Báo cáo Y học: The cold-active lipase of Pseudomonas fragi Heterologous expression, biochemical characterization and molecular modeling

Eur. J. Biochem. 269, 3321–3328 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03012.x The cold-active lipase of Pseudomonas fragi Heterologous expression, biochemical characterization and molecular modeling Claudia Alquati, Luca De Gioia, Gianluca Santarossa, Lilia Alberghina, Piercarlo Fantucci and Marina Lotti Dipartimento di Biotecnologie e Bioscienze, Universita degli Studi di Milano-Bicocca, Milano, Italy A recombinant lipase cloned from Pseudomonas fragi strain IFO 3458 (PFL) was found to retain significant activity at low temperature. In an attempt to elucidate the structural basis of this behaviour, a model of its three-dimensional structure was built by homology and compared with homologous mesophilic lipases, i.e. the Pseudomonas aeruginosa lipase (45% sequence identity) and Burkholderia cepacia lipase (38%). In this model, features common to all knownlipaseshavebeenidentified,suchasthecatalytictriad (S83, D238 and H260) and the oxyanion hole (L17, Q84). Enzymes from psychrotrophic and psychrophilic microor-ganisms have recently received increasing attention, due to theirrelevanceforbothbasicandappliedresearch.Thiseffort has been stimulated by the recognition that cold-adapted enzymesmightoffernovelopportunitiesforbiotechnological exploitation based on their high catalytic activity at low temperatures, low thermostability and unusual specificities. These properties are of interest in different fields such as detergents, textile and food industry, bioremediation and biocatalysis under low water conditions [1,2]. Furthermore, fundamental issues concerning the molecular basis of cold activity and the interplay between flexibility and catalytic efficiencyareofimportanceinthestudyofstructure–function relationships in proteins. Such issues are often approached through comparison with the mesophilic or thermophilic counterparts, if available, and/or mutagenesis [3,4]. In this context, the recent cloning of a few lipases (acylglycerol ester hydrolases, EC 3.1.1.3) active at low temperatureisrelevant[5–7].Becauseoftheirmetabolicand industrial role, lipases have been thoroughly investigated by studies encompassing sequence, structure, regulation of expression, activity and specificity [8]. Among bacterial lipases, a focus has been on enzymes produced by members of the genus Pseudomonas, some of which have been recently reclassified as Burkholderia. A dozen of Correspondence to M. Lotti, Dipartimento di Biotecnologie e Bioscienze, Universita degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy. Fax: + 39 02 64483565, Tel.: + 39 02 64483527, E-mail: marina.lotti@unimib.it Abbreviations: BCL, Burkholderia cepacia lipase; BGL, Burkholderia glumae lipase; MM, molecular mechanics; PAL, Pseudomonas aeruginosa lipase; PFL, Pseudomonas fragi lipase; PCR, polymerase chain reaction; SRC, structural conserved regions. Enzyme: lipase (EC 3.1.1.3). (Received 18 February 2002, revised 17 May 2002, accepted 23 May 2002) Structural modifications recurrent in cold-adaptation, i.e. a large amount of charged residues exposed at the protein surface, have been detected. Noteworthy is the lack of a disulphide bridge conserved in homologous Pseudomonas lipases that may contribute to increased conformational flexibility of the cold-active enzyme. Keywords: lipase; Pseudomonas; cold-active enzymes; modeling; selectivity. lipase-encoding genes have been cloned from different species, and the corresponding proteins have been classified intofamiliesI.1andI.2ofbacteriallipasesaccordingtotheir molecular properties and to the requirement of helper proteins for correct folding and secretion [9]. Crystal structure determinations of Pseudomonas lipases have been reported including B. glumae (BGL) [10], P. aeruginosa (PAL) [11], and B. cepacia (BCL) [12–14]. In this paper, we describe the characterization of a cold-activelipaseclonedfromP. fragi,themainspoilingagentof refrigerated meat and raw milk [15]. This enzyme shares highsequencesimilaritywithPseudomonaslipasesofknown three-dimensional structure, therefore providing a new tool to study the molecular bases of cold-adaptation. EXPERIMENTAL PROCEDURES P. fragi strain IFO3458 (LMG2191T) was obtained from the BCCMTM/LMG bacteria collection (Universiteit Gent, Belgium). As the cloning host, E. coli JM101 (Promega Co, Madison, Wisconsin) was used. Heterologous expression was performed in the E. coli strain SG13009[pREP4] (Qiagen). Cloning and expression DNAmanipulationswereaccordingtoSambrooket al.