ethyl-paraoxon as a substrate, W263M and αsB5 produce a catalytic efficiency improvement of 14 and 67-fold, respectively, while the combination αsB5-W263M increases activity only 11-fold (Table 2). This example of negative epistasis could be due to the mode of action of these mutations. Structures reveal that αsB5 mutations have a destabilizing effect on loop 8, as illustrated by the B factor analysis. The same was observed for W263M in a previous study¹¹. Combining these two destabilizing sets of mutations may have harmed the active site integrity, as previously described with another enzyme as “conformational active site disorder”⁵⁴, and in particular the necessary alignment and pre-organization of the catalytic residues. The destabilizing effect of these mutations can also be observed on the overall enzyme stability, with αsB5, W263M, αsB5-W263M exhibiting T_m values of 70.4 °C, 85.3 °C and 69.1 °C, respectively, as compared to 106 °C for the wt enzyme (Table S4). Here, the effects of the combined 5 mutations harbored by variant αsB5 together with the previously identified positive substitutions at residue W263 were not additive underlining that single beneficial mutations do not necessarily induce positive epistatic effects. Synergistic effects of mutations are often complex to predict and most engineering strategies usually lead to an optimization plateau difficult to overcome⁵⁵.

Improvement of engineered variants over W263 mutation

In a previous report, we noted that mutation of W263 into all the other 19 residues lead to an increase of the promiscuous paraoxonase activity¹¹. Here we note that most of the improved variants (i.e. αsA1, αsA6, αsB5, αsC6 & αsD6) (i) exhibit higher paraoxonase activity than W263L/M, and (ii) foremost are capable of hydrolyzing phosphotriesters that are not substrates for the wild-type enzyme or W263 mutants, such as ethyl-parathion, chlorpyrifos or fensulfothion (Table 2). The activity spectrum of these engineered variants against the tested phosphotriesters is therefore much wider than the wild-type enzyme and W263 mutants.

Relation between active site loop disorder and broad enzymatic specificity

The organophosphorus compound bioremediation potential of several mutants was investigated. In particular, the best variants, αsD6 and αsB5, alongside other improved variants αsB5-W263I/L/M, W263L/M and the wt-enzyme, were characterized for their phosphotriesterase activity with ethyl-paraoxon (I), ethyl-parathion (II), malathion (V), chlorpyrifos (VI), diazinon (VII), fenitrothion (VIII), fensulfothion (IX) and coumaphos (X) (Fig. 2). Interestingly, we found that most mutants were able to hydrolyze fensulfothion, previously reported as an inhibitor of the wild-type enzyme⁴⁶. Fensulfothion was indeed previously shown to bind head-to-tail into the wild-type enzyme active side⁴⁶, a non-productive binding mode. Changes in the active site loop 8 conformational ensemble of engineered mutants might have allowed for the productive binding of fensulfothion.

All mutants selected and used in this study are destabilized as compared to the wt-enzyme (Fig. 7; Table S4). Structures reveal that these mutations, located on loop 8, contribute to increasing the mobility of the loop. Interestingly, the ability of the tested mutants to hydrolyze a wide range of phosphotriesters, including compounds that were inhibitors for the wt-enzyme, correlates with a lower T_m (Fig. 7 ). This correlation suggest that the active site loop 8 degree of disorder may modulate the enzyme’s ability to bind and hydrolyze a variety of phosphotriesters, a wider conformational ensemble of loop 8 being correlated with a broader enzyme specificity. This observation is consistent with previous studies highlighting the importance of the destabilization of active site loops to evolve new activities and new substrate specificity^54,56. Such flexible active site loops involved in enzymatic specificity supports the notion of fold polarity: a portion of the active site (e.g. the loop) is weakly connected to the enzyme scaffold and thereby makes it possible for new functions to evolve with few changes⁵⁷.

Mutant αsD6 is an active, broad spectrum phosphotriester biodecontaminant

Mutant αsD6 was further investigated for bioremediation considerations. Organophosphate-based pesticides are usually spread with concentrations in the millimolar range leading to micromolar-ranged contaminations. We thus investigated the potential of αsD6 for decontaminating pesticide solutions at 250 µM to simulate a contamination of groundwater or runoff waters. We investigated two $\text{[E]/}[S]$ ratios, 10⁻² and 10⁻³ respectively, for decontaminating OP solutions and determined the time required to hydrolyze 95% of pesticide preparations (Fig. 3B; Figure S4). When considering the $\text{[E]/}[S]$ ratio of 10⁻³, 95% degradation was achieved for four substrates (ethyl-paraoxon, diazinon, fenitrothion and coumaphos) within 10 minutes. Two substrates were decontaminated within an hour (ethyl-parathion and chlorpyrifos) and fensulfothion was degraded in three hours.

