Structural insights of a hormone sensitive lipase homologue Est22

Hormone sensitive lipase (HSL) catalyzes the hydrolysis of triacylglycerols into fatty acids and glycerol, thus playing key roles in energy homeostasis. However, the application of HSL serving as a pharmaceutical target and an industrial biocatalyst is largely hampered due to the lack of high-resolution structural information. Here we report biochemical properties and crystal structures of a novel HSL homologue esterase Est22 from a deep-sea metagenomic library. Est22 prefers short acyl chain esters and has a very high activity with substrate p-nitrophenyl butyrate. The crystal structures of wild type and mutated Est22 with its product p-nitrophenol are solved with resolutions ranging from 1.4 Å to 2.43 Å. The Est22 exhibits a α/β-hydrolase fold consisting with a catalytic domain and a substrate-recognizing cap domain. Residues Ser188, Asp287, and His317 comprise the catalytic triad in the catalytic domain. The p-nitrophenol molecule occupies the substrate binding pocket and forms hydrogen bonds with adjacent residues Gly108, Gly109, and Gly189. Est22 exhibits a dimeric form in solution, whereas mutants D287A and H317A change to polymeric form, which totally abolished its enzymatic activities. Our study provides insights into the catalytic mechanism of HSL family esterase and facilitates the understanding for further industrial and biotechnological applications of esterases.

Multi-angle light scattering (MALS) analysis of Est22 on the wtc030s5 column (Wyatt) with the elution buffer (20 mM Tris-HCl, 100 mM NaCl, pH 7.4). The molecular weight of Est22 was 79.1 kD, which showed that Est22 is a dimer in solution. (C) Gel filtration profile of Est22 and mutants on a Superdex 200 16/600 column. Wild type Est22 and other mutants S170A, S188A, S188A/D287A, and S188A/H317A formed dimers in solution, whereas mutants D287A and H317A changed from dimeric to polymeric forms evidenced as they were eluted in void volume on gel filtration profiles. (D) Esterase activity was examined using different substrates, including p-nitrophenyl acetate (C2), p-nitrophenyl butyrate (C4) and p-nitrophenyl hexanoate (C6) at pH 7.5 and 40 °C. Compared with wild type Est22, S170A mutant decreased 10-30% activity with different substrates, whereas other mutants almost abolished the enzyme activities with substrates. (E) Gel filtration profiles of WT Est22 and mutants. The amino acid residues that involved in dimeric surface were mutated to alanine. Mutants K308A, C309A, Q311A, M313A, R331A, D332A, and S336A changed slightly, whereas mutants E285A, R298A, M313A, D328A, D287N, H317F and H317L showed heterogeneity on gel filtration, distributing from dimeric to polymeric states. (F) The enzymatic activities of WT Est22 and mutants were examined with C4 substrate. are 0.4899 mM, 44.02 μ M/min, and 3160 s −1 , respectively. All of these values are significantly higher than previous reported esterases [10][11][12] . The substrate specificity of Est22 was examined using various p-nitrophenyl (p-Np) esters with acyl chain lengths from C2 to C16 in standard conditions. We found that Est22 hydrolyzed p-Np esters up to C8, while the enzymatic activity decreased significantly with increasing chain length of the p-Np esters. Est22 had a maximal enzymatic activity at pH 7.5 and at 40 °C, whereas the enzymatic activity of Est22 slightly decreased to 95% at 35 °C. Thermostability analysis showed that Est22 activity decreased with increasing temperature, and was inert beyond 50 °C (Fig. 2a-d).
As investigation into the tolerance of Est22 to different metal ions, detergents, and organic solvents is critical for industrial applications, therefore, nine divalent cations were utilized during the enzymatic activity test. Zn 2+ and Cu 2+ abolished the enzymatic activity of Est22. Ni 2+ , Co 2+ , and Ba 2+ had decreased activity upwards of 70%, whereas the other four cations (Sr 2+ , Mn 2+ , Mg 2+ , and Ca 2+ ) had little effect. The chelating agent EDTA had no obvious inhibition on enzymatic activity, which indicated this esterase was not a metal enzyme (Fig. 2e). These results suggest that Est22 has the potential to be applied in the industrial environments containing metal ions such as Sr 2+ , Mn 2+ , Mg 2+ , and Ca 2+ . Next, we investigated the influence of organic solvents and detergents to the enzyme activity of Est22 (Fig. 2f). We found that the activity of Est22 was completely abolished in 1% SDS and 15% acetonitrile, while there was a decrease in enzymatic activity (from 20% to 90%) in 15% acetone, alcohol, dimethylformamide (DMF) and isopropanol. However, 15% dimethyl sulfoxide (DMSO), methanol, 1% TritonX-100, Tween 20, or Tween 80 can increase its activity (Fig. 2f).
