Background

Phosphotriesterase-Like Lactonases (PLLs) are natural lactonases (EC 3.1.1.25) (Figure 1C, D, E) with promiscuous phosphotriesterase activity (EC 3.1.8.1) (Figure 1A) [1, 2]. They are structurally closely related to bacterial phosphotriesterases (PTEs) [3–6], such as Brevundimonas diminuta PTE (Bd PTE; ~30% sequence identity) [7]. PTEs naturally hydrolyze neurotoxic organophosphorus (OPs) compounds (Figure 1A) such as paraoxon (the active metabolite of the insecticide parathion) with catalytic constants that approach the diffusion limit (i.e. kcat/KM ~ 108 M−1 s−1) [7]. Because OPs have been massively used as pesticides since the 50′s [8], PTEs are believed to have emerged in few decades from a PLL progenitor [2], providing a new source of phosphorus to bacteria, and consequently a selective advantage [8].

Figure 1
figure 1

Chemical structure of Sac Pox substrates. Chemical structures of (A) phosphotriesters, (B) esters, (C) Acyl-Homoserine Lactones, (D) γ-lactones and (E) δ-lactones are presented. For phosphotriesters, R corresponds to different nature of substituents; LG corresponds to the leaving group. The terminal substituent could be S atom if the molecule is a thionophosphotriester or an O atom if the molecule is an oxonophosphotriester. For esters, R corresponds to different nature of substituent. For AHLs and γ/δ-lactones, R corresponds to different size of acyl chain.

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Both enzyme families exhibit the same (β/α)8-barrel topology [9, 10] and belong to the amidohydrolase superfamily [11, 12]. Their structure consists of 8 β-strands forming a central barrel surrounded by 8 α-helixes. The active site is constituted by a bimetallic center (two metal cations) localized at the C-terminus of the barrel. 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].

The difference in substrate specificities of PLLs and PTEs seems mainly governed by variation in the connecting loops of the barrel [2, 16]. Major differences between PTEs and PLLs reside in the active site loop size and conformation [1, 2]. Indeed, loop 7 is shorter in PLLs than in PTEs whereas the loop 8 is larger, forming a hydrophobic channel that accommodates lactones aliphatic chain [9]. Loop 7/8 length and sequence also differ within the PLL family and led to the identification of two different subfamilies: PLLs-A and PLLs-B [2]. Both subfamilies exhibit different substrate specificities: PLLs-B are exclusively oxo-lactonases (Figure 1DE) whereas PLLs-A hydrolyze efficiently oxo-lactones and Acyl-Homoserine Lactones (AHLs, Figure 1C) [2]. AHLs are messenger molecules involved in a bacterial communication system dubbed quorum sensing (QS) [17]. QS regulates the expression of numerous genes, and enables bacterial population to adopt a “group” behavior, including the expression of virulence factors of some pathogens [18, 19]. The involvement of PLLs-A in quorum sensing has not yet been demonstrated, and these enzymes are often found with no other AHL components, including in archaeal species [20]. However, the fact that they hydrolyze specifically the natural enantiomer of AHL indicates that it may be their native substrate [16].

PLLs are promiscuous enzymes that catalyze two chemical reactions of potential biotechnological interest. Indeed, the inhibition or “quenching” of the QS is seen as a possibly promising strategy to develop innovative therapies [21–25]. Indeed, lactonases such as PLLs can inhibit QS (known as quorum quenching, i.e. QQ) [26, 27] and thereby annihilate the virulence of micro-organisms possessing an AHL-based QS system [28]. Moreover, PLLs are endowed with relatively low phosphotriesterase activity, but might be optimized against OPs and subsequently used for degrading organophosphorus pesticides [3, 5, 6, 9, 29] and nerve agents [30], for which no satisfactory remediation methods are currently available [31].

In addition, several PLLs members are thermostable [3, 4, 6, 32–34]; e.g. PLLs from extremophilic crenarchaeaon sources [3, 4, 16, 34]. These counterparts exhibit industry-compatible properties (e.g. thermal and detergent resistance) [35–37]; making them good starting point for in vitro improvement protocols [37, 38]. Several studies report the engineering of thermostable PLLs and improvement of catalytic efficiency against OPs, including for Sso Pox [16, 39], Dr OPH (Deinococcus radiodurans organophosphorus hydrolase) [6, 40] and Gk L (Geobacillus kaustropilus lactonase) [41] but also for the lactonase activity of Sso Pox [16], MCP (Mycobacterium avium subsp. Paratuberculosis K-10 lactonase) [42] and Gk L [43].