When increasing enzyme concentration 10-fold ($\text{[E]/}[S]$ ratio of 10⁻²), five substrates (ethyl-paraoxon, ethyl-parathion, diazinon, fenitrothion and coumaphos) were decontaminated within two minutes, while fensulfothion needed ~20 minutes and the two other tested (chlorpyrifos and malathion) required 40 minutes. Given both its catalytic proficiency and high thermal stability (T_m = 82.5 °C), αsD6 is a promising candidate for organophosphorus compound bioremediation and, in particular, the decontamination of water runoffs, soils, food products and materials. Moreover, SsoPox variants were previously shown to be compatible with immobilization steps including alginate beads and polyurethane-based coatings^48,58, and could be of prime interest for the development of filtration devices for water treatment purposes.

Methods

Structure-based identification of mutations

Sharing the same (α/β)₈ barrel topology, SsoPox and BdPTE structures (Pdb ID 2vc5 and 1dpm, respectively) were superimposed (Fig. 1) using PyMol⁵⁹, making it possible to identify structurally equivalent residues at their respective active sites. In an effort to reshape the SsoPox active site, side chains that were well superimposed between the two enzymes were mutated using Coot into the residue present in BdPTE⁶⁰, with the exception of V27 which was mutated in Ala (and not Gly) to avoid increased entropy (Fig. 1; Table S1). However, due to the major differences in loop 8 and 7 between PLLs and PTEs, numerous residues were not superimposable. These active site residues (Y99, L228, F229 and W263) were therefore mutated in an effort to mimic the BdPTE active site cavity in terms of shape and chemical nature, as illustrated by the chimeric reconstruction of SsoPox carrying all these mutations (Figure S1 ). Others mutations were also implemented in the dataset, e.g. C258A and C258L and different substitutions of the key position W263 (F/L/M/A) that have been shown to improve the phosphodiesterase activity¹¹.

Synthesis of the combinatorial library

The SsoPox coding gene was amplified from the previously described pET22b-SsoPox plasmid⁴⁶. For each of the 14 mutations, a mutagenesis primer of 30–33 bp (~10–15 bp from each mutation side) was synthesized (Table S2). For close mutations, primers can share several mutations and different primers were synthesized and mixed to keep an equivalent statistical probability for each of the 14 mutations. Typically, 2 pmol of primer mixture and 100 ng of DNaseI (TaKaRa)-generated fragments of the SsoPox gene were assembled as previously described¹⁷, followed by nested PCR with external cloning primers (SsoPox-lib-pET-5′/3′) (Table S2). The PCR product was then cloned into a customized version of the pET32b(-ΔTrx) plasmid and then electroporated into E. cloni cells (Lucigen, USA). After agar-plate growth, plasmid extraction was performed to create the plasmid bank. Variant genes were PCR amplified using T7-prom and pET-RP primers (Table S2) and sequenced. The combinatorial library shows an average of 5.3 ± 3.3 library mutations by sequencing 10 randomly picked colonies, and 0.43 ± 0.53 random mutations per gene.

Screening of the library

The plasmid library was used to transform the Escherichia coli strain BL21(DE₃)-pGro7/GroEL (TaKaRa) to obtain colonies expressing a library of SsoPox variants. Randomly picked clones (184), representing a coverage of 3.8% of the library, were grown on a 96-well plate in 500 µL of ZYP medium as previously described¹¹. Production of proteins and chaperones was induced after five hours of culture at 37 °C by reducing the temperature to 25 °C, adding CoCl₂ (0.2 mM) and adding arabinose (0.2%, w/v). After overnight growth, cell lysate was used to perform activity screening with 100 µM of paraoxon substrate (Figure S2B) after partial purification of the protein with a heating step of 15 minutes incubation at 70 °C^35,49. The screening was performed in 50 mM HEPES pH 8, 150 mM NaCl, 0.2 mM CoCl₂. Kinetics of paraoxon/methyl-parathion hydrolysis were monitored by following absorbance at 405 nm for 10 minutes using a microplate reader (Synergy HT, BioTek, USA) and the Gen5.1 software.

Saturation library

Saturation mutagenesis directed on residue 263 of the αsB5 variant encoding gene, was performed using NNS degenerated primers (W263NNS direct: 5′-TGCACCATTGATNNSGGCACCGCAAAACCG-3′ and W263NNS reverse: 5′-CGGTTTTGCGGTGCCSNNATCAATGGTGCA-3′) and pET32b-ΔTrx carrying αsB5 coding gene as template. PCR amplification was performed with 2.5 U of PfuTurbo DNA polymerase (Agilent) according to the manufacturer’s recommendations [95 °C, 5 minutes; 20x (95 °C, 30 seconds; 55 °C, one minute; 68 °C, 15 minutes); 68 °C, 25 minutes]. DNA was digested with DpnI to remove the methylated parental template. E. coli BL21(DE₃)-pGro7/GroEL (TaKaRa) competent cells were transformed with the plasmid pool and plated on LB-agar supplemented with 100 µg.mL⁻¹ ampicillin and 34 µg.mL⁻¹ chloramphenicol. Over a theoretical diversity of 20 sequences, 181 variants were collected in a microplate containing LB (100 µg.mL⁻¹ ampicillin and 34 µg.mL⁻¹ chloramphenicol) for characterization. This oversampling by a factor of nearly 10-fold ensure a >99% probability to get the 20 possible substitutions.

