Title: Activation of Bacterial Thermoalkalophilic Lipases Is Spurred by Dramatic Structural Rearrangements
Abstract: The bacterial thermoalkalophilic lipases that hydrolyze saturated fatty acids at 60–75 °C and pH 8–10 are grouped as the lipase family I.5. We report here the crystal structure of the lipase from Geobacillus thermocatenulatus, the first structure of a member of the lipase family I.5 showing an open configuration. Unexpectedly, enzyme activation involves large structural rearrangements of around 70 amino acids and the concerted movement of two lids, the α6- and α7-helices, unmasking the active site. Central in the restructuring process of the lids are both the transfer of bulky hydrophobic residues out of the N-terminal end of the α6-helix and the incorporation of short side chain residues to the α6 C-terminal end. All these structural changes are stabilized by the Zn2+-binding domain, which is characteristic of this family of lipases. Two detergent molecules are placed in the active site, mimicking chains of the triglyceride substrate, demonstrating the position of the oxyanion hole and the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acids chains. The combination of structural and biochemical studies indicate that the lid opening is not mediated by temperature but triggered by interaction with lipid substrate. The bacterial thermoalkalophilic lipases that hydrolyze saturated fatty acids at 60–75 °C and pH 8–10 are grouped as the lipase family I.5. We report here the crystal structure of the lipase from Geobacillus thermocatenulatus, the first structure of a member of the lipase family I.5 showing an open configuration. Unexpectedly, enzyme activation involves large structural rearrangements of around 70 amino acids and the concerted movement of two lids, the α6- and α7-helices, unmasking the active site. Central in the restructuring process of the lids are both the transfer of bulky hydrophobic residues out of the N-terminal end of the α6-helix and the incorporation of short side chain residues to the α6 C-terminal end. All these structural changes are stabilized by the Zn2+-binding domain, which is characteristic of this family of lipases. Two detergent molecules are placed in the active site, mimicking chains of the triglyceride substrate, demonstrating the position of the oxyanion hole and the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acids chains. The combination of structural and biochemical studies indicate that the lid opening is not mediated by temperature but triggered by interaction with lipid substrate. Lipases (triacylglycerol lipase EC 3.1.1.3) catalyze the hydrolysis of long-chain triacylglycerides at water/oil interfaces. They are very versatile enzymes because, in vitro, they catalyze both the hydrolysis and the synthesis of a great variety of esters (1Björkling F. Godtfredsen S.E. Kirk O. Trends Biotechnol. 1991; 9: 360-363Abstract Full Text PDF Scopus (227) Google Scholar, 2Bornscheuer U.T. Enzyme Microb. 1995; 17: 578-586Crossref Scopus (201) Google Scholar), the hydrolysis and transesterification of triacylglycerols (3Mukherjee K.D. Biocatalysis. 1990; 3: 277-293Crossref Scopus (165) Google Scholar), or the resolution of racemic mixtures (4Jaeger K.E. Reetz M.T. Trends Biotechnol. 1998; 16: 396-403Abstract Full Text Full Text PDF PubMed Scopus (879) Google Scholar). Most lipases contain a lid domain controlling access to the active site (5Derewenda Z.S. Sharp A.M. Trends Biochem. Sci. 1993; 18: 20-25Abstract Full Text PDF PubMed Scopus (197) Google Scholar). The interaction of the enzyme with lipid aggregates induces the displacement of the lid, which makes the active site accessible to individual substrate molecules and increases the catalytic activity. This phenomenon is known as interfacial activation (5Derewenda Z.S. Sharp A.M. Trends Biochem. Sci. 1993; 18: 20-25Abstract Full Text PDF PubMed Scopus (197) Google Scholar, 6Sarda L. Desnuelle P. Biochim. Biophys. Acta. 1958; 30: 513-521Crossref PubMed Scopus (642) Google Scholar).Much of the interest in extremophiles stems from their surprising properties. There has been extensive research on the