Here we focus on Sac Pox, the PLL from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius (living conditions: 55–85°C, pH 2–3) [44]. Sac Pox was originally isolated and studied for its ability to hydrolyze OP compounds at high temperature [4]. The enzyme shares about 30% of sequence identity with Bd PTE and about 70% with its closest homologues, i.e. Sso Pox from Sulfolobus solfataricus[3] and Sis Lac from Sulfolobus islandicus[33, 45]. 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. In this study, we performed a broad kinetic characterization of Sac Pox at room temperature (25°C) for several OPs, esters (Figure 1B) and lactone molecules including AHLs, γ-lactones and δ-lactones in the aim to evaluate the biotechnological potentialities of this enzyme.

Methods

Sequence alignment

The sequence alignment was performed based on the previously published PLL sequence alignment [2], using the T-coffee server (expresso) [47, 48] and manually improved with the seaview software [49]. It contains 29 different sequences (Additional file 1: Table S1). The sequence alignment was represented using the BioEdit 7.1.3 software [50]. Protein sequence identities were computed using ClustalW server [51]. The phylogenetic tree was performed using PhyML[49] and default parameters.

Protein production and purification

The protein production and subsequent purification steps were performed analogously to previously described [16, 33, 34, 45, 52–54]. In brief, the protein was heterologously produced in Escherichia coli strain BL21(DE3)-pGro7/GroEL (TaKaRa) at 37°C in ZYP medium [55]. When OD600nm reaches 0.8, protein production was induced with addition of arabinose (0.2%, w/v) and CoCl2 (2 mM) and temperature transition to 25°C for 20 hours. Cells were harvested by centrifugation, and pelleted cells were suspended in lysis buffer (50 mM HEPES pH 8, 150 mM NaCl, 0.2 mM CoCl2, lysozyme 25 mg/ml, PMSF 0.1 mM, DNase I 10 mg/ml), stored at −80°C during 2 hours; then sonicated 3 times during 30 seconds (Branson Sonifier 450, 80% intensity and microtype limit of 8) and centrifuged. Taking advantage of the high stability of Sac Pox, the supernatant was heated at 70°C during 30 minutes and centrifuged before proceeding a STREP-TRAP affinity chromatography step (GE Healthcare, Uppsala, Sweden). The sample was then cleaved by the Tobacco Etch Virus protease (TEV, ratio 1:20, w/w [56]) during 20 hours at 30°C prior to be loaded a second time on STREP-TRAP affinity chromatography. The flow through containing the cleaved protein was then concentrated and loaded on a size exclusion column (S75-16-60; GE Healthcare, Uppsala, Sweden). The protein purity and identity were checked by SDS-PAGE and mass spectrometry analysis (MS platform Timone, Marseille, France). The protein concentration was determined using a nanospectrophotometer (Nanodrop, Thermofisher Scientific, France) using its molar extinction coefficient (Sac Pox ϵ280 nm = 35 307.7 M−1 cm−1) calculated by the PROT-PARAM server [57].

Kinetic characterization

General procedures

Catalytic parameters were evaluated at 25°C and recorded with a microplate reader (Synergy HT, BioTek, USA) and the Gen5.1 software as previously explained [16, 33, 52, 54]. The reaction was performed in a 200 μL volume using a 96-well plate with a 6.2 mm path length as previously described [33]. The collected data were subsequently fitted to the Michaelis-Menten (MM) equation [58] using Graph-Pad Prism 5.00 (GraphPad Software, San Diego California USA, http://www.graphpad.com). In cases where Vmax could not be reached, the catalytic efficiency was obtained by fitting the linear part of MM plot to a linear regression using Graph-Pad Prism 5.00 software.