Screening was performed as described above (Figure S2C). Clones were produced in liquid ZYP medium and their activity was screened with 100 µM of paraoxon substrate. The plasmids corresponding to the most interesting variants were extracted and the SsoPox variant encoding genes were sequenced.

Production-purification of SsoPox wild-type and variants

The genes coding for the SsoPox wild-type and its variant were cloned in a pET32b-Δtrx plasmid. Productions were performed using the E. coli BL21(DE₃)-pGro7/GroEL (TaKaRa) chaperone expressing strain. Starter cultures were produced in an auto-inducible ZYP medium (supplemented with 100 µg.mL⁻¹ ampicillin and 34 µg.mL⁻¹ chloramphenicol). When OD_600nm reached a value of 0.8–1, CoCl₂ was added (final concentration 0.2 mM) as well as L-arabinose (final concentration 2 g.L⁻¹) to induce the production of chaperones GroEL/ES and the temperature was decreased to 23 °C for 16–20 hours. Cells were harvested by centrifugation (4400 g, 4 °C, 20 minutes) and resuspended in lysis buffer (50 mM HEPES pH 8.0, 150 mM NaCl, 0.2 mM CoCl₂, 0.25 mg.mL⁻¹ lysozyme, 0.1 mM PMSF and 10 µg.mL⁻¹ DNAseI) and were stored at -80 °C. Cells were thawed and lysed in three steps of 30 seconds of sonication (Qsonica, Q700; Amplitude 45). Cell debris was removed by centrifugation (20000 g, 4 °C, 15 minutes). As SsoPox and its variants are hyperthermostable, a pre-purification step was performed by heating the lysate for 30 minutes at 80 °C. Precipitated host proteins were removed by centrifugation (20000 g, 4 °C, 15 minutes). SsoPox and its variants were collected by ammonium sulfate precipitation (75%) and resuspended in 8 ml of activity buffer (50 mM HEPES pH 8.0, 150 mM NaCl, 0.2 mM CoCl₂). The remaining ammonium sulfate was eliminated by injection on a desalting column (HiPrep 26/10 desalting, GE Healthcare; ÄKTA Avant) and concentrated to 2 mL for separation on exclusion size chromatography (HiLoad 16/600 Superdex^TM 75 pg, GE Healthcare; ÄKTA Avant). Final purity was monitored by SDS-PAGE and protein concentration was measured with a NanoDrop 2000 spectrophotometer (Thermo Scientific).

Kinetic assays

Generalities. Catalytic parameters were evaluated in triplicate at 25 °C, and recorded using a microplate reader (Synergy HT, BioTek, USA) and the Gen5.1 software, in a 6.2 mm path length cell for 200 µL reaction in 96-well plate, as previously explained³⁰. Catalytic parameters were obtained by fitting the data to the Michaelis-Menten (MM) equation using Graph-Pad Prism 6 software. When V_max could not be reached in the experiments, catalytic efficiency was obtained by fitting the linear part of MM plot to a linear regression using Graph-Pad Prism 6 software. For some SsoPox variants, the MM plot was fitted to the substrate inhibition equation using Graph-Pad Prism 6 software enabling us to determine a K_I for several substrates. Consequently, the calculated catalytic efficiencies in these conditions are true only at low substrate concentrations. In some other cases, saturation could not be reached, therefore ${k}_{{cat}}/{K}_{M}$ values were determined using linear regression. For Coumaphos, data were fitted to one-phase decay non-linear regression.

Phosphotriesterase activity characterization

The kinetic assays were carried out in activity buffer. The conditions used for pesticides were as follows: ethyl-paraoxon (I), ethyl-parathion (II), methyl-paraoxon (III) and methyl-parathion (IV) hydrolysis was recorded at 405 nm (ε = 17000 M⁻¹.cm⁻¹). The concentration interval was between 0.05 and 2 mM from 100 mM stock solution in ethanol⁴⁶. Malathion (V) hydrolysis was monitored at 412 nm, (ε = 13400 M⁻¹.cm⁻¹)⁴⁶. The concentration interval was between 0.05 and 2 mM from 100 mM stock solution in DMSO. DTNB was added to the activity buffer. Chlorpyrifos (VI) hydrolysis was recorded at 310 nm (ε = 5562 M⁻¹.cm⁻¹)⁵¹. The concentration interval was between 0.05 and 2 mM from 100 mM stock solution in 1-propanol. Diazinon (VII) hydrolysis was recorded at 228 nm (ε = 3300 M⁻¹.cm⁻¹)²⁷. The concentration interval was between 0.05 and 2 mM from 200 mM stock solution in ethanol. Fenitrothion (VIII) hydrolysis was recorded at 358 nm (ε = 18700 M⁻¹.cm⁻¹)⁵². The concentration interval was between 0.05 and 2 mM from 200 mM stock solution in ethanol. Fensulfothion (IX) hydrolysis was recorded at 284 nm (ε = 8000 M⁻¹.cm⁻¹)²⁷. The concentration interval was between 0.05 and 2 mM from 200 mM stock solution in methanol. Coumaphos (X) hydrolysis was monitored by fluorescence of released chlorferon (excitation 360/40 and emission 460/40)⁵¹. A linear correlation between fluorescence and chlorferon was found (Figure S3). Measurements were taken at 5 µM from a 200 mM stock solution in DMSO. We assumed that ${K}_{M}\gg [S]$, thus enabling us to estimate ${k}_{{cat}}/{K}_{M}$.

