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• W2034972775 abstract "Enzymes play critical roles in cellular function and provide the basis for versatile applications in the food, pharmaceutical, and fine chemical industries. The field of metagenomic gene discovery offers enormous scope and potential for both fundamental microbiology and biotechnological development.1 The metagenome approach thus provides a means to view both the structural and the functional genomics of microbial diversity and to discover novel genes for obtaining newer and more useful industrial alkaline proteolytic enzymes with improved properties.2 Esterases (EC 3.1.1.1) represent a diverse group of hydrolases catalyzing the cleavage of ester bonds and are widely distributed in animals, plants, and microorganisms. In addition to lipases, a considerable number of microbial carboxyl esterases have also been discovered and overexpressed.3 This enzymatic reaction, the hydrolysis of the carboxylic ester, is catalyzed by a functionally versatile group of enzymes with many important applications in biocatalysis.4 On the basis of their amino acid sequence homologies and the occurrence of different conserved motifs, these esterases have been classified into three separate groups: cholinesterases, lipases, and hormone-sensitive lipases (HSLs).5, 6 In particular, HSLs play an important role in the regulation of rodent fat cell lipolysis and are regarded as adipose tissue-specific enzymes whose sole metabolic role is the catalysis of hormone-stimulated lipolysis in mammalian cells.7, 8 Recently, we isolated a novel esterase (estE7) gene from a metagenome library (Accession number DQ842024). Here, we report the characterization and X-ray structural analysis of EstE7 identified from a metagenome library. On the basis of these results, we show that EstE7 belongs to the hormone-sensitive lipase family. Active site residue and dimer arrangement of this protein have dissimilar character with other esterase in HSL family. These structural studies may have applications for the production of value-added products, including pharmaceuticals. The complete estE7 sequence (Accession no. DQ842024) from the MUC-positive clones (pCE92) was cloned into a pET-21a vector and expressed in BL21(DE3). The cell pellet was suspended in ice-cold lysis buffer (50 mM Tris-HCl, pH 8.0, 0.2M NaCl, 2 mM β-mercaptoethanol, and 5 mM imidazole: buffer A) and was disrupted by sonication. The crude lysate was centrifuged at 13,000 rpm for 30 min at 4°C and the cell debris was discarded. The supernatant was loaded onto a His Trap (GE Healthcare) preequilibrated with buffer A. The protein was eluted using a linear gradient of 0.5M imidazole in buffer A, and further purified on a Hi-Load 16/60 Superdex 200 prep-grade column (GE-Healthcare) in buffer composed of 10 mM Tris-HCl, pH 8.0, 50 mM NaCl, and 10 mM DTT. The enzyme assays were carried out with the following p-nitrophenyl derivatives: p-nitrophenyl acetate (pNPC2), p-nitrophenyl butyrate (pNPC4), p-nitrophenyl carproate (pNPC6), p-nitrophenyl caprylate (pNPC8), p-nitrophenyl decanoate (pNPC10), p-nitrophenyl palmitate (pNPC16), and p-nitrophenyl stearate (pNPC18).9 The effects of pH and temperature on the esterase activity were examined using the purified recombinant enzyme. To determine the optimal pH, various buffers were used that covered the pH range of 4.0–9.0. To determine the optimal temperature, the enzyme mixtures were incubated at temperatures ranging from 30 to 50°C for 10–60 min. Thermostability data were obtained by preincubating the enzyme at various temperatures, as aforementioned, and then measuring the residual activities under the standard assay conditions. The purified protein was concentrated to 15 mg/mL and suitable crystals for X-ray diffraction were obtained in 0.1M Bis-Tris propane, pH 7.0, 0.2M ammonium sulfate, 1M lithium sulfate, using the hanging-drop vapor-diffusion method at 22°C. X-ray diffraction data were collected from the cooled crystal with an ADSC Quantum 210 CCD detector at beamline 6C using the Pohang Light Source (PLS, South Korea). Crystals were flash- frozen in a liquid nitrogen stream with 25% (v/v) glycerol as a cryoprotectant. The raw data were processed and scaled using DENZO and SCALEPACK from the HKL2000 program.10 Initial phases were obtained using molecular replacement. The program MOLREP, within the CCP4 program suite was employed along with a model of the heroin esterase structure from Rhodococcus sp. (PDB code 1LZL).11 The structure was refined using simulated annealing, energy minimizations, and