structural proteins and key metabolic enzymes that are responsible for the unusual properties of the organisms. Recent research has focused on the identification of extremozymes relevant for industrial biocatalysis. The bacterial thermoalkalophilic lipases are among the most biocatalytically relevant extremozymes due to their resistance to proteases, detergents, and chaotropic agents together with their extreme stability at elevated temperatures and in organic solvents (7Demirjian D.C. Moris-Varas F. Cassidy C.S. Curr. Opin. Chem. Biol. 2001; 5: 144-151Crossref PubMed Scopus (423) Google Scholar). Thermoalkalophilic lipases are found in several thermophilic aerobic bacteria recently reclassified as the new genus Geobacillus (8Nazina T.N. Tourova T.P. Poltaraus A.B. Novikova E.V. Grigoryan A.A. Ivanova A.E. Lysenko A.M. Petrunyaka V.V. Osipov G.A. Belyaev S.S. Ivanov M.V. Int. J. Syst. Evol. Microbiol. 2001; 51: 433-446Crossref PubMed Scopus (598) Google Scholar), Geobacillus stearothermophilus (9Jeong S.T. Kim H.K. Kim S.J. Chi S.W. Pan J.G. Oh T.K. Ryu S.E. J. Biol. Chem. 2002; 277: 17041-17047Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), Geobacillus thermocatenulatus (10Schmidt-Dannert C. Sztajer H. Stocklein W. Menge U. Schmid R.D. Biochim. Biophys. Acta. 1994; 1214: 43-53Crossref PubMed Scopus (173) Google Scholar), Geobacillus thermoleovorans (11Cho A.R. Yoo S.K. Kim E.J. FEMS Microbiol. Lett. 2000; 186: 235-238Crossref PubMed Google Scholar), and Geobacillus sp. TP10A (12Bell P.J.L. Nevalainen H. Morgan H.W. Bergquist P.L. Biotechnol. Lett. 1999; 21: 1003-1006Crossref Scopus (16) Google Scholar). Thermoalkalophilic lipases show optimal activity at 60–75 °C and pH 8–10, share about 95% amino acid sequence identity among them, and show a significant similarity of 30–35% with the mature lipases from other Gram-positive bacteria Staphylococcus strains. In contrast to the similarity with staphylococcal lipases, the thermoalkalophilic lipases exhibit no sequence similarity with other microbial lipases (13Schmidt-Dannert C. Rua M.L. Schmid R.D. Methods Enzymol. 1997; 284: 194-220Crossref PubMed Scopus (53) Google Scholar). They are also characterized by their significantly larger molecular sizes (40–45 kDa) relative to other microbial lipases (usually under 35 kDa). Thus, the thermoalkalophilic lipases and staphylococcal lipases were originally grouped in one lipase family, named the lipase family I.5 (14Jaeger K.E. Dijkstra B.W. Reetz M.T. Annu. Rev. Microbiol. 1999; 53: 315-351Crossref PubMed Scopus (899) Google Scholar) or Staphylococcus family (13Schmidt-Dannert C. Rua M.L. Schmid R.D. Methods Enzymol. 1997; 284: 194-220Crossref PubMed Scopus (53) Google Scholar). Later, because of their lower level of sequence similarity to the Geobacillus lipases, the Staphylococcus enzymes were re-assigned to family I.6 (15Jaeger K.E. Eggert T. Curr. Opin. Biotechnol. 2002; 13: 390-397Crossref PubMed Scopus (1148) Google Scholar).Crystal structures of two isoforms of the G. stearothermophilus lipase (named L1 and P1), and of the lipase from Geobacillus zaliae (named T1) have been reported (9Jeong S.T. Kim H.K. Kim S.J. Chi S.W. Pan J.G. Oh T.K. Ryu S.E. J. Biol. Chem. 2002; 277: 17041-17047Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 16Tyndall J.D. Sinchaikul S. Fothergill-Gilmore L.A. Taylor P. Walkinshaw M.D. J. Mol. Biol. 2002; 323: 859-869Crossref PubMed Scopus (108) Google Scholar, 17Matsumura H. Yamamoto T. Leow T.C. Mori T. Salleh A.B. Basri M. Inoue T. Kai Y. Rahman R.N. Proteins. 2008; 70: 592-598Crossref PubMed Scopus (66) Google Scholar). In those structures, lipases presented its closed conformation with the active site buried under a long lid helix. The structures exhibited a zinc-binding site in an extra domain accounting for the larger molecular size of the family I.5 enzymes in comparison to other microbial lipases.The thermophile G. thermocatenulatus produces two lipases (BTL1 and BTL2) with different sizes (13Schmidt-Dannert C. Rua M.L. Schmid R.D. Methods Enzymol. 