Lactonase activity characterization

Lactonase kinetics were performed using a previously described protocol³⁰. The time course hydrolysis of lactones were performed in lac buffer (2.5 mM Bicine pH 8.3, 150 mM NaCl, 0.2 mM CoCl₂, 0.25 mM Cresol purple and 0.5% DMSO) over a concentration range 0–2 mM for 3-oxo-C10 AHLs (XI) or 0–5 mM for undecanoic- γ/δ -lactones (XII, XIII). Cresol purple (pK_a 8.3 at 25 °C) is a pH indicator used to follow lactone ring hydrolysis by acidification of the medium. Molar coefficient extinction at 577 nm was evaluated recording absorbance of the buffer over an acetic acid range of concentration 0–0.35 mM.

Bioremediation of pesticide solutions at 250 µM

For all OP substrates but malathion, experiments were performed in triplicate at 25 °C using a microplate reader (Synergy HT, BioTek, USA). Wavelengths were chosen as described in the kinetic assays section. Degradation of malathion was followed by GC/MS in triplicate. OP concentration was fixed at 250 µM. All the substrates were soluble at this concentration. The best variants of SsoPox for each substrate were used, namely αsD6 for all the OPs. Two enzyme-to-substrate ratios $\text{[E]/}[S]$ were used, 10⁻² and 10⁻³, corresponding to enzyme concentrations of 90 µg.ml⁻¹ and 9 µg.ml⁻¹ respectively. The reactions were monitored until the plateau was reached. Experimental measures were obtained using Gen5.1 software, then analyzed with GraphPad Prism 6 software. Curves were then fitted using One-Phase Decay non-linear regression with the equation (1):

$$Y=(Y0-Plateau)\times {e}^{(-kt)}+Plateau$$

(1)

where Y0 = 0% and Plateau = 100%. The rate constant k was determined and the time required to observe a 95% decontamination was calculated accordingly. The curves and results of the fits are shown in Figure S4.

Degradation at 95% was confirmed using a final point by GC/MS for all pesticides with a 10⁻³ enzyme-to-substrate ratio. 100 µL of activity buffer solution was first extracted with 100 µL chloroform. Organic extracts were analyzed by using a Clarus 500 gas chromatograph equipped with a SQ8S MS detector (Perkin Elmer, Courtaboeuf, France). 1 µL of organic extract was volatilized at 220 °C (split 15 mL.min⁻¹) in a deactivated FocusLiner with quartz wool (SGE, Ringwood, Australia) and compounds separated on an Elite-5MS column (30 m, 0.25 mm i.d., 0.25 mm film thickness) for 12 minutes using a temperature gradient (80–280 °C at 30 °C.min⁻¹, five minutes’ hold). Helium flowing at 2 mL.min⁻¹ was used as the carrier gas. The MS inlet line was set at 280 °C and the electron ionization source at 280 °C and 70 eV. Full scan monitoring was performed from 40 to 400 m/z in order to identify chemicals by spectral database search using MS Search 2.0 operated with the Standard Reference Database 1 A (National Institute of Standards and Technology, Gaithersburg, MD, USA). m/z is the mass-to-charge ratio of the base peak fragment detected for each molecule. In the case of weak pesticide signals, extracted ion chromatograms were generated with base peak ions to confirm the presence of chemicals (Paraoxon (I) and Parathion ethyl (II) m/z 109; Malathion (V) and Fenitrothion (VIII) m/z 125; Diazinon (VII) m/z 137; Coumaphos (X) m/z 97; Fensulfothion (IX) m/z 293; and Chlorpyrifos (VI) m/z 197). Selected Ion Recording using base peaks ions was applied in order to specifically monitor pesticides and collect peak areas for kinetics. All samples were analyzed over short periods of time to avoid signal drift. All data were processed using Turbomass 6.1 (Perkin Elmer).

Crystallization

Crystallization assays were performed as previously described^42,46. Crystallization was performed using the hanging drop vapor diffusion method in 96-well plates (Greiner Microplate, 96 well, PS, F-bottom) on ViewDrop II seals (TPP Labtech). Equal volumes (0.5 µL) of protein and reservoir solutions were mixed, and the resulting drops were equilibrated against a 150 µL reservoir solution containing 20–30% (w/v) PEG 8000 and 50 mM Tris-HCl buffer (pH 8). Crystals appeared after few days at 4 °C.