individual isotropic B factor refinement from the CNS.12 The adjustment of the model was carried out into sigma A weighted 2Fo-Fc, Fo-Fc using the program Coot.13 The final models were validated with PROCHECK.14 Molecular graphic figures were generated using PyMOL.15 Crystal diffraction data and refinement statistics for the structure are displayed in Table I. The coordinate and structure factors for the hormone-sensitive lipase from metagenome library have been deposited in the RCSB Protein Data Bank with the accession code 3DNM. The activity of the purified esterase was tested using a large number of p-nitrophenyl derivative compounds as substrates [Fig. 1(a)]. Although the enzyme efficiently hydrolyzed pNPC2 (90%), pNPC4 (100%), pNPC6 (60%), and pNPC8 (50%), a lower level of hydrolysis was observed for pNPC10 (30%), pNPC16 (2%), and pNPC18 (2%). These results indicated that EstE7 preferentially hydrolyzes short-chain ester compounds. The kinetic parameters, Km and Vmax, for purified EstE7 were obtained using pNPC4 as a substrate over a concentration range of 0.1–5 mM. The Km values were determined by analyzing the slopes of the Michaelis–Menten equation using GraphPad software (GraphPad, USA), which revealed a linear substrate response over the concentration range. The Km and Vmax values of the EstE7 enzyme for pNPC4 were 0.39 mM and 222 μM/min, respectively. The temperature dependence of EstE7 activity toward pNPC4 was determined by measuring the degradation of pNPC4 at various temperatures at pH 7.5, and the maximal activity was observed at 40°C. Thermostability data were obtained by preincubating EstE7 at various temperatures, including the optimal temperature of 30°C, and then measuring the residual hydrolyzing activity using the standard pNPC4 assay. Similarly, the effect of pH on EstE7 activity toward pNPC4 was determined at 4°C in various buffers ranging from pH 4.0 to 9.0. The maximal pNPC4 degradation activity was observed at pH 5.0, and more than 50% of the enzymatic activity was retained within a pH range of 6.0–9.0. The stability test at different pH levels showed that the purified enzyme was broadly stable within a pH range of 5.5–9.0. Biological and structural analysis of EstE7. (a) Relative activity of recombinant EstE7 toward p-nitrophenyl esters. Esterase activity was determined photometrically in a 50 mM Tris-HCl buffer (pH 7.5) using various p-nitrophenyl esters as substrates. (b) The EstE7 monomer structure has two domains: an α/β domain (green) and a regulatory domain (blue). The catalytic triad residues are shown in ball-and-stick representations. (c) Nucleophile Ser157 in molecule A interact with β-mercaptoethanol. The catalytic site consists of three residues: Ser157 (nucleophile), Glu251 (charge-relay network), and His281 (proton carrier). β-mercaptoethanol is shown in ball-and-stick representations. σA weighted electron density maps (2Fo − Fc) contoured at 1σ in the vicinity of catalytic residues and β-mercaptoethanol. (d) Substrate-binding tunnel. This tunnel of the hydrophobic pocket has an ovoidal shape with approximate depth of ∼16 Å. The nucleophile Ser157 residue is shown in ball-and-stick representations in the area of the orange dotted circle. The EstE7 exists in solution as a monomer and dimer during size-exclusion chromatography (data not shown). We have tried to grow up the crystal using the monomer EstE7 protein. And then, the monomer EstE7 protein was crystallized. The crystal belongs to the orthorhombic space group I212121 with unit-cell dimensions of a = 117.321, b = 126.828, and c = 233.393 Å, with four monomers occupying the asymmetric unit. N-terminal ∼14 amino acids (Met1-Thr14) have lack of the electron density map. When these four monomers are superimposed, the rms deviation for the Cα atom from residues 15 to 308 is about 0.5. The EstE7 exists as a tightly associated dimer in the crystal structure, the β-strand (β8) of each monomer participates in twofold pseudosymmetry main-chain interactions with that of the adjacent molecules. Six residues are involved in the dimerization, including three hydrophobic and three hydrophilic residues (Val271, Glu272, Lue273, Lys274, Ile275, and Trp276). The EstE7 protein consists of two domains, a regulatory domain and a catalytic domain [Fig. 1(b)]. The cap domain, located in the N-terminus of proteins of the HSL family, contributes to several aspects of enzyme function such as activity, specificity, regioselectivity, thermophilicity, and thermostability.16 The catalytic domain shows the typical α/β hydrolase fold, with a central antiparallel β-sheet