OP hydrolase and esterase kinetics

Standard assays for organophosphates (Figure 1A) and esters (Figure 1B) were performed in activity buffer (50 mM HEPES pH 8, 150 mM NaCl, 0.2 mM CoCl2) by measuring the p-nitrophenolate release over time at 405 nm (ϵ405 nm = 17 000 M−1 cm−1). For ethyl-paraoxon (Additional file 1: Figure S1I), the activity buffer has also been supplemented with SDS (w/v) at 0.01% or 0.1% for detergent essays. Malathion (Additional file 1: Figure S1V) hydrolysis was followed at 412 nm in activity buffer added of 2 mM DTNB to follow the release of free thiols (ϵ412 nm = 13 700 M−1 cm−1). The time course hydrolysis of dihydrocoumarin (Additional file 1: Figure S1X), CMP-coumarin (Additional file 1: Figure S1VI) and phenyl-acetate (Additional file 1: Figure S1VII) were respectively monitored at 270 nm (ϵ270 nm = 1 400 M−1 cm−1), 412 nm (ϵ412 nm = 37 000 M−1 cm−1) and 270 nm (ϵ270 nm = 1 400 M−1 cm−1).

Lactonase kinetics

Kinetics monitoring the lactone hydrolysis were performed according to a previously described protocol [33]. The lactone hydrolysis was monitored in the lactonase buffer (2.5 mM Bicine pH 8.3, 150 mM NaCl, 0.2 mM CoCl2, 0.25 mM Cresol purple and 0.5% DMSO) with different AHLs (Figure 1C) [i.e. C4-AHL (r), C6-AHL (r), C8-AHL (r), 3-oxo-C8-AHL (l), 3-oxo-C10-AHL (l)] (Additional file 1: Figure S1XI-XVI) and oxo-lactones (Figure 1D,E) [i.e. ϵ-caprolactone, γ-heptanolide (r), Nonanoic-γ-lactone (r), Nonanoic-δ-lactone (r), Undecanoic-γ-lactone (r), Undecanoic-δ-lactone (r), Dodecanoic-γ-lactone (r) and Dodecanoic-δ-lactone (r)] (Additional file 1: Figure S1XVII-XXIV). Cresol purple (pKa 8.3 at 25°C) is a pH indicator (577 nm) used to monitor the acidification of the medium following lactone ring hydrolysis (ϵ577nm = 5 500 M−1 cm−1).

Structural modeling and structural analysis

The Sac Pox structure was modelled using the ESyPred3D server using Sac Pox protein sequence as query and Sso Pox structure (2VC5) as template [59]. Structures were analyzed and figure made using PyMol [60].

Results

First classified within the bacterial PTEs, Sac Pox shares in fact only 33.8% sequence identity with Bd PTE (Additional file 1: Table S2). Sac Pox indeed belongs to the PLLs-A (Figure 2A) [2]: it shares 76.1% of sequence identity with its closest homologues Sso Pox and Sis Lac, and only 30.6% identity with the PLL-B Dr OPH. Together with Sis Lac and Sso Pox, Sac Pox comprises the creanarcheal clade of the PLLs-A (Figure 2A). The sequence alignment highlights the strict conservation of essential active site residues between the different clades (Figure 2B).

Figure 2
figure 2

Phylogenetic analysis of the PLL family. A. Phylogenetic tree of PLLs, PTEs, and close homologues. Members of PLL-B are colored in green while within the PLL-As, mesophilic and archaeal PLLs are respectively colored in red and orange. The clades of PHPs, PTEs and RTXs were collapsed for clarity. All the sequences used for this tree are listed in Additional file 1: Table S1. B. Sequence alignment of Bd PTE from B. diminuta, Sso Pox from S. solfataricus, Sac Pox from S. acidocaldarius and Dr OPH from D. radiodurans. Conserved amino acid residues are highlighted in black and similar residues in grey. Conserved active site residues involved in metals coordination are highlighted by red stars. Secondary structures are represented according to Sso Pox structure (with pink arrows depicting β-sheets and red cylinders depicting α-helixes).

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Enzymatic characterization

Phosphotriesterase activity

Sac Pox ability to hydrolyze insecticides ethyl/methyl-paraoxon, ethyl/methyl-parathion and malathion has been evaluated (Table 1). The best Sac Pox phosphotriester substrate, methyl-paraoxon is processed with moderate catalytic efficiency (kcat/KM = 1.10(±0.17)×103 M−1.s−1), low rate (kcat = 0.307 s−1) and low KM (278.3 μM). Very similar catalytic efficiencies were recorded for Sso Pox and Sis Lac: kcat/KM of 1.27×103 M−1.s−1 and 4.26×103 M−1.s−1, respectively [33, 52]. Ethyl-paraoxon comprise a slower substrate, (kcat/KM = 2.81×102 M−1.s−1), highlighting the enzyme preference for OP substrates with small substituents. No hydrolysis could be measured for ethyl-parathion and malathion, whereas a low catalytic efficiency was recorded for methyl-parathion (kcat/KM = 4.31 M−1.s−1). This specificity profile illustrates the clear preference of Sac Pox for oxono-phosphotriesters rather than thiono-phosphotriesters; as previously observed for Sso Pox [52] and Sis Lac [33]. Moreover, whereas anionic detergents like SDS can significantly stimulate Sso Pox phosphotriesterase activity [52], the same treatment on Sac Pox yields only a 2-fold increase in catalytic efficiency with ethyl-paraoxon as substrate. Finally, we show that Sac Pox hydrolyzes CMP-coumarin (kcat/KM = 4.38 × 102 M−1.s−1), albeit with 20-fold lower catalytic efficiency than Sso Pox [52].

