- Research article
- Open Access
Sac Pox from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius is a proficient lactonase
- Janek Bzdrenga†1,
- Julien Hiblot†1,
- Guillaume Gotthard1,
- Charlotte Champion1,
- Mikael Elias2Email author and
- Eric Chabriere1Email author
© Bzdrenga et al.; licensee BioMed Central Ltd. 2014
Received: 4 February 2014
Accepted: 27 May 2014
Published: 3 June 2014
Sac Pox, 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, Sac Pox 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.
Here, we present a comprehensive and broad enzymatic characterization of the natural lactonase and promiscuous organophosphorus hydrolase activities of Sac Pox, as well as a structural analysis using a model.
Kinetic experiments show that Sac Pox is a proficient lactonase, including at room temperature. Moreover, we discuss the observed differences in substrate specificity between Sac Pox and its closest homologues Sso Pox and Sis Lac together with the possible structural causes for these observations.
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 . 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 . 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 . 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) . AHLs are messenger molecules involved in a bacterial communication system dubbed quorum sensing (QS) . 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 . However, the fact that they hydrolyze specifically the natural enantiomer of AHL indicates that it may be their native substrate .
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 . 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 , for which no satisfactory remediation methods are currently available .
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)  but also for the lactonase activity of Sso Pox , MCP (Mycobacterium avium subsp. Paratuberculosis K-10 lactonase)  and Gk L .
Here we focus on Sac Pox, the PLL from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius (living conditions: 55–85°C, pH 2–3) . Sac Pox was originally isolated and studied for its ability to hydrolyze OP compounds at high temperature . 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 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  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.
The sequence alignment was performed based on the previously published PLL sequence alignment , using the T-coffee server (expresso) [47, 48] and manually improved with the seaview software . It contains 29 different sequences (Additional file 1: Table S1). The sequence alignment was represented using the BioEdit 7.1.3 software . Protein sequence identities were computed using ClustalW server . The phylogenetic tree was performed using PhyML 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 . 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 ) 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 .
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 . The collected data were subsequently fitted to the Michaelis-Menten (MM) equation  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).
Kinetics monitoring the lactone hydrolysis were performed according to a previously described protocol . 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 . Structures were analyzed and figure made using PyMol .
Phosphotriesterase kinetic parameters
0.12 ± 0.01
434 ± 54
2.81 (±0.38) × 102
Paraoxon 0.01% SDS
0.28 ± 0.01
537 ± 48
5.22 (±0.51) × 102
Paraoxon 0.1% SDS
0.25 ± 0.01
405 ± 21
6.10 (±0.34) × 102
0.31 ± 0.02
278 ± 40
1.10 (±0.17) × 103
4.31 ± 0.20
0.28 ± 0.02
642 ± 89
4.38 (±0.68) × 102
Esterase kinetic parameters
0.35 ± 0.05
8 181 ± 1750
42.3 ± 11.1
0.13 ± 0.01
2 107 ± 313
60.1 ± 9.9
Lactonase kinetic parameters
0.94 ± 0.02
178 ± 26
5.28 (±0.77) × 103
3-oxo C6 AHL
3-oxo C8 AHL
0.89 ± 0.07
836 ± 178
1.07 (±0.25) × 103
3-oxo C10 AHL
1.03 ± 0.04
213 ± 33
4.88 (±0.77) × 103
10.25 ± 0.50
388 ± 62
2.64 (±0.44) × 104
2.64 ± 0.07
109 ± 19
2.44 (±0.44) × 104
0.34 ± 0.01
578 ± 78
5.89 (±0.84) × 102
0.53 ± 0.03
242 ± 60
2.21 (±0.57) × 103
4.55 ± 0.21
348 ± 53
1.31 (±0.21) × 104
1.05 ± 0.05
168 ± 37
6.22 (±1.40) × 103
3.34 ± 0.07
185 ± 27
1.81 (±0.27) × 104
15.04 ± 0.47
1 031 ± 83
1.46 (±0.13) × 104
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  (~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).
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 . 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 .
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 . 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.
To conclude, we here demonstrate that albeit being initially isolated, characterized, and named after its ability to degrade the insecticide paraoxon (pox; ), 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.
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.
