# SacPox from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius is a proficient lactonase

Janek Bzdrenga, Julien Hiblot, Guillaume Gotthard, Charlotte Champion,

Mikael Elias, Eric Chabriere

► To cite this version:

Janek Bzdrenga, Julien Hiblot, Guillaume Gotthard, Charlotte Champion, Mikael Elias, et al.. SacPox from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius is a proficient lactonase. BMC Research Notes, 2014, 7 (1), 10.1186/1756-0500-7-333. doi:10.1186/1756-0500-7-333

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# SacPox from the thermoacidophilic crenarchaeon *Sulfolobus acidocaldarius* is a proficient lactonase

Janek Bzdrenga<fnref n="1" />†, Julien Hiblot<fnref n="1" />†, Guillaume Gotthard<fnref n="1" />, Charlotte Champion<fnref n="1" />, Mikael Elias<fnref n="2" />* and Eric Chabriere<fnref n="1" />*

## Abstract

**Background:** SacPox, an enzyme from the extremophilic crenarchaeal *Sulfolobus acidocaldarius* (*Sac*), was isolated by virtue of its phosphotriesterase (or paraoxonase; Pox) activity, *i.e.* its ability to hydrolyze the neurotoxic organophosphorus insecticides. Later on, *SacPox* was shown to belong to the Phosphotriesterase-Like Lactonase family that comprises natural lactonases, possibly involved in *quorum* sensing, and endowed with promiscuous, phosphotriesterase activity.

**Results:** Here, we present a comprehensive and broad enzymatic characterization of the natural lactonase and promiscuous organophosphorus hydrolase activities of *SacPox*, as well as a structural analysis using a model.

**Conclusion:** Kinetic experiments show that *SacPox* is a proficient lactonase, including at room temperature. Moreover, we discuss the observed differences in substrate specificity between *SacPox* and its closest homologues *SsoPox* and *SisLac* together with the possible structural causes for these observations.

**Keywords:** Lactonase, PLL, *Quorum* sensing, Phosphotriesterase, Extremophile, Thermoacidophile

## 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 (*BdPTE*; ~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.* $k_{cat}/K_M \sim 10^8 M^{-1} s^{-1}$) [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].

Both enzyme families exhibit the same $(\beta/\alpha)_8$-barrel topology [9,10] and belong to the amidohydrolase super-family [11,12]. Their structure consists of 8 $\beta$-strands forming a central barrel surrounded by 8 $\alpha$-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

<footnote>* Correspondence: mikael.elias@gmx.fr; eric.chabriere@univ-amu.fr</footnote>

<footnote>†Equal contributors</footnote>

<footnote n="2">²Weizmann Institute of Science, Biological Chemistry, Rehovot, Israel</footnote>

<footnote n="1">¹URMITE UMR CNRS-IRD 6236, IFR48, Faculté de Médecine et de Pharmacie, Université de la Méditerranée, Marseille, France</footnote>
<figure><img src="image_2.png" /><figcaption>**Figure 1** Chemical structure of *SacPox* 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.</figcaption></figure>

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 crenarchaeon 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 *SsoPox* [16,39], *DrOPH* (*Deinococcus radiodurans* organophosphorus hydrolase) [6,40] and *GkL* (*Geobacillus kaustro-pilus* lactonase) [41] but also for the lactonase activity of *SsoPox* [16], *MCP* (*Mycobacterium avium* subsp. Paratuberculosis K-10 lactonase) [42] and *GkL* [43].

Here we focus on *SacPox*, the PLL from the thermoacidophilic crenarchaeon *Sulfolobus acidocaldarius* (living conditions: 55–85°C, pH 2–3) [44]. *SacPox* 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 *BdPTE* and
about 70% with its closest homologues, i.e. *SsoPox* from *Sulfolobus solfataricus* [3] and *SisLac* from *Sulfolobus islandicus* [33,45]. Being an enzyme from a hyperthermophile, *SacPox* is however less stable than *SsoPox* (half-life of 5 min at 90°C [4] and of 4 h at 95°C [3,46], respectively). The kinetic characterizations performed on *SacPox* 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 *SacPox* 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 [16,33,45,52-54]. 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 *SacPox*, 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 (SacPox 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, 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-
<figure><img src="image_3.png" /><figcaption>**Figure 2** (See legend on next page.)</figcaption></figure>
(See figure on previous page.)