1997; 284: 194-220Crossref PubMed Scopus (53) Google Scholar). BTL2 is a 43-kDa protein (predicted from the DNA sequence) that showed high stability at medium temperatures (50 °C), alkaline pH (9.0–11.0), and in organic solvents (2-propanol, acetone, methanol) (18Schmidt-Dannert C. Rua M.L. Atomi H. Schmid R.D. Biochim. Biophys. Acta. 1996; 1301: 105-114Crossref PubMed Scopus (189) Google Scholar). Furthermore, it has been reported that this lipase presents a high specificity toward very different substrates with a great selectivity in the resolution of key intermediates in the synthesis of drugs (19Palomo J.M. Penas M.M. Fernandez-Lorente G. Mateo C. Pisabarro A.G. Fernandez-Lafuente R. Ramirez L. Guisan J.M. Biomacromolecules. 2003; 4: 204-210Crossref PubMed Scopus (91) Google Scholar). Here, we report the crystal structure of the BTL2 in an open conformation with two molecules of Triton detergent present in the active site. Activation involves dramatic concerted structural rearrangements in which the zinc-binding domain plays a critical role. The combination of crystallographic and biochemical studies has allowed us to gain unprecedented insights into the activation events that lead to the catalytic turnover processes. This knowledge is central to understanding how interfacial activation is triggered in thermoalkalophilic lipases and paves the way for engineering lipases with biotechnological purposes.EXPERIMENTAL PROCEDURESCloning, Expression, and Purification of BTL2—The gene corresponding to the mature lipase from G. thermocatenulatus BTL2 was cloned into pT1 expression vector as previously described (18Schmidt-Dannert C. Rua M.L. Atomi H. Schmid R.D. Biochim. Biophys. Acta. 1996; 1301: 105-114Crossref PubMed Scopus (189) Google Scholar). Cells carrying the recombinant plasmid pT1BTL2 were grown at 30 °C, and overexpression was induced by raising the temperature to 42 °C for 20 h. The enzyme was purified from Escherichia coli crude extract using a sequential chromatography step procedure in batch (octyl-Sepharose, PEI-agarose, and Q-Sepharose) as previously described (22Carrasco-López C. Godoy C. de las Rivas B. Fernández-Lorente G. Palomo J.M. Guisán J.M. Fernández-Lafuente R. Martínez-Ripoll M. Hermoso J.A. Acta Crystallogr F. 2008; 64: 1043-1045Crossref PubMed Scopus (10) Google Scholar). In this purification procedure, a final washing step containing Triton X-100 (10 nm in distilled water) before enzyme elution from Q-Sepharose, permitted BTL2 activation for crystallization experiments.Immobilization of BTL2—BTL2 immobilization on cyanogen bromide-agarose (CNBr) was performed as previously described (20Palomo J.M. Ortiz C. Fuentes M. Fernandez-Lorente G. Guisan J.M. Fernandez-Lafuente R. J. Chromatogr A. 2004; 1038: 267-273Crossref PubMed Scopus (114) Google Scholar). This preparation (prepared in the presence of detergent but later thoroughly washed with distilled water) permitted the immobilization of the monomeric form of the lipase, avoiding any intermolecular lipase-lipase interactions that could interfere with the properties of soluble lipases (21Fernandez-Lorente G. Godoy C.A. Mendes A.A. Lopez-Gallego F. Grazu V. de Las Rivas B. Palomo J.M. Hermoso J. Fernandez-Lafuente R. Guisan J.M. Biomacromolecules. 2008; 9: 2553-2561Crossref PubMed Scopus (94) Google Scholar).BTL2 Activity Measurements—Lipase activity was measured by absorbance variation at 348 nm of p-nitrophenol because of p-nitrophenylphosphate butyrate (pNPB) 3The abbreviations used are: pNPB, p-nitrophenylphosphate butyrate; D-pNP, diethyl p-nitrophenylphosphate; MPD, 2-methyl-2,4-pentanediol; r.m.s.d., root mean square deviation; PAL, P. aeruginosa lipase; PDB, Protein Data Bank. hydrolysis (0.4 mm) in buffer M (sodium phosphate, 25 mm, pH 7.0) at 25 °C, using a thermostatted cuvette with continuous magnetic stirring.BTL2 Activation Experiments—To test the detergent's effect in BTL2 activation, increasing concentrations of Triton X-100 were added to purified BTL2 (soluble and immobilized) in 25 mm sodium phosphate at pH 7 and 25 °C at a concentration of 0.1 mg of BTL/ml.To study lipase inhibition kinetics, D-pNP (0.6 μm) was added to purified BTL2 (soluble and immobilized) at a concentration of 0.1 mg of BTL/ml in 25 mm sodium phosphate at