[16] and according to manufacturer’s instructions for the enzymes and materials employed. Chromosomal DNA was extracted as described previ-ously [17] with minor modifications from a P. fragi culture grown to the late exponential phase at 25 °C in 1% bacto tryptone, 0.5% bacto yeast extract and 0.5% NaCl. The lipase-encoding gene was amplified from chromoso-mal DNA by PCR with oligonucleotide primers designed based on the sequence of the homologous lipase from strain IFO 12049 (AC X14033). Forward primer: 5¢-CACCCTG CGAGATTGAACATG-3¢(nucleotides)18to+3);reverse primer:5¢-AAGCTTGATTACAGGCTACAAG-3¢(+938 3322 C. Alquati et al. (Eur. J. Biochem. 269) to +924), where a restriction site for HindIII was inserted. Reactionwascarriedoutinatotalvolumeof100 lLandwas catalyzedby2.5 UofPfuTurboTMpolymerase(Stratagene, CA, USA). The amplification program was as follows: 3 min94 °Cfollowedby25cyclesof30 s94 °C,45 s55 °C, 1 min 72 °C, the final elongation step was 5 min 72 °C and 15 min 10 °C. The amplified fragment, purified from a 0.8% agarose gel, was automatically sequenced by M-Medical (Firenze, Italy). The lipase-encoding gene was inserted in the BamHI and HindIII cloning sites of pQE30 (Qiagen), a plasmid designed for the regulated expression in E. coli of foreign genes. Recombinant proteins are produced as fusion with a His6 tagattheN-terminus.Thegenewasamplifiedasbeforeand modified by: (a) introducing a BamHI site and deleting the starting ATG codon and (b) introducing a HindIII cleavage site at the 3¢ end. Primers were as follows: forward primer: 5¢-GGATCCGACGATTCGGTAAAT-3¢; reverse primer: 5¢-AAGCTTGATTACAGGCTACAAG-3¢. E. coliSG13009transformedwiththeexpressionplasmid was grown overnight at 27 °C in Luria–Bertani medium supplemented with 100 lgÆmL)1 ampicillin and 25 lgÆmL)1 kanamycin. Isopropyl thio-b-D-galactoside was added to a concentration of 0.4 mM and cultivation was continued for an additional 4 h. Cells were collected by centrifugation (30 min, 8000 g, 4 °C) and resuspended in 50 mM NaH PO pH 8.0, 300 mM NaCl and 10 mM imidazole. After the addition of 1 mgÆmL)1 lysozyme, the suspension wasincubatedonicefor30 minandthensonicatedsixtimes for 10 s. The cell lysate was centrifuged for 20 min at 4 °C, 25 000 g. The His6-tagged lipase was purified at 4 °C on 50% Ni-nitrilotriacetic acidresin (Qiagen). Two mililiters of clear lysate were added to 1 mL resin, mixed by gentle shaking at 4 °C for 60 min and loaded on a column. After washing with 4 mL of 50 mM NaH2PO4 pH 8.0, 300 mM NaCl and 20 mM imidazole, the recombinant lipase was eluted at pH 7.5 with 2 mL of 50 mM NaH2PO4, 300 mM NaCl and 250 mM imidazole. Purification of the recombin-ant protein was monitored spectrophotometrically follow-ing the increase in absorbance at 410 nm due to hydrolysis of p-nitro-phenylpalmitate. The reaction mixture (1 mL) contained 0.8 mM p-nitro-phenylpalmitate in 50 mM Na2HPO4Æ2H2O, KH2PO4 with 2% arabic gum. Protein characterization SDS PAGE [18] was performed using 12% acrylamide gels with GELCODE staining (Pierce). The protein concentra-tionwasdeterminedaccordingtoBradford[19]withBSAas the standard. Lipase activity was determined in a pH-stat assay by titrating fatty acids released from triacylglycerols with 0.01 M sodium hydroxide using a 718 STAT TITRINO (Metrhom). Emulsions of 20 mM triacylglycerols with 2% arabic gum were used as the substrate. Tricaprylin was the substrate for activity determination, if not otherwise stated. The pH and temperature optima were investigated in the range of 4–9 and 25–40 °C, respectively. Substrate speci-ficity was determined at 29 °C, pH 8.0 on triacylglycerol substrates with chain lengths ranging from C4 to C18. The effect of temperature on stability