The monomer structure of Est22. The crystal structures of wild type and mutated Est22 and with its associated product p-Np, were solved with resolutions ranging from 1.4 Å to 2.43 Å. Given that the overall architecture is very similar among these structures, we will discuss below the product p-Np bound Est22 structure (Fig. 3a,b). The crystal structure of Est22 with its product p-Np was refined to 1.4 Å resolution with a satisfied R factor and R free factor values of 14.27% and 16.68%, respectively (PDB ID: 5HC0). The crystallographic statistics for data collection and structure refinement are summarized in Table 1. The Est22 exhibits a classical α /β -fold hydrolase structure, which is composed of 11 α -helices and 8 β -sheets (Fig. 3a). There are two p-Np molecules in this structure. One is outside of the cap domain, which could be due to non-specific binding. The other is in the substrate-binding pocket, but it does not occupy the active site (Fig. 3a). The calculated surface electrostatic potential of Est22 is negatively charged (Fig. 3d).
The substrate binding pocket and active sites of Est22. The substrate-binding pocket of Est22 forms a deep and narrow gorge, which is distant from the dimeric interface. It can be divided into three regions, including the bottom, the central and the entrance part. The bottom region is composed of the loop region and the catalytic triad residues Ser188, His317, and Asp287 (Fig. 4a). Several large hydrophobic regions in the middle part are formed by the side chains of residues Leu113, Tyr219, Leu240, Leu289, Ile321, and Phe322. At the entrance of the binding pocket, Arg49, Phe55 (α 3 helix), and Asp241 (in the loop between α 7 and α 8 helices) could facilitate the entry of substrate via hydrophobic and electrostatic interactions. Five acidic residues were found in the entrance, including Glu43, Glu44, Asp50 and Asp57 on the α 3 helix, and Glu187 on the bottom of binding pocket.
In the catalytic sites of Est22, Ser188 is a nucleophile residue, His317 is the proton acceptor/donor, while Asp287 helps to stabilize the His317 residue. Within the conserved penta-peptide sequence motif Gly-X-Ser-X-Gly 16 , Ser188 is located at the apex of the nucleophile elbow 1 , a sharp turn connecting β 5 and α 6. Ser188 is ~25 Å away from the protein surface, which could protect the active site from being exposed to water. The Ser188 conformation is stabilized by a hydrogen bond between the O γ atom of Ser188 and the N ε2 atom of His317. Asp287 and His317 are stabilized by the hydrogen bond network between the carboxyl edge of β 7 and β 8.
One oxyanion hole was found in Est22, which was composed of residues Gly108, Gly109, and Gly189. Gly108 and Gly109, located in the His-Gly-Gly-Gly motif (residues 106-109), is usually conserved in HSL family (Fig. 4a). The main-chain nitrogen atoms of the oxyanion hole donate hydrogen to the cleaved substrate, which obtained at 40 °C is shown as 100%. (D) Thermostability of the recombinant Est22. (E) Effects of metal ions on the activity of Est22. An enzymatic assay was performed at 20 °C in100 mM Tris-HCl buffer (pH 7.5) with substrate C2. Metal ions (Zn 2+ , Sr 2+ , Ni 2+ , Mn 2+ , Cu 2+ , Co 2+ , Ca 2+ , Mg 2+ , and Ba 2+ ) and EDTA were added at the final concentration of 10 mM. (F) Effects of detergents and organic solvents on the activity of Est22. Organic solvents were added at the concentration of 15%. Detergents (Tween 20, Tween 80, Triton X-100, and SDS) were added at the concentration of 1%. The value obtained with no additives in the reaction mixture is shown as 100%. Enzyme activity was measured under standard condition.
stabilizes the negative charges on the tetrahedral intermediates arising from the nucleophilic attack of Ser188 to substrate during hydrolysis 14 . The hydrogen bond network contributes to the configuration of Gly109 and Gly189. For example, Gly109 interacts with Gln111 and Ser112 via hydrogen bonds between the O atom of Gly109 and the N atoms of Gln111 and Ser112.