- Afriat L, Roodveldt C, Manco G, Tawfik DS: The latent promiscuity of newly identified microbial lactonases is linked to a recently diverged phosphotriesterase. Biochemistry. 2006, 45: 13677-13686.PubMedView ArticleGoogle Scholar
- Afriat-Jurnou L, Jackson CJ, Tawfik DS: Reconstructing a missing link in the evolution of a recently diverged phosphotriesterase by active-site loop remodeling. Biochemistry. 2012, 51: 6047-6055.PubMedView ArticleGoogle Scholar
- Merone L, Mandrich L, Rossi M, Manco G: A thermostable phosphotriesterase from the archaeon Sulfolobus solfataricus: cloning, overexpression and properties. Extremophiles. 2005, 9: 297-305.PubMedView ArticleGoogle 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. 2007, 89: 625-636.PubMedView ArticleGoogle Scholar
- Xiang DF, Kolb P, Fedorov AA, Meier MM, Fedorov LV, Nguyen TT, Sterner R, Almo SC, Shoichet BK, Raushel FM: Functional annotation and three-dimensional structure of Dr0930 from Deinococcus radiodurans, a close relative of phosphotriesterase in the amidohydrolase superfamily. Biochemistry. 2009, 48: 2237-2247.PubMedPubMed CentralView ArticleGoogle Scholar
- Hawwa R, Larsen SD, Ratia K, Mesecar AD: Structure-based and random mutagenesis approaches increase the organophosphate-degrading activity of a phosphotriesterase homologue from Deinococcus radiodurans. J Mol Biol. 2009, 393: 36-57.PubMedView ArticleGoogle Scholar
- Dumas DP, Caldwell SR, Wild JR, Raushel FM: Purification and properties of the phosphotriesterase from Pseudomonas diminuta. J Biol Chem. 1989, 264: 19659-19665.PubMedGoogle Scholar
- Singh BK: Organophosphorus-degrading bacteria: ecology and industrial applications. Nat Rev Microbiol. 2009, 7: 156-164.PubMedView ArticleGoogle Scholar
- Elias M, Dupuy J, Merone L, Mandrich L, Porzio E, Moniot S, Rochu D, Lecomte C, Rossi M, Masson P, Manco G, Chabriere E: Structural basis for natural lactonase and promiscuous phosphotriesterase activities. J Mol Biol. 2008, 379: 1017-1028.PubMedView ArticleGoogle Scholar
- Benning MM, Kuo JM, Raushel FM, Holden HM: Three-dimensional structure of phosphotriesterase: an enzyme capable of detoxifying organophosphate nerve agents. Biochemistry. 1994, 33: 15001-15007.PubMedView ArticleGoogle Scholar
- Seibert CM, Raushel FM: Structural and catalytic diversity within the amidohydrolase superfamily. Biochemistry. 2005, 44: 6383-6391.PubMedView ArticleGoogle Scholar
- Roodveldt C, Tawfik DS: Shared promiscuous activities and evolutionary features in various members of the amidohydrolase superfamily. Biochemistry. 2005, 44: 12728-12736.PubMedView ArticleGoogle Scholar
- Porzio E, Di Gennaro S, Palma A, Manco G: Mn(2+) modulates the kinetic properties of an archaeal member of the PLL family. Chem Biol Interact. 2013, 203: 251-256.PubMedView ArticleGoogle Scholar
- Xue B, Chow JY, Baldansuren A, Yap LL, Gan YH, Dikanov SA, Robinson RC, Yew WS: Correction to structural evidence of a productive active site architecture for an evolved quorum-quenching GKL lactonase. Biochemistry. 2012, 51: 10120-View ArticleGoogle Scholar
- Bigley AN, Raushel FM: Catalytic mechanisms for phosphotriesterases. Biochim Biophys Acta. 2013, 1834: 443-453.PubMedPubMed CentralView ArticleGoogle Scholar
- Hiblot J, Gotthard G, Elias M, Chabriere E: Differential active site loop conformations mediate promiscuous activities in the lactonase pox. PLoS One. 2013, 8: e75272-PubMedPubMed CentralView ArticleGoogle Scholar
- Waters CM, Bassler BL: Quorum sensing: cell-to-cell communication in bacteria. Annu Rev Cell Dev Biol. 2005, 21: 319-346.PubMedView ArticleGoogle Scholar
- Popat R, Crusz SA, Diggle SP: The social behaviours of bacterial pathogens. Br Med Bull. 2008, 87: 63-75.PubMedView ArticleGoogle Scholar
- Boyen F, Eeckhaut V, Van Immerseel F, Pasmans F, Ducatelle R, Haesebrouck F: Quorum sensing in veterinary pathogens: mechanisms, clinical importance and future perspectives. Vet Microbiol. 2009, 135: 187-195.PubMedView ArticleGoogle Scholar
- Elias M, Tawfik DS: Divergence and convergence in enzyme evolution: parallel evolution of paraoxonases from quorum-quenching lactonases. J Biol Chem. 2012, 287: 11-20.PubMedPubMed CentralView ArticleGoogle Scholar