was determined by preincubating samples of purified rPFL at 10°, 27° and 50 °C before determining residual activity. Ó FEBS 2002 The effect of calcium ions on activity was investigated under standard assay conditions by measuring enzymatic activity in the presence of 5 mM EDTA at free calcium concentrations varying between 0 and 50 mM. Molecular modeling Multiple sequence alignments were carried out with the program CLUSTALX [20] using the following lipase sequences: P. aeruginosa (PAL, Swissprot code: P26876), B. glumae (BGL, Swissprot code: O05489), B. cepacia (BCL, Swissprot code: P22088), P. fragi (PFL, this work). The alignment featuring the highest score was obtained using the Blosum matrix [21] and standard CLUSTALX parameters. The atomic coordinates of PAL and BCL in their open conformation [11,13] were obtained from the Brookhaven Protein Data Bank. The PFL three-dimensional model was built according to the following procedure: (a) protein regions charac-terized by high similarity, as identified by sequence alignment and featuring very similar secondary structure, as derived from experimental data or as predicted by the PHD algorithm [22], were chosen as structurally con-served regions (SCR); (b) the atomic coordinates of the backbone atoms inside the SCR regions were transferred from the reference X-ray structure to the model; (c) fragments connecting the scaffold elements (usually loops) were modeled scanning the Brookhaven Protein Databank for protein structures of a predefined length that would fit properly into the model protein between two SCRs. The search was carried out by comparing the a-carbon distance matrix of the flanking SCR peptides with a precalculated matrix for all known proteins that have the same number of flanking residues and an intervening peptide segment of the given length. The following strategy has been adopted to optimize the structure of the model: (a) all atoms, except those corresponding to the fragments connecting SCRs, were kept fixed during the first Molecular Mechanics (MM) energy minimization. This allowed the nonSCR elements to re-arrange their conformation without affecting the global folding of the more conserved regions. (b) In a second MM optimization step, only the backbone atoms of the SCRs were kept fixed. (c) Only the a-carbons of the SCR regions were constrained to their initial position and (d) as the final step, the whole model was subjected to MM optimization without constraints. The optimized structure was subjected to the program PROCHECK [23], which allowed confirming its structural reliability. RESULTS AND DISCUSSION Sequence analysis LipaseshavebeenclonedfromafewP. fragistrains[24–26]. Out of them, the short lipase sequence (135 residues) from strain IFO3458 present in the database ([24]; AC: M14604) is probably truncated atits3¢halfasitlacksfunctionalsites. The coding sequence was therefore amplified from chro-mosomal DNA with oligonucleotide primers designed based on the sequences immediately upstream and down-stream the coding sequence of the lipase from the related strain IFO12049 (S02005) [25]. Ó FEBS 2002 Cold-active Pseudomonas fragi lipase (Eur. J. Biochem. 269) 3323 Sequencing revealed an ORF of 879 bp encoding a polypeptide of 293 residues. Comparison with M14604 evidences a sequencing error at bp 354 where a missing nucleotide causes a reading frameshift followed by an early stop codon after few amino acids. The revised sequence has been assigned accession number AJ250176. Total GC content (59.3%) and the predicted codon usage, with 72.2% of the codons ending with G or C, are characteristic of Pseudomonas genes. Analysis of the deduced amino-acid sequence is consistent with a protein of Mr 32.086 and an isoelectric point of 9.33. A single cysteine residue is present at position 39, thus ruling out the presence of a disulphide bridge, characteristic of most Pseudomonas lipases. A leader sequence for secretion could not be unambiguously identi-fied at the N-terminal end. The amino-acid sequence shares 97% identity with IFO 12049 lipase from which it only diverges in its C-terminal 22 residues (highlighted in Fig. 1). Scanning of protein