Data collection and structure refinement

Crystals were first transferred to a cryoprotectant solution consisting of the reservoir solution and 20% (v/v) glycerol. Crystals were then flash-cooled in liquid nitrogen. X-ray diffraction data were collected at 100 K using synchrotron radiation at the ID23-1 beam line (ESRF, Grenoble, France) and using an ADSC Q315r detector. X-ray diffraction data were integrated and scaled with the XDS package (Table 1)⁶¹. The phases were obtained using the native structure of SsoPox (PDB code 2vc5) as a starting model, performing a molecular replacement with MOLREP or PHASER ^62,63. The models were built with Coot and refined using REFMAC ^60,64. We note that three structures presented in this work exhibit one disordered monomer (and low corresponding electronic density): αsB5 (monomer D, total of 4 monomers), αsD6 (monomer C, out of a total of 4 monomers) and αsC6 (monomer C, total of 4 monomers). Structure illustrations were performed using PyMOL ⁴⁸.

Relative B-factor analysis

The occupancies of all residues in all tested structures were set to 1 for this analysis. For residues with alternate conformations, the sums of occupancies were set to 1. Structures were re-refined with REFMAC ^60,64. The relative B-factor values were obtained by normalizing the B-factor values of each residue by the average B-factor of the whole structure as previously described^11,54,57.