- Amara N, Krom BP, Kaufmann GF, Meijler MM: Macromolecular inhibition of quorum sensing: enzymes, antibodies, and beyond. Chem Rev. 2011, 111: 195-208.PubMedView ArticleGoogle Scholar
- Hentzer M, Wu H, Andersen JB, Riedel K, Rasmussen TB, Bagge N, Kumar N, Schembri MA, Song Z, Kristoffersen P, Manefield M, Costerton JW, Molin S, Eberl L, Steinberg P, Kjelleberg S, Høiby N, Givskov M: Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors. EMBO J. 2003, 22: 3803-3815.PubMedPubMed CentralView ArticleGoogle Scholar
- O’Loughlin CT, Miller LC, Siryaporn A, Drescher K, Semmelhack MF, Bassler BL: A quorum-sensing inhibitor blocks Pseudomonas aeruginosa virulence and biofilm formation. Proc Natl Acad Sci U S A. 2013, 110: 17981-17986.PubMedPubMed CentralView ArticleGoogle Scholar
- Wu H, Song Z, Hentzer M, Andersen JB, Molin S, Givskov M, Hoiby N: Synthetic furanones inhibit quorum-sensing and enhance bacterial clearance in Pseudomonas aeruginosa lung infection in mice. J Antimicrob Chemother. 2004, 53: 1054-1061.PubMedView ArticleGoogle Scholar
- Christensen LD, van Gennip M, Jakobsen TH, Alhede M, Hougen HP, Hoiby N, Bjarnsholt T, Givskov M: Synergistic antibacterial efficacy of early combination treatment with tobramycin and quorum-sensing inhibitors against Pseudomonas aeruginosa in an intraperitoneal foreign-body infection mouse model. J Antimicrob Chemother. 2012, 67: 1198-1206.PubMedView ArticleGoogle Scholar
- Dong YH, Wang LH, Xu JL, Zhang HB, Zhang XF, Zhang LH: Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase. Nature. 2001, 411: 813-817.PubMedView ArticleGoogle Scholar
- Dong YH, Wang LY, Zhang LH: Quorum-quenching microbial infections: mechanisms and implications. Philos Trans R Soc Lond B Biol Sci. 2007, 362: 1201-1211.PubMedPubMed CentralView ArticleGoogle Scholar
- Ng FS, Wright DM, Seah SY: Characterization of a phosphotriesterase-like lactonase from Sulfolobus solfataricus and its immobilization for quorum quenching. Appl Environ Microbiol. 2011, 77: 1181-1186.PubMedPubMed CentralView ArticleGoogle Scholar
- Krieger RI: Handbook of Pesticide Toxicology. 2001, San Diego: Academic Press, 2Google Scholar
- Gupta RC: Handbook of Toxicology of Chemical Warfare Agents. 2009, San Diego: Elsevier IncGoogle Scholar
- LeJeune KE, Wild JR, Russell AJ: Nerve agents degraded by enzymatic foams. Nature. 1998, 395: 27-28.PubMedView ArticleGoogle Scholar
- Hawwa R, Aikens J, Turner RJ, Santarsiero BD, Mesecar AD: Structural basis for thermostability revealed through the identification and characterization of a highly thermostable phosphotriesterase-like lactonase from Geobacillus stearothermophilus. Arch Biochem Biophys. 2009, 488: 109-120.PubMedView ArticleGoogle Scholar
- Hiblot J, Gotthard G, Chabriere E, Elias M: Structural and enzymatic characterization of the lactonase SisLac from Sulfolobus islandicus. PLoS One. 2012, 7: e47028-PubMedPubMed CentralView ArticleGoogle 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 Crystallogr Sect F Struct Biol Cryst Commun. 2013, 69: 1235-1238.PubMedPubMed CentralView ArticleGoogle Scholar
- Vieille C, Zeikus GJ: Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev. 2001, 65: 1-43.PubMedPubMed CentralView ArticleGoogle Scholar
- Demirjian DC, Moris-Varas F, Cassidy CS: Enzymes from extremophiles. Curr Opin Chem Biol. 2001, 5: 144-151.PubMedView ArticleGoogle Scholar
- Burton SG, Cowan DA, Woodley JM: The search for the ideal biocatalyst. Nat Biotechnol. 2002, 20: 37-45.PubMedView ArticleGoogle Scholar
- Singh RK, Tiwari MK, Singh R, Lee JK: From protein engineering to immobilization: promising strategies for the upgrade of industrial enzymes. Int J Mol Sci. 2013, 14: 1232-1277.PubMedPubMed CentralView ArticleGoogle Scholar
- Merone L, Mandrich L, Porzio E, Rossi M, Muller S, Reiter G, Worek F, Manco G: Improving the promiscuous nerve agent hydrolase activity of a thermostable archaeal lactonase. Bioresour Technol. 2010, 101: 9204-9212.PubMedView ArticleGoogle Scholar