**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 BdPTE from *B. diminuta*, SsoPox from *S. solfataricus*, SacPox from *S. acidocaldarius* and DrOPH 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 SsoPox structure (with pink arrows depicting β-sheets and red cylinders depicting α-helixes).

lactone (*r*), Dodecanoic-γ-lactone (*r*) and Dodecanoic-δ-lactone (*r*)] (Additional file 1: Figure S1XVII-XXIV). Cresol purple (pK<sub>a</sub> 8.3 at 25°C) is a pH indicator (577 nm) used to monitor the acidification of the medium following lactone ring hydrolysis (ε<sub>577nm</sub> = 5 500 M<sup>-1</sup> cm<sup>-1</sup>).

Structural modeling and structural analysis

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

Results

First classified within the bacterial PTEs, *SacPox* shares in fact only 33.8% sequence identity with *BdPTE* (Additional file 1: Table S2). *SacPox* indeed belongs to the PLLs-A (Figure 2A) [2]: it shares 76.1% of sequence identity with its closest homologues *SsoPox* and *SisLac*, and only 30.6% identity with the PLL-B *DrOPH*. Together with *SisLac* and *SsoPox*, *SacPox* 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).

Enzymatic characterization

Phosphotriesterase activity

*SacPox* ability to hydrolyze insecticides ethyl/methyl-paraoxon, ethyl/methyl-parathion and malathion has been evaluated (Table 1). The best *SacPox* phosphotriester substrate, methyl-paraoxon is processed with moderate catalytic efficiency (k<sub>cat</sub>/K<sub>M</sub> = 1.10(±0.17)×10<sup>3</sup> M<sup>-1</sup>.s<sup>-1</sup>), low

<table><caption>Table 1 Phosphotriesterase kinetic parameters</caption><thead><tr><th></th><th>k<sub>cat</sub> (s<sup>-1</sup>)</th><th>K<sub>M</sub> (µM)</th><th>k<sub>cat</sub>/K<sub>M</sub> (M<sup>-1</sup>.s<sup>-1</sup>)</th></tr></thead><tbody><tr><td>Paraoxon</td><td>0.12 ± 0.01</td><td>434 ± 54</td><td>2.81 (±0.38) × 10<sup>2</sup></td></tr><tr><td>Paraoxon 0.01% SDS</td><td>0.28 ± 0.01</td><td>537 ± 48</td><td>5.22 (±0.51) × 10<sup>2</sup></td></tr><tr><td>Paraoxon 0.1% SDS</td><td>0.25 ± 0.01</td><td>405 ± 21</td><td>6.10 (±0.34) × 10<sup>2</sup></td></tr><tr><td>Methyl Paraoxon</td><td>0.31 ± 0.02</td><td>278 ± 40</td><td>1.10 (±0.17) × 10<sup>3</sup></td></tr><tr><td>Parathion</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>Methyl Parathion</td><td>ND</td><td>ND</td><td>4.31 ± 0.20</td></tr><tr><td>Malathion</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>CMP-Coumarin</td><td>0.28 ± 0.02</td><td>642 ± 89</td><td>4.38 (±0.68) × 10<sup>2</sup></td></tr></tbody></table>

ND correspond to Not Detected hydrolysis. Results have been obtained with cobalt as cofactor.