surrounded by α-helices. The catalytic domain contains the catalytic triad of EstE7. It is comprised of Ser157, Glu251, and His281. In the EstE7 structure, the coordinates for residue Ser157 are ill-defined within the conserved G-X-S-X-G sequence.5 This residue is located between β5 and α5 and is called the “nucleophile elbow,”6 with an approximate torsion of Φ of 60° and ψ of −120°, respectively. In general, its conformation is stabilized by an intricate hydrogen bond formed between the Nδ atom of His281 (proton carrier) and Oδ atoms of Glu251 (charge-relay). In the crystal structure of EstE7, catalytic Ser157 is stabilized by a covalent bond to the β-mercaptoethanol in molecules A and C [Fig. 1(c)]. The EstE7 enzyme has a hydrophobic tunnel with a approximate depth of ∼16 Å on the catalytic triad [Fig. 1(d)]. Structural and functional studies of this region allowed the identification of the tunnel and the cleft as the appropriate enzyme acyl- and alcohol-binding pockets, respectively.6 We hypothesize that the depth of this tunnel influences the length of substrate chains, based on our biological assays. Interestingly, sequence analysis results indicated that this enzyme contains additional lipase motifs (GDXG_His; Ile83-His99). However, these regions contain a shallow cavity and have a high negative charge, therefore limiting their selectivity and activity. A structural homologue search using the program SCOP identified several proteins homologous to the esterase and revealed that the three-dimensional structure of EstE7 is related to proteins in the carboxyesterase family.17 The closest homologue was carboxylesterase ESTE1 (2C7B) from metagenomic library (rmsd of 1.53 Å and Z score of 11.0), a member of the HSL group within the esterase family. We generated an alignment of esterases from Alicyclobacillus acidocaldarius, Archaeoglobus fulgidus, and Bacillus subtilis and found that EstE7 has sequence identity with EST2 (in a 283-residue overlap), AFEST (in a 272-residue overlap), and BFAE (in a 265-residue overlap) of 29, 26, and 22%, respectively. On the basis of the sequence alignment results, we determined that the EstE7 catalytic region is conserved but not the 80 residues of the N-terminus, which exhibit low homology to other HSL proteins. Nevertheless, the three-dimensional structure is strongly conserved; the superposition of atomic coordinates of the EstE7 monomer with other HSL family members including EST2 (PDB code 1QZ3), AFEST (1JJI), and BFAE (1JKM) result in Cα rms deviations of 1.93, 1.66, and 1.77 Å, respectively. The hyperthermophilic BFAE contains 189 intramolecular hydrogen bonds between the main-chain, whereas mesophilic EstE7 contains 154 hydrogen bonds. Thus, we consider that intermolecular hydrogen bond interactions contribute to the thermophilic character of proteins. In general, HSL family members have a classical catalytic triad consisting of Ser (nucleophile), Asp (charge-relay network), and His (proton carrier), with all residues located on the top of a β-sheet in the C-terminal region that forms part of a canonical α/β hydrolase fold.5, 6, 16, 18 In contrast, the catalytic triad of EstE7 has Glu instead of Asp; however, the structural-based functions are the same for the charge-relay network. On the other hand, EstE7 dimer formation is different from that of other HSLs family members, and EstE7 dimer formations have been reported to have an unexpected function.18 On the basis of our size exclusion chromatography results, we consider that dimer formation by members of the HSL family involves crystallographic packing, or that these proteins exist in various oligomeric states. In summary, the EstE7 protein, identified from a metagenome library, effectively hydrolyzed short-chain ester compounds, and our kinetic studies revealed the optimal pH and temperature of hydrolysis to be pH 7.5 and 30°C (mesophilic), respectively. On the basis of the structural analysis, we defined the active site and the binding pocket. This structure belongs to the hormone-sensitive lipase group. Our biological and structural analyses provide insights into how this enzyme might be used for the production of value-added products, including pharmaceuticals. The authors thank Dr. H.S. Lee, K.H. Kim, and K.J. Kim for assistance during data collection at beamline 6C of the Pohang Light Source, Korea." @default.
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• W2034972775 title "Structural and functional analysis of a novel hormone‐sensitive lipase from a metagenome library" @default.
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