sequence databases by FASTA [27] revealed a high degree of similarity with the P. fluorescens strain C9 (O68310, 48.1% identity over 297 amino acids), Proteus vulgaris (Q52614, 47.9% over 286 amino acids), Pseudomonas sp. (Q9X512, 47.2% over 290 amino acids), P. aeruginosa (Q9L6C7, 45.1% over 288 amino acids) and Vibrio cholerae (P15493,48,2%over288aminoacids)lipases.Highidentity is also shared with other Pseudomonas lipases of known three-dimensional structure, i.e. B. cepacia (37.7% over 318 amino acids), and B. glumae (37.9% over 322 amino acids). The similarity with P. fluorescens lipases is restricted to a 30-kDa enzyme recently isolated from milk [15] and does not extend to the 50-kDa P. fluorescens lipases described so far. Surprisingly, the remarkable similarity to the lipase from Proteus vulgaris K80 [28], that in addition shares with PFL the absence of cysteine residues possibly involved in intramolecular disulphide bond formation. On the other hand, the lipase sequence does not display obvious sequence similarity with either a lipase cloned from an alaskan psychrotrophic Pseudomonas [5] or with other cold-adapted lipases from different microorganisms [6,7]. Expression and purification Expression of rPFL carrying a His6 tag at its N-terminus was obtained as described above. Culture growth and inductionwasperformedat27 °Ctocopewithbothenzyme thermolability and the optimal growth temperature for the E. coli expression system. Under these conditions, rPFL was partly obtained in a soluble, active form suitable for further characterization. About 2 mg of pure recombinant rPFL per g wet weight cells were recovered by a one-step purification method involving metal-chelating chromatog-raphy (Fig. 2). In order to exclude any influence of the His6 tag on the recombinant lipase activity, a control plasmid wasconstructedwherethetagwasfollowedbyarecognition site for Tevprotease. Enzyme obtained by protease digestion did not show any difference in activity (not shown). Several Pseudomonas lipases have been reported to requireachaperone(orhelper)proteinforefficientsecretion and folding of the active lipase [9]. Therefore, coexpression of the lipase- and helper-encoding genes has been success-fully exploited as a tool to obtainhigh levels ofrecombinant active lipase in heterologous bacterial hosts [29]. However, coexpression of rPFL with the foldase of P. aeruginosa did Fig. 1. Alignment of the PFL sequence with Pseudomonas lipases of known three-dimensional structure. The sequence is identical to that of the lipase from strain IFO12049 except for the C-terminal 22 amino acids which are shown in bold. ¼ amino acid forming the catalytic triad, °¼ amino acid forming the LID, §¼ amino acid involved in calcium binding. PAL ¼ P. aeruginosa lipase, PFL ¼ P. fragi lipase, BCL ¼ B. cepacia lipase and BGL ¼ B. glumae lipase. 3324 C. Alquati et al. (Eur. J. Biochem. 269) Fig. 2. Electrophoretic analysis showing the purification of recombinant PFL.(1)Molecularmassstandards;(2)2 lgrPFLafterpurificationby metal-chelating chromatograpy; (3) total soluble extract of E. coli expressing the recombinant protein. Ó FEBS 2002 Fig. 3. Effect of temperature on the enzyme stability as measured at 10 °C (d), 27 °C (r) and 50 °C (m). not produce significant improvements in the fraction of soluble lipase. The apparent ineffectiveness of foldase together with the lack of a signal peptide at the N-terminal suggests the hypothesis that PFL might be secreted by a signal peptide-independent pathway and calls for further investigation on this subject. Enzyme activity and specificity In the standard tricaprylin hydrolysis assay, rPFL displayed highest activity at 29 °C and pH 8.0, in good agreement with results reported for the wild type enzyme [30]. Interestingly, the enzyme lost most of its activity at 50 °C but retained about 60% of its specific activity at 10 °C (Table 1). Under the same conditions, the activity of the homologous lipase from Burkholderia cepacia was reduced to 10% (not shown). The stability of rPFL was further investigated. Incubation of the enzyme at different temper-atures showed that at 10 °C rPFL retains for several hours most