rate (k<sub>cat</sub> = 0.307 s<sup>-1</sup>) and low K<sub>M</sub> (278.3 µM). Very similar catalytic efficiencies were recorded for *SsoPox* and *SisLac*: k<sub>cat</sub>/K<sub>M</sub> of 1.27×10<sup>3</sup> M<sup>-1</sup>.s<sup>-1</sup> and 4.26×10<sup>3</sup> M<sup>-1</sup>.s<sup>-1</sup>, respectively [33,52]. Ethyl-paraoxon comprise a slower substrate, (k<sub>cat</sub>/K<sub>M</sub> = 2.81×10<sup>2</sup> M<sup>-1</sup>.s<sup>-1</sup>), 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 (k<sub>cat</sub>/K<sub>M</sub> = 4.31 M<sup>-1</sup>.s<sup>-1</sup>). This specificity profile illustrates the clear preference of *SacPox* for oxono-phosphotriesters rather than thionophosphotriesters; as previously observed for *SsoPox* [52] and *SisLac* [33]. Moreover, whereas anionic detergents like SDS can significantly stimulate *SsoPox* phosphotriesterase activity [52], the same treatment on *SacPox* yields only a 2-fold increase in catalytic efficiency with ethyl-paraoxon as substrate. Finally, we show that *SacPox* hydrolyzes CMP-coumarin (k<sub>cat</sub>/K<sub>M</sub> = 4.38 × 10<sup>2</sup> M<sup>-1</sup>.s<sup>-1</sup>), albeit with 20-fold lower catalytic efficiency than *SsoPox* [52].

Esterase activity

The ability of *SacPox* to hydrolyze phenyl-acetate, pNP-acetate and pNP-decanoate (Additional file 1: Figure S1VII-IX) has been evaluated (Table 2). While no activity could be detected against pNP-decanoate, *SacPox* exhibits low catalytic efficiencies against both phenyl-acetate and pNP-acetate (k<sub>cat</sub>/K<sub>M</sub> ≈ 50 M<sup>-1</sup>.s<sup>-1</sup>). This weak activity against classical esters differs from previous studies on the close homologues *SsoPox* and *SisLac*, for which activity has only been recorded on pNP-acetate [33].

Lactonase activity

The catalytic parameters of *SacPox* for various lactone substrates have been measured, including against oxo-lactones (lipophilic aroma), AHLs and dihydrocoumarin (Table 3). Our results indicate a preference of *SacPox* for oxo-lactone substrates; i.e. γ-heptanolide and nonanoic-γ-lactone (k<sub>cat</sub>/K<sub>M</sub> ≈ 2.5×10<sup>4</sup> M<sup>-1</sup>.s<sup>-1</sup>), while AHLs are about 10 times worse substrates (i.e.; C8 AHLs, k<sub>cat</sub>/K<sub>M</sub> ≈ 5×10<sup>3</sup> M<sup>-1</sup>.s<sup>-1</sup>). Furthermore, it seems that *SacPox* prefers AHLs vs 3-oxo-AHLs since the K<sub>M</sub> 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 *SacPox*. Interestingly, this preference is not retained for oxo-lactones, for which molecules with short
<table><caption>Table 2 Esterase kinetic parameters</caption><thead><tr><th></th><th>k<sub>cat</sub> (s<sup>-1</sup>)</th><th>K<sub>M</sub> (μM)</th><th>k<sub>cat</sub>/K<sub>M</sub> (M<sup>-1</sup>.s<sup>-1</sup>)</th></tr></thead><tbody><tr><td>Phenyl-acetate</td><td>0.35 ± 0.05</td><td>8 181 ± 1750</td><td>42.3 ± 11.1</td></tr><tr><td>pNP-acetate</td><td>0.13 ± 0.01</td><td>2 107 ± 313</td><td>60.1 ± 9.9</td></tr><tr><td>pNP-decanoate</td><td>ND</td><td>ND</td><td>ND</td></tr></tbody></table>

ND correspond to Not Detected hydrolysis. Results have been obtained with cobalt as cofactor.

or without aliphatic chain are efficiently hydrolyzed ($k_{cat}$/K$_M$ ≈ $10^4$ M$^{-1}$.s$^{-1}$). As previously observed for SsoPox and SisLac [16,33], this feature may reveal a potential alternative binding mode of these compounds in SacPox active site. Finally, contrary to SsoPox and SisLac [16,33], SacPox does not hydrolyze dihydrocoumarin.