Table 1 Phosphotriesterase kinetic parameters
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Esterase activity

The ability of Sac Pox to hydrolyze phenyl-acetate, p NP-acetate and p NP-decanoate (Additional file 1: Figure S1VII-IX) has been evaluated (Table 2). While no activity could be detected against p NP-decanoate, Sac Pox exhibits low catalytic efficiencies against both phenyl-acetate and p NP-acetate (kcat/KM ≈ 50 M−1.s−1). This weak activity against classical esters differs from previous studies on the close homologues Sso Pox and Sis Lac, for which activity has only been recorded on p NP-acetate [33].

Table 2 Esterase kinetic parameters
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Lactonase activity

The catalytic parameters of Sac Pox for various lactone substrates have been measured, including against oxo-lactones (lipophilic aroma), AHLs and dihydrocoumarin (Table 3). Our results indicate a preference of Sac Pox for oxo-lactone substrates; i.e. γ-heptanolide and nonanoic-γ-lactone (kcat/KM ≈ 2.5×104 M−1.s−1), while AHLs are about 10 times worse substrates (i.e.; C8 AHLs, kcat/KM ≈ 5×103 M−1.s−1). Furthermore, it seems that Sac Pox prefers AHLs vs 3-oxo-AHLs since the KM for C8 aliphatic chains is 5-fold lower than that for 3-oxo-C8 AHLs. Overall, long aliphatic chain substrates AHLs are better substrates for the enzyme. Indeed, short aliphatic chain AHLs are not hydrolyzed by Sac Pox. Interestingly, this preference is not retained for oxo-lactones, for which molecules with short or without aliphatic chain are efficiently hydrolyzed (kcat/KM ≈ 104 M−1.s−1). As previously observed for Sso Pox and Sis Lac [16, 33], this feature may reveal a potential alternative binding mode of these compounds in Sac Pox active site. Finally, contrary to Sso Pox and Sis Lac [16, 33], Sac Pox does not hydrolyze dihydrocoumarin.

Table 3 Lactonase kinetic parameters
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Structural analysis

Numerous attempts to crystallize Sac Pox were made, with no success (Elias, Hiblot, Gotthard & Chabriere, unpublished). A previous structural model was generated by homology modeling based on Bd PTE structure [4] (~33.8% sequence identity with Sac Pox), but yielded little insights given the moderate sequence identity with the template and the very significant differences in the active site loops between these two representatives of distinct enzyme families [1, 9, 16]. Here we generated a homology-based model using the structure of Sso Pox as template (76.1% of sequence identity; Additional file 1: Table S2).

As expected, the Sac Pox model structure almost perfectly superimposes to the Sso Pox crystal structure (Figure 3A). Residues forming the active site are all conserved and residues involved in loops 7 and 8 occupy nearly identical conformation in Sac Pox and Sso Pox but also in Sis Lac structures (Figure 3B). Noteworthy, loop 8 is partially structured into an α-helix, as seen in X-ray structures of Sso Pox and Sis Lac. A substitution (I266 in Sac Pox; T265 in Sso Pox and Sis Lac) in loop 8 may slightly alter the shape of the aliphatic channel. But overall, the active site of Sac Pox and Sso Pox are nearly identical (Figure 2B). Furthermore, four other substitutions between Sac Pox and its close homologues can be seen in loop 8: Sac Pox exhibits a K at position 268, instead of an R residue (R267 in Sis Lac), Y271 instead of L (L270 in Sis Lac), K278 instead of R (R277 in both Sis Lac and Sso Pox), and M281 instead of I (I280 in Sso Pox) (Additional file 1: Figure S2). While the structural model suggests that these substitutions are not affecting directly the binding cleft of Sac Pox, they might modulate loop 8 conformation and its dynamics. Indeed, it was shown in the close homologue Sso Pox that a single substitution in loop 8 (W263 in Sso Pox, equivalent to W264 in Sac Pox) increases the conformational flexibility of loop 8, thereby conferring higher promiscuity to the enzyme [16]. The effect is in fact so dramatic that the substitution in Sso Pox of W263 by any of the 19 other natural amino acids yields a variant with improved phosphotriesterase activity [16]. Additionally, loop 8 being involved in the accommodation of the aliphatic substituent of lactones substrates [9], mutations in this loop can also affect the lactonase activity [16].