References

Wolfenden, R. Benchmark Reaction Rates, the Stability of Biological Molecules in Water, and the Evolution of Catalytic Power in Enzymes. Annu. Rev. Biochem. 80, 645–667 (2011).
Article CAS PubMed Google Scholar
Singh, R. K., Tiwari, M. K., Singh, R. & Lee, J.-K. From Protein Engineering to Immobilization: Promising Strategies for the Upgrade of Industrial Enzymes. Int. J. Mol. Sci. 14, 1232–1277 (2013).
Article CAS PubMed PubMed Central Google Scholar
Bar-Even, A. et al. The Moderately Efficient Enzyme: Evolutionary and Physicochemical Trends Shaping Enzyme Parameters. Biochemistry (Mosc.) 50, 4402–4410 (2011).
Article CAS Google Scholar
Tokuriki, N. et al. Diminishing returns and tradeoffs constrain the laboratory optimization of an enzyme. Nat. Commun. 3, 1257 (2012).
Article PubMed Google Scholar
Burton, S. G., Cowan, D. A. & Woodley, J. M. The search for the ideal biocatalyst. Nat. Biotechnol. 20, 37–45 (2002).
Article CAS PubMed Google Scholar
Gupta, R. D. & Tawfik, D. S. Directed enzyme evolution via small and effective neutral drift libraries. Nat. Methods 5, 939–942 (2008).
Article CAS PubMed Google Scholar
Bommarius, A. S. Biocatalysis: A Status Report. Annu. Rev. Chem. Biomol. Eng. 6, 319–345 (2015).
Article CAS PubMed Google Scholar
Currin, A., Swainston, N., Day, P. J. & Kell, D. B. Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently. Chem Soc Rev 44, 1172–1239 (2015).
Article CAS PubMed Google Scholar
Reetz, M. T. Directed Evolution of Selective Enzymes: Catalysts for Organic Chemistry and Biotechnology. (Wiley-VCH Verlag GmbH & Co. KGaA, 2016), https://doi.org/10.1002/9783527655465.
Directed Enzyme Evolution: Advances and Applications. (Springer International Publishing, 2017). https://doi.org/10.1007/978-3-319-50413-1.
Hiblot, J., Gotthard, G., Elias, M. & Chabriere, E. Differential Active Site Loop Conformations Mediate Promiscuous Activities in the Lactonase SsoPox. PLoS ONE 8, e75272 (2013).
Article ADS CAS PubMed PubMed Central Google Scholar
Reetz, M. T. Laboratory Evolution of Stereoselective Enzymes: A Prolific Source of Catalysts for Asymmetric Reactions. Angew. Chem. Int. Ed. 50, 138–174 (2011).
Article CAS Google Scholar
Daudé, D., Topham, C. M., Remaud‐Siméon, M. & André, I. Probing impact of active site residue mutations on stability and activity of Neisseria polysaccharea amylosucrase. Protein Sci. 22, 1754–1765 (2013).
Article PubMed PubMed Central Google Scholar
Alcolombri, U., Elias, M. & Tawfik, D. S. Directed Evolution of Sulfotransferases and Paraoxonases by Ancestral Libraries. J. Mol. Biol. 411, 837–853 (2011).
Article CAS PubMed Google Scholar
Gonzalez, D. et al. Ancestral mutations as a tool for solubilizing proteins: The case of a hydrophobic phosphate-binding protein. FEBS Open Bio 4, 121–127 (2014).
Article CAS PubMed PubMed Central Google Scholar
Hawwa, R., Larsen, S. D., Ratia, K. & Mesecar, A. D. Structure-Based and Random Mutagenesis Approaches Increase the Organophosphate-Degrading Activity of a Phosphotriesterase Homologue from Deinococcus radiodurans. J. Mol. Biol. 393, 36–57 (2009).
Article CAS PubMed Google Scholar
Herman, A. & Tawfik, D. S. Incorporating Synthetic Oligonucleotides via Gene Reassembly (ISOR): a versatile tool for generating targeted libraries. Protein Eng. Des. Sel. 20, 219–226 (2007).
Article CAS PubMed Google Scholar
Rochu, D., Chabrière, E. & Masson, P. Human paraoxonase: A promising approach for pre-treatment and therapy of organophosphorus poisoning. Toxicology 233, 47–59 (2007).
Article CAS PubMed Google Scholar
Krieger, R. Handbook of Pesticide Toxicology, Two-Volume Set: Principles and Agents. (Academic Press, 2001).
Gupta, R. C. Handbook of Toxicology of Chemical Warfare Agents. (Academic Press, 2009).
Sapozhnikova, Y. et al. Evaluation of pesticides and metals in fish of the Dniester River, Moldova. Chemosphere 60, 196–205 (2005).
Article ADS CAS PubMed Google Scholar
Sapozhnikova, Y., Bawardi, O. & Schlenk, D. Pesticides and PCBs in sediments and fish from the Salton Sea, California, USA. Chemosphere 55, 797–809 (2004).
Article ADS CAS PubMed Google Scholar
LeJeune, K. E., Wild, J. R. & Russell, A. J. Nerve agents degraded by enzymatic foams. Nature 395, 27–28 (1998).
Article ADS CAS PubMed Google Scholar
Jacquet, P. et al. Current and emerging strategies for organophosphate decontamination: special focus on hyperstable enzymes. Environ. Sci. Pollut. Res. 1–19 https://doi.org/10.1007/s11356-016-6143-1 (2016).
Poirier, L., Jacquet, P., Elias, M., Daudé, D. & Chabrière, E. Decontamination of organophosphorus compounds: Towards new alternatives. Ann. Pharm. Fr. https://doi.org/10.1016/j.pharma.2017.01.004 (2017).
Afriat, L., Roodveldt, C., Manco, G. & Tawfik, D. S. The Latent Promiscuity of Newly Identified Microbial Lactonases Is Linked to a Recently Diverged Phosphotriesterase†. Biochemistry (Mosc.) 45, 13677–13686 (2006).
Article CAS Google Scholar
Dumas, D. P., Caldwell, S. R., Wild, J. R. & Raushel, F. M. Purification and properties of the phosphotriesterase from Pseudomonas diminuta. J. Biol. Chem. 264, 19659–19665 (1989).
CAS PubMed Google Scholar
Seibert, C. M. & Raushel, F. M. Structural and Catalytic Diversity within the Amidohydrolase Superfamily†. Biochemistry (Mosc.) 44, 6383–6391 (2005).
Article CAS Google Scholar
Roodveldt, C. & Tawfik, D. S. Shared Promiscuous Activities and Evolutionary Features in Various Members of the Amidohydrolase Superfamily. Biochemistry (Mosc.) 44, 12728–12736 (2005).
Article CAS Google Scholar
Hiblot, J., Gotthard, G., Chabriere, E. & Elias, M. Structural and Enzymatic characterization of the lactonase SisLac from Sulfolobus islandicus. PLoS ONE 7, e47028 (2012).
Article ADS CAS PubMed PubMed Central Google Scholar
Porzio, E., Merone, L., Mandrich, L., Rossi, M. & Manco, G. A new phosphotriesterase from Sulfolobus acidocaldarius and its comparison with the homologue from Sulfolobus solfataricus. Biochimie 89, 625–636 (2007).
Article CAS PubMed Google Scholar
Merone, L., Mandrich, L., Rossi, M. & Manco, G. A thermostable phosphotriesterase from the archaeon Sulfolobus solfataricus: cloning, overexpression and properties. Extremophiles 9, 297–305 (2005).