Structural analysis

Numerous attempts to crystallize SacPox were made, with no success (Elias, Hiblot, Gotthard & Chabriere, unpublished). A previous structural model was generated by homology modeling based on BdPTE structure [4] (~33.8% sequence identity with SacPox), 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 SsoPox as template (76.1% of sequence identity; Additional file 1: Table S2).

As expected, the SacPox model structure almost perfectly superimposes to the SsoPox 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 SacPox and SsoPox but

<table><caption>Table 3 Lactonase kinetic parameters</caption><thead><tr><th></th><th>k<sub>cat</sub> (s<sup>-1</sup>)</th><th>K<sub>M</sub> (μM)</th><th>k<sub>cat</sub>/K<sub>M</sub> (M<sup>-1</sup>.s<sup>-1</sup>)</th></tr></thead><tbody><tr><td>C4 AHL</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>C6 AHL</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>C8 AHL</td><td>0.94 ± 0.02</td><td>178 ± 26</td><td>5.28 (±0.77) × 10<sup>3</sup></td></tr><tr><td>3-oxo C6 AHL</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>3-oxo C8 AHL</td><td>0.89 ± 0.07</td><td>836 ± 178</td><td>1.07 (±0.25) × 10<sup>3</sup></td></tr><tr><td>3-oxo C10 AHL</td><td>1.03 ± 0.04</td><td>213 ± 33</td><td>4.88 (±0.77) × 10<sup>3</sup></td></tr><tr><td>γ heptanolide</td><td>10.25 ± 0.50</td><td>388 ± 62</td><td>2.64 (±0.44) × 10<sup>4</sup></td></tr><tr><td>Nonanoic-γ-lactone</td><td>2.64 ± 0.07</td><td>109 ± 19</td><td>2.44 (±0.44) × 10<sup>4</sup></td></tr><tr><td>Undecanoic-γ-lactone</td><td>0.34 ± 0.01</td><td>578 ± 78</td><td>5.89 (±0.84) × 10<sup>2</sup></td></tr><tr><td>dodecanoic-γ-lactone</td><td>0.53 ± 0.03</td><td>242 ± 60</td><td>2.21 (±0.57) × 10<sup>3</sup></td></tr><tr><td>Nonanoic-δ-lactone</td><td>4.55 ± 0.21</td><td>348 ± 53</td><td>1.31 (±0.21) × 10<sup>4</sup></td></tr><tr><td>Undecanoic-δ-lactone</td><td>1.05 ± 0.05</td><td>168 ± 37</td><td>6.22 (±1.40) × 10<sup>3</sup></td></tr><tr><td>Dodecanoic-δ-lactone</td><td>3.34 ± 0.07</td><td>185 ± 27</td><td>1.81 (±0.27) × 10<sup>4</sup></td></tr><tr><td>ε caprolactone</td><td>15.04 ± 0.47</td><td>1 031 ± 83</td><td>1.46 (±0.13) × 10<sup>4</sup></td></tr><tr><td>Dihydrocoumarine</td><td>ND</td><td>ND</td><td>ND</td></tr></tbody></table>

ND correspond to Not Detected hydrolysis. Results have been obtained with cobalt as cofactor.

also in SisLac structures (Figure 3B). Noteworthy, loop 8 is partially structured into an α-helix, as seen in X-ray structures of SsoPox and SisLac. A substitution (I266 in SacPox; T265 in SsoPox and SisLac) in loop 8 may slightly alter the shape of the aliphatic channel. But overall, the active site of SacPox and SsoPox are nearly identical (Figure 2B). Furthermore, four other substitutions between SacPox and its close homologues can be seen in loop 8: SacPox exhibits a K at position 268, instead of an R residue (R267 in SisLac), Y271 instead of L (L270 in SisLac), K278 instead of R (R277 in both SisLac and SsoPox), and M281 instead of I (I280 in SsoPox) (Additional file 1: Figure S2). While the structural model suggests that these substitutions are not affecting directly the binding cleft of SacPox, they might modulate loop 8 conformation and its dynamics. Indeed, it was shown in the close homologue SsoPox that a single substitution in loop 8 (W263 in SsoPox, equivalent to W264 in SacPox) 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 SsoPox 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].

