Cloning, Mutation, Protein Expression and Purification
Croceicoccus marinus E4A9T was previously isolated from a deep-sea sediment sample, and was stored in our lab. Altererythrobacter indicus DSM 18604T was isolated from mangrove-associated wild rice, and was purchased from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. The genes crme10 and aline4 were cloned into plasmid pSMT3 to produce the N-terminal His-Sumo tagged fusion proteins from genomic DNA of C. marinus E4A9T and A. indicus DSM 18604T, respectively. Point mutants were generated by site-directed mutagenesis using wild-type plasmids as templates for PCR. After digested with DpnI enzyme, the products were transformed into E. coli DH5α cells and determined by DNA sequencing. CrmE10-W1 contains wild-type CrmE10, ribosomal binding Site (RBS) and CrmE10-D178A in the pSMT3 plasmid, whereas the polycistronic CrmE10-W2 plasmid contains wild-type CrmE10, ribosome biding sites, and CrmE10-D178A-S29A.
The wide-type and mutated proteins were expressed in E. coli BL21 (DE3) cells. The cells were induced by adding 0.5 mM isopropyl-β-D-thiogalactoside (IPTG), when OD600 came to 0.6-0.8. After cultivation at 16 °C for 20 h, cells were harvested by centrifugation at 6,000 rpm for 15 min at 4 °C, resuspended in start buffer (50 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole, 5% glycerol, pH 8.0), and disrupted by French press homogenizer (JNBio, China). Cell debris was removed by centrifugation and the supernatant was incubated with Ni sepharose (GE, USA) for 1 h. After washing with buffer 1 (50 mM Tris-HCl, 500 mM NaCl, 50 mM imidazole, 5% glycerol, pH 8.0), the recombinant protein was eluted with buffer 2 (50 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, 5% glycerol, pH 8.0). Subsequently, the His-Sumo tag was removed by overnight digestion with ULP1 enzyme. The recombinant target proteins were further purified by gel filtration by using the Superdex 200 10/300 column (GE, USA) in buffer (20 mM Tris-HCl, 100 mM NaCl, 2 mM DTT, pH 7.4). The fractions of elution were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). And the protein concentrations were determined by Bradford method with bovine serum albumin (BSA) as standard.
Multiple alignment of amino acid sequences was performed using ClustalX v.2 program. Secondary structure alignment was generated by DSSP v.2.0 and ESpript v.3.0 (http://espript.ibcp.fr/ESPript/ESPript/). The phylogenetic tree was constructed by neighbor-joining method using MEGA (Molecular Evolutionary Genetics Analysis) v.7.0 software.
Multi-Angle Light Scattering (MALS) Analysis
MALS analysis was performed in the National Center for Protein Science Shanghai (NCPSS). 20 μl of 1 mg/ml purified target protein was subjected to SEC-MALS using a WTC-030S5 size-exclusion column (Wyatt, USA) with elution buffer (20 mM Tris-HCl, pH 7.4, 100 mM NaCl) and passed in tandem through a Wyatt DAWN HELEOS II light scattering instrument (Wyatt, USA) and an Optilab rEX refractometer (Wyatt, USA). Data collection and analysis were performed with Astra 6 software (Wyatt, USA).
Enzymatic Activity Assays
Esterase activity assays were performed using a spectrophotometric method with the appropriate amount of purified enzyme in standard reaction buffer containing 100 mM Tris-HCl (pH 7.5), and 1 mM p-nitrophenyl (p-NP) hexanoate (for CrmE10 and its mutants, TCI, Japan) or p-NP butyrate (for AlinE4 and its mutants, Sigma-Aldrich, USA). The enzymatic activity was determined at 20 °C (for CrmE10 and its mutants) or 40 °C (for AlinE4 and its mutants) by measuring the amount of releasing p-nitrophenol using Beckman Coulter DU 800 UV/Visible spectrophotometer (Beckman, USA) at 405 nm. All values were measured in triplicates and corrected for autohydrolysis of the substrates. One unit of enzymatic activity was defined as the amount of enzyme required for releasing 1 μmol of p-nitrophenol per minute from the p-nitrophenyl ester. The kinetic parameters (Km and Vmax) were calculated from enzymatic activity measurements with p-NP hexanoate (for CrmE10) or p-NP butyrate (for AlinE4) ranging from 0.05 mM to 2 mM. Initial reaction velocities measured at various concentrations were fitted to the Lineweaver-Burk transformation of Michaelis-Menten equation.