Structural based mutation study of Est22. In order to investigate the relationship between key residues and the hydrolysis activity of Est22, we performed mutagenesis analysis for the catalytic triad residues Ser188, His317, and Asp287, and residue Ser170 that is located on α 5 helix. The native Est22 and mutants Ser188Ala, Ser170Ala, Ser188Ala/Asp287Ala, and Ser188A/His317A were eluted as a dimer on gel-filtration profile, whereas Asp287Ala, His317Ala, Asp287Asn, His317Phe and His317Leu shifted from dimeric to polymeric form (Fig. 1c,e). As immobilized enzymes (aggregates) can be served as highly efficient and specific catalysts in industry application 9,17 , we compared the enzymatic activities of the mutants with wild type Est22. Esterase activity was examined using different substrates, including p-nitrophenyl acetate (C2), p-nitrophenyl butyrate (C4) and p-nitrophenyl hexanoate (C6) at pH 7.5 and 40 °C (Fig. 1d,f). A significant decrease in enzymatic activity was observed for mutants of residues in active center, while the activity of Ser170Ala decreased 40% on C4 but increased 10% on C6 than native Est22. These results clearly show that the three amino acids (Ser188, His317, and Asp287) are active in the center and are essential for the biological function of Est22.
As the hydrolysis process is superfast, it is difficult to get to the intermediate state by crystallization. However, we successfully obtained several types of Est22 crystal structures, including the p-Np bound Est22 (PDB ID: 5HC0), the p-Np bound mutant S188A (PDB ID: 5HC2), S188A (PDB ID: 5HC5), and S170A (PDB ID: 5HC3) ( Table 1). Structure superimposition of the wild type Est22 with its mutants revealed that the structures are almost identical, as the RMSD of Cα atom values are 0.163 Å (p-Np bound Est22), 0.156 Å (S188A), 0.196 Å (p-Np bound S188A), and 0.190 Å (S170A), respectively. In the wild type Est22, His317 forms hydrogen bonds with Ser188 and Asp287, while the bond is abolished in the mutant S188A structure (Fig. 4a-d). Meanwhile, the configuration and space site of the product p-Np is totally different between the wild type and mutant structures. The product p-Np is located in the substrate-binding pocket, but does not occupy the active site, which indicates that the product transferred out of the active site after hydrolysis was done (Fig. 4b,e). In the p-Np bound mutant S188A structure, one p-Np located on the active center, which could result from the enzymatic activity abolishment (Fig. 1d). The p-NP in S188A mutant was stabilized by hydrogen-bonding interactions with three amino acids of oxyanion hole, Gly108, Gly109, and Gly189, whereas the p-NP in wild type Est22 only interacted with Ser188 via hydrogen bond (Fig. 4a-d).
To investigate the interaction between the Est22 and the substrate, the complex structure was simulated by AUTODOCK program 18 using the configuration of wild type protein structure and substrate p-nitrophenyl butyrate (C4). Ten configuration models were obtained with the most probable configuration found at the lowest binding energy. In this configuration, the oxygen atoms on C4 interact with the amino acids (Gly108, Gly109, Gly189, Ser188, and His317) of the active center and oxyanion hole via hydrogen and covalent bonds (Fig. 4f). When the four structures were superimposed together (Fig. 4e), it clearly showed that the product p-Np orientation changed between the wild type and mutants Est22. Combined with the conformations in p-Np bound wild type Est22 or mutant S188A, it was proposed that His317 could be protonated by Ser188, thus, remaining Ser188 could act as a nucleophile to attack the carbonyl carbon atoms of the ester, forming a tetrahedral shape of intermediates. The intermediate was stabilized by an oxygen hole, which in turn leads to the carbonyl of the tetrahedral intermediate to transfer the proton to N atom of His317. Finally, the carboxylate moiety leaves the enzyme when the hydrolysis is finished 14 .