Article CAS PubMed Google Scholar
Hawwa, R., Aikens, J., Turner, R. J., Santarsiero, B. D. & Mesecar, A. D. Structural basis for thermostability revealed through the identification and characterization of a highly thermostable phosphotriesterase-like lactonase from Geobacillus stearothermophilus. Arch. Biochem. Biophys. 488, 109–120 (2009).
Article CAS PubMed PubMed Central Google Scholar
Xiang, D. F. et al. Functional Identification of Incorrectly Annotated Prolidases from the Amidohydrolase Superfamily of Enzymes. Biochemistry (Mosc.) 48, 3730–3742 (2009).
Article CAS Google Scholar
Chow, J. Y. et al. Directed Evolution of a Thermostable Quorum-quenching Lactonase from the Amidohydrolase Superfamily. J. Biol. Chem. 285, 40911–40920 (2010).
Article CAS PubMed PubMed Central Google Scholar
Hiblot, J., Gotthard, G., Champion, C., Chabriere, E. & Elias, M. Crystallization and preliminary X-ray diffraction analysis of the lactonase VmoLac from Vulcanisaeta moutnovskia. Acta Crystallograph. Sect. F Struct. Biol. Cryst. Commun. 69, 1235–1238 (2013).
Article CAS Google Scholar
Hiblot, J., Bzdrenga, J., Champion, C., Chabriere, E. & Elias, M. Crystal structure of VmoLac, a tentative quorum quenching lactonase from the extremophilic crenarchaeon Vulcanisaeta moutnovskia. Sci. Rep. 5, 8372 (2015).
Article ADS PubMed PubMed Central Google Scholar
Bzdrenga, J. et al. SacPox from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius is a proficient lactonase. BMC Res. Notes 7, 333 (2014).
Article PubMed PubMed Central Google Scholar
Bzdrenga, J. et al. Biotechnological applications of quorum quenching enzymes. Chem. Biol. Interact. https://doi.org/10.1016/j.cbi.2016.05.028 (2016).
Rémy, B., Plener, L., Elias, M., Daudé, D. & Chabrière, E. Des enzymes pour bloquer la communication bactérienne, une alternative aux antibiotiques? Ann. Pharm. Fr. https://doi.org/10.1016/j.pharma.2016.06.005 (2016).
Vieille, C. & Zeikus, G. J. Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability. Microbiol. Mol. Biol. Rev. 65, 1–43 (2001).
Article CAS PubMed PubMed Central Google Scholar
Elias, M. et al. Structural Basis for Natural Lactonase and Promiscuous Phosphotriesterase Activities. J. Mol. Biol. 379, 1017–1028 (2008).
Article CAS PubMed Google Scholar
Hoque, M. A. et al. Stepwise Loop Insertion Strategy for Active Site Remodeling to Generate Novel Enzyme Functions. ACS Chem. Biol. https://doi.org/10.1021/acschembio.7b00018 (2017).
Meier, M. M. et al. Molecular Engineering of Organophosphate Hydrolysis Activity from a Weak Promiscuous Lactonase Template. J. Am. Chem. Soc. 135, 11670–11677 (2013).
Article CAS PubMed PubMed Central Google Scholar
Elias, M. et al. Crystallization and preliminary X-ray diffraction analysis of the hyperthermophilic Sulfolobus solfataricus phosphotriesterase. Acta Crystallograph. Sect. F Struct. Biol. Cryst. Commun. 63, 553–555 (2007).
Article CAS Google Scholar
Hiblot, J., Gotthard, G., Chabriere, E. & Elias, M. Characterisation of the organophosphate hydrolase catalytic activity of SsoPox. Sci. Rep. 2 (2012).
Del Giudice, I. et al. An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination. Biotechnol. Bioeng. 113, 724–734 (2016).
Article PubMed Google Scholar
Rémy, B. et al. Harnessing hyperthermostable lactonase from Sulfolobus solfataricus for biotechnological applications. Sci. Rep. 6 (2016).
Vecchio, P. D. et al. Structural determinants of the high thermal stability of SsoPox from the hyperthermophilic archaeon Sulfolobus solfataricus. Extremophiles 13, 461–470 (2009).
Article CAS PubMed Google Scholar
Afriat-Jurnou, L., Jackson, C. J. & Tawfik, D. S. Reconstructing a Missing Link in the Evolution of a Recently Diverged Phosphotriesterase by Active-Site Loop Remodeling. Biochemistry (Mosc.) 51, 6047–6055 (2012).
Article CAS Google Scholar
Jackson, C. J. et al. Anomalous scattering analysis of Agrobacterium radiobacter phosphotriesterase: the prominent role of iron in the heterobinuclear active site. Biochem. J. 397, 501 (2006).
Article CAS PubMed PubMed Central Google Scholar
Tawfik, D. S. Accuracy-rate tradeoffs: how do enzymes meet demands of selectivity and catalytic efficiency? Curr. Opin. Chem. Biol. 21, 73–80 (2014).
Article CAS PubMed Google Scholar
Xiang, D. F. et al. Functional Annotation and Three-Dimensional Structure of Dr0930 from Deinococcus radiodurans: A Close Relative of Phosphotriesterase in the Amidohydrolase Superfamily. Biochemistry (Mosc.) 48, 2237–2247 (2009).
Article CAS Google Scholar
Dellus-Gur, E. et al. Negative Epistasis and Evolvability in TEM-1 β-Lactamase—The Thin Line between an Enzyme’s Conformational Freedom and Disorder. J. Mol. Biol. 427, 2396–2409 (2015).
Article CAS PubMed PubMed Central Google Scholar
Reetz, M. T. The Importance of Additive and Non-Additive Mutational Effects in Protein Engineering. Angew. Chem. Int. Ed. 52, 2658–2666 (2013).
Article CAS Google Scholar
Campbell, E. et al. The role of protein dynamics in the evolution of new enzyme function. Nat. Chem. Biol. 12, 944–950 (2016).
Article CAS PubMed Google Scholar
Dellus-Gur, E., Toth-Petroczy, A., Elias, M. & Tawfik, D. S. What Makes a Protein Fold Amenable to Functional Innovation? Fold Polarity and Stability Trade-offs. J. Mol. Biol. 425, 2609–2621 (2013).
Article CAS PubMed Google Scholar
Guendouze, A. et al. Effect of quorum quenching lactonase in clinical isolates of Pseudomonas aeruginosa and comparison with quorum sensing inhibitors. Front. Microbiol. 8 (2017).
DeLano, W. L. The PyMOL molecular graphics system (2002).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
Article PubMed Google Scholar
Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993).
Article CAS Google Scholar
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Article CAS PubMed PubMed Central Google Scholar
Vagin, A. & Teplyakov, A. An approach to multi-copy search in molecular replacement. Acta Crystallogr. D Biol. Crystallogr. 56, 1622–1624 (2000).
Article CAS PubMed Google Scholar
Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of Macromolecular Structures by the Maximum-Likelihood Method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).
Article CAS PubMed Google Scholar