Figure 3
figure 3

Structural model of Sac Pox. A. Structural superposition of Sso Pox structure (2VC5; grey) and the Sac Pox model (green). Cobalt, iron and the catalytic water molecule are respectively represented by pink, orange and red spheres. Bimetallic center coordinating residues are represented as sticks. B. Active site view of superimposed Sso Pox structure (grey) and the Sac Pox model (green). Several active site residues are represented as sticks. Numbering is made according to Sac Pox sequence.

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Discussion

Here we show that Sac Pox is a proficient lactonase (~104 M−1.s−1) and can hydrolyze both oxo-lactones and AHLs. Nevertheless, Sac Pox have a slightly different substrate specificity than its close homologues [16, 33]. Indeed, Sac Pox exhibits slightly lower catalytic efficiencies, prefers AHLs over 3-oxo-AHLs and does not show any activity against dihydrocoumarin. Interestingly, as noted for Sis Lac and Sso Pox [16, 33], Sac Pox clearly prefers long chain AHLs, but can efficiently hydrolyze short chain or oxo-lactones without aliphatic substituents. This feature could reflect a putatively different binding mode of AHLs and oxo-lactones into PLLs active sites. We note that the biological role of lactonases such as PLLs is yet unclear, especially in extremophilic archaea where no AHL-based quorum sensing systems have been identified so far.

Sac Pox also exhibits promiscuous esterase and phosphotriesterase activities, a common feature of PLLs. Similarly to Sso Pox and Sis Lac [33, 52], Sac Pox prefers OPs with small substituents. Moreover, Sac Pox also shows a clear preference for oxono-phosphotriesters, rather than thiono-phosphotriesters, a feature previously dubbed thiono-effect [52]. Interestingly, Sso Pox, Sis Lac and Sac Pox exhibit similar catalytic efficiencies against OPs (102–3 M−1.s−1) at 25°C, efficiencies that are close to those measured at much higher temperatures [4].

The structural model shows that Sac Pox structure is very close to that of Sso Pox (Figure 2A). Most critically, the active sites of both enzymes are essentially identical (Figure 2B), with the exception of position 266 (I in Sac Pox, T in Sso Pox and Sis Lac). This substitution might partly account for the observed differences in substrates specificity between these enzymes, and would thereby represent an interesting target for future mutagenesis studies. But four other substitutions in loop 8 between these close homologues might be involved as well, and comprise also interesting options for mutagenesis studies (K268R, Y27IL, K278R and M281I). A recent study on Sso Pox highlighted how profound the effect on catalysis of a single substitution on loop 8 (W263) can be [16]. Therefore, substitution T266I, and/or the four others on loop 8, might contribute to the observed differences between Sac Pox and Sso Pox in substrate specificity, in combination with other factors that cannot be assessed by a structural model such as subtle changes in active site loops conformation and dynamics [16, 33]. Indeed, the observed differences in the detergent stimulation between both enzymes (Sac Pox is only weakly stimulated by SDS, as compared to Sso Pox) could well be a manifestation of different dynamics of their respective active site loops.

Conclusions

To conclude, we here demonstrate that albeit being initially isolated, characterized, and named after its ability to degrade the insecticide paraoxon (pox; [4]), Sac Pox is putatively a native lactonase, capable of hydrolyzing these compounds with significant catalytic efficiencies at 25°C (up to 104 M−1.s−1). The extensive kinetic characterization reveals some substrate specificity differences between Sac Pox and its close homologues Sis Lac and Sso Pox, and the proposed structural model of Sac Pox suggests putative candidates (e.g. I266) that could account for these observations. Such positions might constitute interesting targets for future engineering studies, with the aim of improving or altering the catalytic properties of Sac Pox.