The dimeric interface of Est22. The wild type Est22 has four molecules in the asymmetric unit. The four monomers in an asymmetric unit were arranged as a dimer of dimers (Fig. 5a). Each dimer in the asymmetric unit was related by two-fold noncrystallographic symmetry, which shared similar horseshoe-shaped cap domain with previous reported Est25, whereas differing from other esterases in its topological confirmation (Figs 5a and 6a). The monomers in the asymmetric unit were almost identical, as the pairwise root-mean-square deviation Data collection Est22 Est22+ p-Np Est22 (S188A) Est22 (S188A) + p-Np Est22 (S170A) The Est22 dimer was formed by edge-to-edge interactions via two core β -sheets of each monomer with two-fold symmetry. Its interface was formed largely by α 10, α 11 helices, and β 8 stand, which included 2 salt bridges and 11 hydrogen bonds. One salt bridge was between residues Lys308 (at β 8 sheet) and Asp328 (at α 11 helix), while the other was between Glu285 (at the loop between β 7 and α 10) and Arg298 (at α 10 helix). 11 hydrogen bonds located between the residues of Glu285, Arg298, Lys308, Cys309, Gln311, Met313, Arg331, Asp332, Asp328, Ser336, and Gly344 (Fig. 5b). The residues Phe280, Ile294, Tyr297, Arg310, Gly312, and Val329 appeared in non-bonded contacts at the dimeric interface. To achieve catalytic activity and maintain thermostability, dimerization of two monomers is essential in HSL family enzymes [19][20][21] . In addition, we mutated 11 amino acid residues that are involved in the dimeric surface (E285A, R298A, K308A, C309A, Q311A, M313A, D328A, R331A, D332A, and S336A) (Fig. 1e,f). The gel filtration profiles and enzymatic activities of these mutants clearly explain the reason why Est22 has hyperthermostability and very high enzymatic activity. Notably, disruption of dimerization has successfully been employed as a strategy for the development of effective inhibitors 22,23 .
Structural comparison of Est22 with other esterases. The structural similarity analysis was carried out using DALI server search, which showed that Est22 has significant structural homologies to previously reported esterases, including Est25 from metagenomic library (PDB code 4J7A), BFAE from B. subtilis (PDB code: 1JKM), PcEst from P. calidifontis (PDB code 3ZWQ), and PestE from P. calidifontis (PDB code 2YH2) 10,16,24 . However, the primary sequence between Est22 and these esterases are low, which ranges from 27% to 39%, the RMSD values of Cα atom are 1.7 Å (for 323 residues in Est25), 2.2 Å (for 326 residues in BFAE), 2.2 Å (for 295 residues in PcEst), and 2.1 Å (for 294 residues in PestE), respectively. This clearly shows that Est22 shares high similarity with other members of α /β -hydrolase fold family. Superimposing Est22 on the structures of Est25, BFAE, PcEst, and PestE revealed similar features of overall folds between these HSL family esterases (Fig. 6a,b). The core residues of the α /β -hydrolase including the catalytic triad are highly conserved (Figs 6c and 7). Several conserved sequence motifs, such as 106 HGGG 109 , 186 GXSXG 190 and 287 DPXXD 291 , widely exist in the catalytic center of HSL family members, suggesting Est22 shares the same catalytic mechanism with that of HSL family members. The structural differences are largely in the three loop regions, which are located between α 1 and α 2, β 4 and α 5, β 6 and α 7, respectively (Fig. 7). Several reported mutants (P146Q and a 19bp frameshift deletion in exon 9) are located on the outside of the alpha/beta hydrolase domain of HSL, which could function via regulating the enzymatic activity of HSL 8,25 .

Conclusions
Here we report the structural and functional analysis of an HSL family esterase Est22 from the deep sea. The analysis of the biochemical properties and the structural information of Est22 and its mutants greatly improve our understanding of the catalytic mechanism of the α /β -hydrolase fold family. As Est22 shares conserved active sites and essential motifs for hydrolysis of HSL family, our study could provide a novel way for developing new HSL inhibitors and broadening the applications as a biocatalyst in environment and industry.