Download references

Acknowledgements

This work was granted by DGA, France (REI. 2009 34 0045). G.G. and P.J. are PhD students granted by DGA. Financial support was also obtained from the MnDrive Initiative (M.E.) to support this work. This work was supported by “Investissements d’avenir” program (Méditerranée Infection 10-IAHU-03) of the French Agence Nationale de la Recherche (ANR).

Author information

Julien Hiblot
Present address: MPI for Medical Research, Chemical Biology department (EPFL), Heidelberg, Germany
Pauline Jacquet, Julien Hiblot and David Daudé contributed equally to this work.

Authors and Affiliations

CNRS UMR 7278, IRD198, INSERM U1095, APHM, Institut Hospitalier Universitaire Méditerranée-Infection, Aix-Marseille Université, 19-21 Bd Jean Moulin, 13005, Marseille, France
Pauline Jacquet, Julien Hiblot, Céline Bergonzi, Guillaume Gotthard, Nicholas Armstrong & Eric Chabrière
Gene&GreenTK, IHU Méditerranée Infection, 19-21 Bd Jean Moulin, 13005, Marseille, France
David Daudé
University of Minnesota, Department of Biochemistry, Molecular Biology and Biophysics & Biotechnology Institute, St. Paul, MN, 55108, USA
Céline Bergonzi & Mikael Elias

Authors

Pauline Jacquet
View author publications
You can also search for this author in PubMed Google Scholar
Julien Hiblot
View author publications
You can also search for this author in PubMed Google Scholar
David Daudé
View author publications
You can also search for this author in PubMed Google Scholar
Céline Bergonzi
View author publications
You can also search for this author in PubMed Google Scholar
Guillaume Gotthard
View author publications
You can also search for this author in PubMed Google Scholar
Nicholas Armstrong
View author publications
You can also search for this author in PubMed Google Scholar
Eric Chabrière
View author publications
You can also search for this author in PubMed Google Scholar
Mikael Elias
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

P.J., J.H., D.D., C.B., N.A., G.G. performed the experiments, P.J., J.H., D.D., C.B., N.A., G.G., E.C., M.E. analyzed the data, P.J., J.H., D.D., C.B., M.E. wrote the manuscript and prepared the figures, E.C., D.D. and M.E. designed the experiments.

Corresponding authors

Correspondence to Eric Chabrière or Mikael Elias.

Ethics declarations

Competing Interests

M.E. and E.C. have a patent WO2014167140 A1 licensed to Gene&GreenTK. D.D. and E.C. report personal fees from Gene&GreenTK during the conduct of the study.

Additional information

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Jacquet, P., Hiblot, J., Daudé, D. et al. Rational engineering of a native hyperthermostable lactonase into a broad spectrum phosphotriesterase. Sci Rep 7, 16745 (2017). https://doi.org/10.1038/s41598-017-16841-0

Download citation

Received: 29 August 2017
Accepted: 16 November 2017
Published: 01 December 2017
DOI: https://doi.org/10.1038/s41598-017-16841-0

This article is cited by

An integrative approach to improving the biocatalytic reactions of whole cells expressing recombinant enzymes
- Kyung-Chul Shin
- Deok-Kun Oh
World Journal of Microbiology and Biotechnology (2021)
Organophosphorus poisoning in animals and enzymatic antidotes
- Laetitia Poirier
- Pauline Jacquet
- Eric Chabrière
Environmental Science and Pollution Research (2021)
Enzymatic decontamination of paraoxon-ethyl limits long-term effects in planarians
- Laetitia Poirier
- Laure Plener
- Eric Chabrière
Scientific Reports (2020)
Structural and Biochemical Characterization of AaL, a Quorum Quenching Lactonase with Unusual Kinetic Properties
- Celine Bergonzi
- Michael Schwab
- Mikael Elias
Scientific Reports (2018)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.