of its activity, whereas its half-life at 27 °C is about 5 h. Activity dramatically drops after a few minutes at 50 °C (Fig. 3). This is an important characteristic for PFL identification as a cold-active enzyme and well fits with the structural features evidenced by the three-dimensional model, as reported below. Specificity towards triglycerides was tested in a pH-stat assay using six triacylglycerol substrates of different chain length (C4 to C18). As shown in Fig. 4, rPFL activity decreases going from short- to long-chain substrates and triolein is a poor substrate. Therefore, PFL differs in chain length selectivity from both PAL, with broad substrate specificity on triglycerides [31] and BCL which shows a high preference for the hydrolysis of triglycerides with a chain Fig. 4. Activity of rPFL on triacylglycerols with different chain length. C4, C8, C12, C16, C18 and C18* are tributyrin, tricaprylin, trilaurin, tripalmitin, tristearin and triolein, respectively. length ‡8 [32]. No activity was detected in a phospholipase assay using phosphatidylcholine as the substrate. Finally, experiments have been carried out to elucidate the influence of Ca2+ ions on the catalytic activity. rPFL activity measured in the presence of 5 mM EDTA was by 55% lower than the control. Variation of the calcium concentration resulted in a saturation curve with a plateau between 10 mM and 20 mM calcium (Fig. 5). Table 1. Rate of hydrolysis of tricaprylin by rPFL at different temper-atures. Temperature of hydrolysis (°C) 10 29 50 Relative hydrolysis (%) 59 100 15 Fig. 5. Enzymatic activity of rPFL as a function of calcium concentra- tion. Ó FEBS 2002 Cold-active Pseudomonas fragi lipase (Eur. J. Biochem. 269) 3325 Model building In order to derive a three-dimensional model of the PFL structure, its sequence was aligned with those of Pseudo-monaslipasesofknownthree-dimensionalstructure,namely B. cepacia (BCL), B. glumae (BGL) and P. aeruginosa (PAL). As can be observed in Fig. 1, sequence similarity extends along all the protein sequence with the exception of the 177–213 region (PFL numbering) where the BCL and BGL sequences are characterized by peptide insertions. Use of secondary structure data allowed to better define SCR in regions where sequence similarity alone provided ambigu-ous results. Using this approach, 11 SCRs were defined that constitutethestructural scaffoldupon which thePFL three-dimensional model was built. These SCRs span the peptide segments 5–23, 31–49, 54–68, 76–120, 129–148, 155–181, 191–201, 214–218, 237–242, 245–267 and 277–293 of PFL. It is interesting to note that most nonSCRs are peptide fragments forming loop regions in the experimentally derived three-dimensional structures, supporting our choice of SCRs. On the other hand, regions forming important structural and/or functional portions in the selected lipases, such as the catalytic triad, the oxyanion hole and the calcium binding site are all located in SCRs. In this work, PAL was chosen as structural reference as this enzyme shows the highest degree of similarity to PFL. Furthermore, the computed structure was validated against the BCL structure. We observed that our PFL model might overlap to a high degree with both structures, as expected from the overall structural similarity relating PAL and BCL [11]. On these bases, the structural features of the PFL were analysed in deeper detail (Figs 6–8). Fig. 6. Overall three-dimensional structure of PFL, as obtained by homology modeling. For clarity, only arginine side chains are explicitly shown. Fig. 7. Schematic representation of the Ca-coordination environment in PFL. The conserved residues D217 and D262 are coordinated to the metal ion by one carboxylic oxygen atom belonging to the side chain. The Ca ion is coordinated also by the backbone carbonyl groups of H266 and R269. Bond distances and angles involving the metal atom andthecoordinatingaminoacidsareverysimilartothecorresponding values observed in PAL (data not shown). Fig. 8. Schematic representation of the two aromatic residues of PFL (W184 and F244) that structurally correspond to the disulphide bridge in PAL, shown in grey. ... - tailieumienphi.vn