Materials and Methods
Protein expression and purification. cDNA fragment encoding full length Est22 (1-344 aa) was amplified by PCR method from a deep-sea metagenomic library 15 . The cDNA of Est22 was cloned into pET28b vector with a N-terminal 6xHis tag. The wild type and mutants of Est22 plasmids were transformed into E. coli Rosetta (DE3) cells for protein expression. E. coli cells were cultured in LB medium with 50 ug/ml kanamycin and 34 ug/ml chloramphenicol at 37 °C. Isopropyl 1-thio-β -D-galactopyranoside (IPTG, 0.5 mM) was added into cells to induce protein expression at 25 °C for 8 h when the OD 600 reached 0.6-0.8. Then the cells were harvested by centrifugation at 6000 rpm for 10 min at 4 °C. The cell pellets were resuspended in a lysis buffer (50 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole, 5% Glycerol, 2 mM β ME, pH 8.0) and disrupted using a high-pressure homogenizer (JNBIO, China). The cell debris was removed by centrifugation at 17000 rpm for 60 min at 4 °C. The supernatant was purified using a His Trap TM HP column (GE Healthcare) and was eluted in a buffer (50 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, 5% Glycerol, pH 8.0). The protein was further purified by gel filtration using a Superdex 200 16/600 column (GE Healthcare) in a gel filtration buffer (20 mM Tris-HCl, 100 mM NaCl, 2 mM Mutagenesis. Mutants S170A, S188A, D287A, H317A, D287A/S188A, S188A/H317A, A287N, H317F, Biochemical characterization of Est22. The standard reaction was carried out with the appropriate amount of purified Est22 in 1 ml buffer containing 100 mM Tris-HCl (pH7.5) and 1 mM p-nitrophenyl acetate 27 . The activity of the enzyme was determined by measuring the amount of released p-nitrophenol at 40 °C at 405 nm using DU800 UV/Visible spectrophotometer (Beckman, USA). All samples were measured in triplicate and corrected for autohydrolysis of the substrate. The molar extinction coefficient of p-nitrophenyl is 15,000 M −1 cm −1 at 405 nm. The kinetic parameters were determined by using p-nitrophenyl acetate as substrate at different concentrations ranging from 0.05 to 4 mM. The values of Km and Vmax were analyzed by the Michaelis-Menten equation using GraphPad Software (GraphPad Inc., USA).
Crystallization and data collection. Est22 was crystallized by hanging-and sitting-drop vapour-diffusion methods by mixing 1 μ l of 20 mg ml −1 protein with 1 μ l reservoir solution at 4 °C and 20 °C respectively. The diffraction quality crystals of Est22 were grown in a reservoir solution containing 0.1 M HEPES sodium (pH 7.5) and 1.5 M Lithium sulfate monohydrate. The pNp bound Est22 crystals were grown in the condition of 0.1 M MES/sodium hydroxide (pH 5.5), 0.2 M calcium acetate and 15% PEG8000. The S188A mutant with or without pNp-bound crystals were both grown in the condition of 1 M sodium citrate (pH 8.0), 0.1 M imidazole. The   1JKM from B. subtilis), PcEst (PDB code 3ZWQ from P. calidifontis), PestE (PDB code 2YH2 from P. calidifontis), and human HSL (hHSL). Four helices (α 1, α 2, α 3, and α 8) are shown in black boxes labeled with cap domain at the bottom. Identical and highly conserved residues are colored in red and white, respectively. Enzyme activity site triad S188, D287 and H317 are labeled with asterisk. Three highly conserved motifs are depicted by light blue box and labeled with CD-1, CD-2 and CD-3 at the bottom. S170A crystals were grown in the condition of 0.2 M sodium tartrate dehydrate (pH 7.3), 20% PEG3350. All of the crystals were briefly soaked in a cryoprotectant solution consisting of 25% (v/v) glycerol dissolved in their corresponding mother liquors before being flash-cooled directly in a liquid-nitrogen stream at 100 K. The X-ray diffraction data were collected at the BL17U and BL19U beamlines of Shanghai Synchrotron Radiation Facility (Shanghai, China). Intensity data were integrated and scaled using HKL2000 28 or HKL3000.
Structure determination and refinement. The crystal structure of wild type Est22 was determined by molecular replacement using the crystal structure of Est25 (PDB code 4J7A) 10 as the search model. The other structures were determined by molecular replacement using Est22 structure as the search model. Cycles of refinement and model building were carried out by using REFMAC 29 and COOT 30 programs until the crystallography R-factor and free R-factory values reached to satisfied range. The quality of the final model was evaluated with PROCHECK 31 . All of the structures were displayed and analyzed using PyMOL program 32 . The simulation of Est22 with its substrate was carried out with AUTODOCK program 18 . The collected data and refinement statistics are summarized in Table 1.