pH 7 and 25 °C in the absence of Triton X-100, and in increasing concentrations of Triton X-100. Enzyme activity was checked at different times using the assay described above.Crystallization and Data Collection—Native crystals of BTL2 were grown using the hanging drop vapor diffusion method as previously reported (22Carrasco-López C. Godoy C. de las Rivas B. Fernández-Lorente G. Palomo J.M. Guisán J.M. Fernández-Lafuente R. Martínez-Ripoll M. Hermoso J.A. Acta Crystallogr F. 2008; 64: 1043-1045Crossref PubMed Scopus (10) Google Scholar). Good quality diffracting crystals, showing rice-grain shapes, were found in 0.05 m sodium citrate, pH 5.6, MPD (13%), and ammonium acetate (0.2 m). Native data sets were collected using synchrotron radiation source at the ESRF (Grenoble) on BM16 beamline using an ADSC Reverse PHI detector and a wavelength of 0.979234 Å. Collected images were processed and scaled using MOSFLM and SCALA programs, respectively from the CCP4 package (Collaborative Computational Project, Number 4, 1994). BTL2 crystals belong to the orthorhombic I222 space group (a = 73.07 Å, b = 129.08 Å, c = 127.49 Å.).Structure Determination and Refinement—The BTL2 structure was solved by the molecular replacement method using the MOLREP program from the CCP4 package with the P1 lipase structure from G. stearothermophilus (PDB code 1ji3) as the initial model. The model was subjected to successive refinement cycles with the CNS program (23Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D. Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16930) Google Scholar), and intensive manual model building used the software package O (24Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar). Excellent density maps were obtained for the BTL2 structure except for the first two residues of the polypeptide chain. Two fragments of Triton X-100 used in the purification procedure were found in the catalytic groove of the BTL2 structure and three MPD molecules on the protein molecular surface involved in crystal packing interactions. Water molecules were gradually added with the waterpick routine of the CNS program.RESULTS AND DISCUSSIONOverall Structure—The crystal structure of BTL2 has been solved at 2.2-Å resolution (structural determination parameters and refinement statistics are summarized in Table 1). The three-dimensional structure of BTL2 (Fig. 1) comprises 389 residues and consists of an irregular (α/β) hydrolase fold formed by a central β-sheet of seven strands (β3-β9) surrounded by α-helices (α1 and α13 on one side and α2, α4, and α10 on the other side). An extra domain involved in Zn2+ coordination is present in this family of lipases consisting of helices α3 and α5 and strands b1 and b2, as previously described (9Jeong S.T. Kim H.K. Kim S.J. Chi S.W. Pan J.G. Oh T.K. Ryu S.E. J. Biol. Chem. 2002; 277: 17041-17047Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). No significant variations are observed in the Zn2+ coordination in BTL2 versus B. stearothermophilus lipases (Lipases used in BTL2 structural analysis are summarized in Table 2). Zn2+ coordination bonds are formed with His-82 and His-88 (2.29 Å and 2.21 Å, respectively) from the extra domain, and Asp-62 (2.11 Å and 2.96 Å) and Asp-239 (2.13 Å and 2.71 Å) from the (α/β) hydrolase core domain. A Ca2+ ion is also found to form hydrogen bonds with regions comprised between b4 and α11, and β9 and α13 (Fig. 1). The coordination includes interactions with carboxyl oxygen atoms of Glu-361 (2.96 Å) and Asp-366 (2.89 Å), and with two main chain carbonyl oxygen atoms of Gly-287 (2.61 Å) and Pro-367 (2.58 Å). The BTL2 structure has been solved in the open (active) conformation with two Triton X-100 detergent molecules placed at the active site (Fig. 2 and supplemental Fig. S2).TABLE 1BTL2 structure determination and refinement statisticsBTL2Data collection statisticsSpace groupI222Unit cell parameters a, Å73.07 b, Å129.08 c, Å127.49T (K)120Wavelength (Å)0.979234Resolution (Å)63.75 (2.36)-2.2Total no. of reflections285455No. of unique reflections37406Redundancy4.1 (4.5)aValues in parentheses correspond to the highest resolution shellCompleteness (%)99.8 (100)I/σ8.9 (2.5)RmergebRsym = Σ| I – Iav|/ΣI, where the summation is over symmetry equivalent reflections0.14 (0.48)Refinement statisticsResolution range (Å)45.18–2.2Protein non-hydrogen atoms3056Ligand non-hydrogen atoms Detergent36 MPD24Water molecules534Metals ions2Rfactor (%)18.15RfreecRfree = value calculated for 7% of data excluded from the refinement (%)22.55R.m.s.d. bond length (Å)0.005R.m.s.d. bond angles (°)1.2Average B-factor (Å2)29.92a Values in parentheses correspond to the highest resolution shellb Rsym = Σ| I – Iav|/ΣI, where the summation is over symmetry equivalent reflectionsc Rfree = value calculated for 7% of data excluded from the refinement Open table in a new tab TABLE 2Lipase structures used in BTL2 structural analysisEnzyme (PDB code)SpeciesSequence identity (%)Lipase familyConformationLigandRef.BTL2 (2W22)G. thermocatenulatus-I.5.OpenTriton X-100L1 (1KU0)G. stearothermophilus95I.5.Closed-(9Jeong S.T. Kim H.K. Kim S.J. Chi S.W. Pan J.G. Oh T.K. Ryu S.E. J. Biol. Chem. 2002; 277: 17041-17047Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar)P1 (1IJ3)G. stearothermophilus95I.5.Closed-(16Tyndall J.D. Sinchaikul S. Fothergill-Gilmore L.A. Taylor P. Walkinshaw M.D. J. Mol. Biol. 2002; 323: 859-869Crossref PubMed Scopus (108) Google Scholar)PAL (1EX9)P. aeruginosa21I.1.OpenRC-Trioctyl Inhibitor(26Nardini M. Lang D.A. Liebeton K. Jaeger K.E. Dijkstra B.W. J. Biol. Chem. 2000; 275: 31219-31225Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar) Open table in a new tab FIGURE 2The active site (cleft) of BTL2. A, stereo view of the catalytic crevice with two chains of Triton X-100 (white sticks). The catalytic triad (Ser-114, His-359, and Asp-318) is colored in blue. Residues lining the hydrophobic groove are drawn as yellow sticks. B, residues of BTL2 involved in the catalytic machinery and oxyanion stabilization are colored in orange; main differences with the closed structure (L1 lipase from B. stearothermophilus) are highlighted in blue. Residues coordinating Zn2+ cation (yellow sphere) are also labeled. Broken lines indicate main interactions.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Catalytic Machinery of BTL2—The catalytic machinery of thermoalkalophilic lipases is formed by the catalytic triad and the oxyanion hole. The active site of BTL2 contains the catalytic triad residues (Ser-114, His-359, and Asp-318) located at their canonical positions in the α/β hydrolase fold (Fig. 2A). The catalytic serine is in the Ala-X-Ser-X-Gly motif characteristic of the thermoalkalophilic lipases (versus the Gly-X-Ser-X-Gly motif of most lipases and serine proteases) (25Kim H.K. Park S.Y. Lee J.K. Oh T.K. Biosci. Biotechnol. Biochem. 1998; 62: 66-71Crossref PubMed Scopus (169) Google Scholar). The catalytic serine is exposed to solvent and substrate. In the closed state of L1 lipase, it has been observed that catalytic serine is in tight side-chain packing with some residues of the active site (His-113, Phe-17, Ile-320, Thr-270, and Met-326) resulting in the stabilization of the serine loop and contributing to the lipase thermostability (9Jeong S.T. Kim H.K. Kim S.J. Chi S.W. Pan J.G. Oh T.K. Ryu S.E. J. Biol. Chem. 2002; 277: 17041-17047Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In the BTL2 open structure, this tight packing is also maintained except for the Phe-17 (Fig. 2B). This residue changes its conformation allowing the access of the substrate to the catalytic Ser that changes its conformation, reorienting its Oγ group to the substrate. The movement of Phe-17 involves a change in its χ1 side-chain torsion angle (Δχ1 = 100°) from the closed to the open state. The conformer change of catalytic serine from the closed to the open forms also involves changes in the hydrogen-bonding pattern. In the closed state, the serine side chain Oγ atom points to His-113 Nδ1 atom (2.89 Å) and is 3.36 Å from the catalytic histidine N∊2 atom. However, in the active state, the serine side chain Oγ atom is 2.96 Å from the catalytic histidine N∊2 atom and 5.08 Å from the His-113 Nδ1 atom. No changes are observed in the hydrogen bonds between catalytic residues His-359 (Nδ1 atom) and