- Meier MM, Rajendran C, Malisi C, Fox NG, Xu C, Schlee S, Barondeau DP, Hocker B, Sterner R, Raushel FM: Molecular engineering of organophosphate hydrolysis activity from a weak promiscuous lactonase template. J Am Chem Soc. 2013, 135: 11670-11677.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhang Y, An J, Ye W, Yang G, Qian ZG, Chen HF, Cui L, Feng Y: Enhancing the promiscuous phosphotriesterase activity of a thermostable lactonase (GkaP) for the efficient degradation of organophosphate pesticides. Appl Environ Microbiol. 2012, 78: 6647-6655.PubMedPubMed CentralView ArticleGoogle Scholar
- Chow JY, Wu L, Yew WS: Directed evolution of a quorum-quenching lactonase from Mycobacterium avium subsp. paratuberculosis K-10 in the amidohydrolase superfamily. Biochemistry. 2009, 48: 4344-4353.PubMedView ArticleGoogle Scholar
- Chow JY, Xue B, Lee KH, Tung A, Wu L, Robinson RC, Yew WS: Directed evolution of a thermostable quorum-quenching lactonase from the amidohydrolase superfamily. J Biol Chem. 2010, 285: 40911-40920.PubMedPubMed CentralView ArticleGoogle Scholar
- Auernik KS, Cooper CR, Kelly RM: Life in hot acid: pathway analyses in extremely thermoacidophilic archaea. Curr Opin Biotechnol. 2008, 19: 445-453.PubMedPubMed CentralView ArticleGoogle Scholar
- Gotthard G, Hiblot J, Elias M, Chabriere E: Crystallization and preliminary X-ray diffraction analysis of the hyperthermophilic Sulfolobus islandicus lactonase. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011, 67: 354-357.PubMedPubMed CentralView ArticleGoogle Scholar
- Del Vecchio P, Elias M, Merone L, Graziano G, Dupuy J, Mandrich L, Carullo P, Fournier B, Rochu D, Rossi M, Masson P, Chabriere E, Manco G: Structural determinants of the high thermal stability of SsoPox from the hyperthermophilic archaeon Sulfolobus solfataricus. Extremophiles. 2009, 13: 461-470.PubMedView ArticleGoogle Scholar
- Notredame C, Higgins DG, Heringa J: T-Coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol. 2000, 302: 205-217.PubMedView ArticleGoogle Scholar
- Poirot O, O’Toole E, Notredame C: Tcoffee@igs: a web server for computing, evaluating and combining multiple sequence alignments. Nucleic Acids Res. 2003, 31: 3503-3506.PubMedPubMed CentralView ArticleGoogle Scholar
- Gouy M, Guindon S, Gascuel O: SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol. 2010, 27: 221-224.PubMedView ArticleGoogle Scholar
- Hall TA: BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series. 1999, 41: 95-98.Google Scholar
- Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG: Clustal W and Clustal X version 2.0. Bioinformatics. 2007, 23: 2947-2948.PubMedView ArticleGoogle Scholar
- Hiblot J, Gotthard G, Chabriere E, Elias M: Characterisation of the organophosphate hydrolase catalytic activity of SsoPox. Sci Rep. 2012, 2: 779-PubMedPubMed CentralView ArticleGoogle Scholar
- Gotthard G, Hiblot J, Gonzalez D, Chabriere E, Elias M: Crystallization and preliminary X-ray diffraction analysis of the organophosphorus hydrolase OPHC2 from Pseudomonas pseudoalcaligenes. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013, 69: 73-76.PubMedPubMed CentralView ArticleGoogle Scholar
- Gotthard G, Hiblot J, Gonzalez D, Elias M, Chabriere E: Structural and enzymatic characterization of the phosphotriesterase OPHC2 from Pseudomonas pseudoalcaligenes. PLoS One. 2013, 8: e77995-PubMedPubMed CentralView ArticleGoogle Scholar
- Studier FW: Protein production by auto-induction in high density shaking cultures. Protein Expr Purif. 2005, 41: 207-234.PubMedView ArticleGoogle Scholar
- van den Berg S, Lofdahl PA, Hard T, Berglund H: Improved solubility of TEV protease by directed evolution. J Biotechnol. 2006, 121: 291-298.PubMedView ArticleGoogle Scholar
- Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, Williams KL, Appel RD, Hochstrasser DF: Protein identification and analysis tools in the ExPASy server. Methods Mol Biol. 1999, 112: 531-552.PubMedGoogle Scholar
- Copeland RA: Enzymes, A Practical Introduction to Structure, Mechanism, and Data Analysis. 2000, New York, Chichester, Weiheim, Brisbane, Singapore, Toronto: WILEY-VCH, 2Google Scholar
- Lambert C, Leonard N, De Bolle X, Depiereux E: ESyPred3D: prediction of proteins 3D structures. Bioinformatics. 2002, 18: 1250-1256.PubMedView ArticleGoogle Scholar
- DeLano W: The PyMOL Molecular Graphics System. 2002, San Carlos, CA, USA: DeLano ScientificGoogle Scholar
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