The optimum pH of esterases CrmE10 and AlinE4 was determined over the pH range from 3.0 to 9.5. The buffers included citrate buffer (100 mM, pH 3.0-6.0), phosphate buffer (100 mM, pH 6.0-7.5), Tris-HCl buffer (100 mM, pH 7.5-8.5 or 7.5-9.0), CHES-NaOH buffer (50 mM, pH 8.5-10.5 or 9.0-10.5). The enzymatic activity was measured under 348 nm. The effects of temperature on esterases CrmE10 and AlinE4 were measured over a range of 15-60 °C. To study the thermostability, the residual activity of CrmE10 and AlinE4 was determined after incubation at various temperatures ranging from 10 °C to 100 °C for 1 h. And the thermostability of AlinE4 was further determined after incubation at 90 °C, 95 °C and 100 °C for 0-2.5 h.
Various chain lengths of p-NP esters, including p-NP acetate (C2), p-NP butyrate (C4), p-NP hexanoate (C6) (TCI, Japan), p-NP octanoate (C8), p-NP decanoate (C10), p-NP dodecanoate (C12), myristate (C14) and p-NP palmitate (C16) (Sigma-Aldrich, USA, unless otherwise stated) were added into the reaction buffer with final concentration of 1 mM for determining substrate specificity.
The effects of NaCl on CrmE10 and AlinE4 activity were evaluated by adding 0-5 M NaCl to the assay mixture. The effects metal ions were measured using various divalent cations, namely Zn2+, Sr2+, Ni2+, Mn2+, Mg2+, Co2+, Ca2+ and Ba2+, at final concentration of 10 mM. The effect of the chelating agent ethylenediaminetetraacetic acid (EDTA) was determined at a final concentration of 10 mM. The effects of organic solvents were determined using acetone, acetonitrile, ethanol, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), glycerol, isopropanol and methanol, at final concentration of 15 % (v/v).
Crystallization and X-ray Data Collection
CrmE10 and AlinE4 were applied to crystallization trials carried out at 20 °C by hanging- and sitting-drop vapour-diffusion methods by mixing 30 mg/ml protein with equal volume reservoir solution. The crystals of native CrmE10 were grown in a reservoir solution containing 150 mM calcium acetate, 100 mM imidazole-HCl (pH 8.0) and 10 % PEG 8000. The crystals were briefly soaked in 25 % (v/v) glycerol dissolved in their reservoir solution, as a cryoprotectant solution, before being flash-frozen directly in liquid-nitrogen. The CrmE10-D178A crystals were grown in the condition of 250 mM calcium acetate, 100 mM imidazole-HCl (pH 8.5), 5 % PEG 1,000 and 3 % 1,6-Hexanediol. The cryoprotectant solution of the CrmE10-D178A crystals was 20 % (v/v) PEG 400. The crystals of native AlinE4, AlinE4-S13A and AlinE4-D178A were grown in 1 M NaAc, 100 mM HEPES and 50 mM CdSO4. All X-ray diffraction datasets were collected at BL17U1, BL18U1, and BL19U1 beamlines of Shanghai Synchrotron Radiation Facility (SSRF, China). Diffraction data was integrated and scaled using software HKL2000.
Structure Analysis and Refinement
The crystal structures of wild-type (WT) CrmE10 and AlinE4 were determined by molecular replacement using esterase TesA (PDB code: 4jgg) as the search model. The mutant protein structures were solved using the WT structure as the search model. After cycles of refinement and model building processed using program REFMAC5[57, 58] of CCP4i and software COOT, the crystallography R-free and R-factor values reached to satisfied range. PROCHECK of PDBsum was used to evaluate the quality of the final 3D-structures. The other homologous structures were identified using DALI sever[61, 62] and blast program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Substrate docking studies were performed using AutoDockTools4 program. All the 3D-structures were analyzed and displayed using the PyMOL molecular graphics system (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC). CrmE10, CrmE10-D178A, AlinE4, AlinE4-D162A and AlinE4-S13A were deposited to Protein Data Bank with accession codes 6IQ7, 6IQ8, 6IQ9, 6IQA and 6IQB, respectively. Data collection and refinement parameters are listed in Table S1.