Asp-318 (Oδ2 atom) in both closed and open states (2.68 Å and 2.81 Å, respectively) (Fig. 2B).The oxyanion produced in the tetrahedral intermediate is stabilized by interactions in the oxyanion hole, which in families I.1 and I.2 of bacterial lipases has been identified to be formed by the main chain amide groups of a couple of residues (Met-16 and His-83 in the I.1 family, and Leu-17 and Gln-88 in the I.2 family) (26Nardini M. Lang D.A. Liebeton K. Jaeger K.E. Dijkstra B.W. J. Biol. Chem. 2000; 275: 31219-31225Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). The equivalent atoms in BTL2 are Phe-17 and Gln-115 that occupy virtually identical positions. The Phe-17 and Gln-115 backbone nitrogen atoms localize the oxyanion-binding pocket in a position that is very well conserved within the α/β hydrolase fold enzymes (27Ollis D.L. Cheah E. Cygler M. Dijkstra B. Frolow F. Franken S.M. Harel M. Remington S.J. Silman I. Schrag J. Sussman J.L. Verschueren K.H.G. Goldman A. Protein Eng. 1992; 5: 197-211Crossref PubMed Scopus (1830) Google Scholar, 28Nardini M. Dijkstra B.W. Curr. Opin. Struct. Biol. 1999; 9: 732-737Crossref PubMed Scopus (668) Google Scholar, 29Cygler M. Schrag J.D. Methods Enzymol. 1997; 284: 3-27Crossref PubMed Scopus (82) Google Scholar). Phe-17 is the second residue of the tetrapeptide motif Gly-Hyd-X-Gly (Hyd = Phe, Leu, or Ile) located between strand β3 and helix α4 and is highly conserved in bacterial lipases from the I.5 family. No relevant differences are observed in the backbone of this region between the closed and open forms, and therefore it seems that formation of the oxyanion-binding pocket only requires the movement of the side chain of the Phe-17 residue.In families I.1 and I.2 of bacterial lipases, a totally buried Arg residue has been found connecting, via hydrogen bonds, the oxyanion hole tetrapeptide motif to the loop between strand β3 and helix α2 (26Nardini M. Lang D.A. Liebeton K. Jaeger K.E. Dijkstra B.W. J. Biol. Chem. 2000; 275: 31219-31225Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). The variation of the hydrogen-bonding pattern of this Arg between the closed and open forms of I.1 and I.2 lipases has been predicted to play an important stabilizing role during the opening of the lid of these lipases (26Nardini M. Lang D.A. Liebeton K. Jaeger K.E. Dijkstra B.W. J. Biol. Chem. 2000; 275: 31219-31225Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). An Arg residue is also found in the equivalent positions of BTL2 (Arg-63) connecting His-15 (through a hydrogen bond between its carbonyl oxygen atom and the Arg-63 NH2 atom) to Gly-55 (through a hydrogen bond between its carbonyl oxygen atom and the Arg-63 NH1 atom) (Fig. 2B). A comparison with the closed form of the L1 lipase reveals that there is no variation in the hydrogen-bonding pattern of this Arg residue in agreement with the preformed oxyanion hole found in BTL2. Interestingly, Arg-63 is placed just after Asp-62, which is directly involved in Zn2+ coordination. Therefore, in the I.5 family of bacterial lipases, the Arg-63 might play a stabilizing role in the oxyanion-binding pocket, a role that should be reinforced by the Zn2+-binding domain, which is characteristic of this family of lipases.The Active Site Cleft and Binding Interactions with the Substrate—Two molecules of Triton detergent have been identified occupying the hydrophobic active site cleft (Fig. 2A). This cleft is 14-Å deep and has an ovoid shape with approximate dimensions of 18 × 25 Å, with an area of 847.9 Å2 and a volume of 1183.1 Å3 (as calculated by CASTp). Its walls are lined mostly with hydrophobic/aromatic side chains (Ala-241, Ile-320, Ile-363, Leu-171, Leu-184, Leu-189, Leu-209, Leu-245, Leu-360, Leu-57, Met-174, Phe-17, Phe-182, Phe-291, Pro-165, Tyr-30, Val-172, Val-175, Val-188, Val-234, Val-295, Val-321, and Val-365) to achieve a perfect stabilization of the lipid substrate. At the base of the cleft, the Phe-17 side chain divides it in two parts, thus giving a boomerang-like shape to the active site as also described for other lipases (Fig. 2A), with four binding pockets, an oxyanion hole, and three pockets for