Discussion

Here we show that SacPox is a proficient lactonase (~$10^4$ M$^{-1}$.s$^{-1}$) and can hydrolyze both oxo-lactones and AHLs. Nevertheless, SacPox have a slightly different substrate specificity than its close homologues [16,33]. Indeed, SacPox exhibits slightly lower catalytic efficiencies, prefers AHLs over 3-oxo-AHLs and does not show any activity against dihydrocoumarin. Interestingly, as noted for SisLac and SsoPox [16,33], SacPox 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.

SacPox also exhibits promiscuous esterase and phosphotriesterase activities, a common feature of PLLs. Similarly to SsoPox and SisLac [33,52], SacPox prefers OPs with small substituents. Moreover, SacPox also shows a clear preference for oxono-phosphotriesters, rather than thionophosphotriesters, a feature previously dubbed thiono-effect [52]. Interestingly, SsoPox, SisLac and SacPox exhibit similar catalytic efficiencies against OPs ($10^{2-3}$ M$^{-1}$.s$^{-1}$) at 25°C, efficiencies that are close to those measured at much higher temperatures [4].
<figure><img src="image_4.png" /><figcaption>**Figure 3** Structural model of *SacPox*. A. Structural superposition of SsoPox structure (2VC5; grey) and the SacPox 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 SsoPox structure (grey) and the SacPox model (green). Several active site residues are represented as sticks. Numbering is made according to SacPox sequence.</figcaption></figure>

The structural model shows that *SacPox* structure is very close to that of *SsoPox* (Figure 2A). Most critically, the active sites of both enzymes are essentially identical (Figure 2B), with the exception of position 266 (I in *SacPox*, T in *SsoPox* and *SisLac*). 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 *SsoPox* 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 *SacPox* and *SsoPox* 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 (SacPox is only weakly stimulated by SDS, as compared to SsoPox) 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]), SacPox is putatively a native lactonase, capable of hydrolyzing these compounds with significant catalytic efficiencies at 25°C (up to $10^4 M^{-1}.s^{-1}$). The extensive kinetic characterization reveals some substrate specificity differences between Sac-Pox and its close homologues SisLac and SsoPox, and the proposed structural model of SacPox 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 SacPox.

## Additional file

**Additional file 1: Figure S1.** Chemical structure of phosphoesters (I-VI), esters (VII-IX) and lactones (X-XXIV). **Figure S2.** Superposition of SsoPox, SisLac and SacPox structural models. **Table S1.** Accession numbers of the sequences used in the phylogeny study. **Table S2.** Sequence identity matrix.

## Competing interests

The authors declare that they have no competing interests.

## Authors' contributions

JH, GG and ME planed the experiments. JB, CC performed the experiments. JH, JB, ME and EC analysed the results. JB, JH and ME wrote the paper. All the authors offered a critical review of the paper.

## Acknowledgements

We are grateful to Dr. Moshe Goldsmith for the kind gift of CMP-coumarin. This work was granted by DGA, France (REI. 2009 34 0045). J.B. is a PhD student granted by DGA. J.H. and C.C. are founded by DGA, France. G.G. is founded by APHM, France.

Received: 4 February 2014 Accepted: 27 May 2014

Published: 3 June 2014

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doi:10.1186/1756-0500-7-333

Cite this article as: Bzdrenga et al.: SacPox from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius is a proficient lactonase. BMC Research Notes 2014 7:333.

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