the different branches of the triacylglycerol substrate.The Pseudomonas aeruginosa lipase (PAL) belonging to the I.1 family of bacterial lipases has been crystallized in complex with a triglyceride-like inhibitor (26Nardini M. Lang D.A. Liebeton K. Jaeger K.E. Dijkstra B.W. J. Biol. Chem. 2000; 275: 31219-31225Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar) (Table 2). The complex has demonstrated, for the I.1 family, the position of the oxyanion hole and of the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acid chains. Despite the structural differences between PAL and BTL2, superimposition of the (α/β) hydrolase core of both enzymes yields a well fitted triglyceride analog into the active site cleft of the BTL2 lipase (Fig. 3A). Remarkably, superimposition reveals that Triton X-100 moieties found in the BTL2 structure are coincident with two acyl chains (sn-1 and sn-3) of the triacylglycerol substrate in PAL. Therefore, structural analysis of PAL and BTL2 offers the opportunity of direct identification of the three acyl-binding pockets of the triglyceride substrate in the I.5 family of bacterial lipases. The Triton X-100 molecule TRT1 found in BTL2 is accommodated in a large groove formed by the side chains of Phe-17, Leu-184, Val-188, Leu-189, Leu-57, Leu-209, Leu-214, and Trp-212 (Fig. 3). The long aliphatic chain of 20 atoms of the detergent perfectly fits in this cleft and is bound via van der Waals interactions. This cavity in PAL corresponds to the acyl chain pocket A (HA). As in the PAL structure, the alcohol-binding pocket is separated from the acyl pocket by the side chain of Phe-17 (Met-16 in PAL); however the acyl pocket groove is larger in BTL2 (about 9.0 Å × 22 Å) than in PAL (about 8.5 Å × 9.0 Å) pointing to longer chain substrates in the I.5 family than in the I.1 family of bacterial lipases.FIGURE 3Putative binding of a triacylglycerol molecule to I.5 lipases. A, stereo view of the surface representation of the substrate binding site of BTL2. The two detergent moieties (TRT1 and TRT2) are represented as white sticks. The structure of the triglyceride-like inhibitor (blue sticks) of the P. aeruginosa lipase complex (PDB code 1EX9) is represented as a direct superimposition of the (α/β) hydrolase core of both enzymes. The identified three acyl chain-binding pockets (HA, HB, and HH) are labeled. B, stereo view of the substrate-binding mode in BTL2. The triglyceride-like inhibitor (blue) and Triton X-100 molecules (white), the catalytic triad (brown), and the residues that line the three acyl chain-binding pockets, HH (purple), HA (green), and HB (yellow) are labeled.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The acyl chain-binding pocket corresponding to the sn-2 moiety of the triglyceride analog (HH pocket) in PAL corresponds in BTL2 to a hydrophobic pocket lined by Ile-363, Trp-20, Phe-28, Met-25, Leu-360, and Val-365 (Fig. 3). The latter residues are part of the calcium-binding loop (residues 358–367). The HH pocket is similar in PAL than in BTL2, and there is space for about 8–10 fatty acid carbon atoms. The acyl chain-binding pocket, the sn-1 moiety of the triglyceride analog (HB pocket) in PAL, corresponds in BTL2 to a hydrophobic pocket lined by Ile-320, Val-321, Leu-171, Val-175, Leu-184, Met-174, Phe-291, and Val-295 (Fig. 3). The hydrophobic nature of the HB pocket is similar in both BTL2 and PAL lipases; however in BTL2 the HB pocket tightly interacts with the TRT2 Triton X-100 molecule, while in PAL this pocket is more exposed to the solvent. The three pockets (HA, HH, and HB) identified in BTL2 should also be present in the rest of the members of the I.5 family of bacterial lipases considering their high degree of sequence similarity.Activation Mechanism—The existence of an adjustable loop at only one end of the α6-helix and the bipartite distribution of hydrophobic resides in that α6-helix, with bulky side chains at the N-terminal-half and short side chain residues at the C-terminal-half, led to the proposed model of a unidirectional opening mechanism of the lid (9Jeong S.T. Kim H.K. Kim S.J. Chi S.W. Pan J.G. Oh T.K. Ryu S.E. J. Biol. Chem. 2002; 277: