Computational protocol: Structural Insights into the Recovery of Aldolase Activity in N-Acetylneuraminic Acid Lyase by Replacement of the Catalytically Active Lysine with γ-Thialysine by Using a Chemical Mutagenesis Strategy

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[…] Cloning and mutagenesis: The nanA gene was amplified from S. aureus NCTC 8325 genomic DNA (0.1 ng μL−1) by PCR with Pfu polymerase using primers designed to incorporate a His6 tag at the N terminus of the protein and including EcoRI and PstI restriction sites to allow subcloning (see Table S2 for primer sequences). The PCR product of around 900 bp was gel purified, restricted with EcoRI and PstI and ligated into pKK223-3 (Pharmacia) cut with the same enzymes. The resulting expression vector was designated pKSAnanA. Site-directed mutagenesis was carried out using QuikChange Lightning or QuikChange multisite-directed mutagenesis kits (Stratagene, UK) according to the manufacturer–s guidelines. Primers were designed by using the recommended QuikChange Primer Design Program (Stratagene, UK).Expression and purification of NAL: Wild-type and variant E. coli NAL were expressed from the plasmid pKnanA-His6 as previously described. S. aureus NAL (wild-type and variants) was expressed from E. coli BL21 (DE3) cells transformed with the plasmid pKSAnanA harbouring the wild-type or mutant S. aureus NAL gene, by using the method previously described. Cells were grown in 2YT medium (5 mL) containing ampicillin (50 μg mL−1) and inoculated into 2YT medium (2 L) containing ampicillin (50 μg mL−1) and IPTG (0.4 mm) and grown for 16 h at 37 °C. Cells were lysed in a Cell Disruption Systems apparatus at 19 000 psi in Tris⋅HCl (50 mm, pH 7.4), imidazole (20 mm) and NaCl (0.5 m). The lysed cells were then centrifuged at 12 000 g for 40 min. The supernatant, containing the soluble protein fraction, was transferred onto nickel-loaded chelating sepharose fast flow resin (10 mL) in a 50 mL Falcon tube. The resin was gently agitated for 20 min at 4 °C to allow the His6-tagged protein to bind. Centrifugation (4000 g, 7 min) yielded the load supernatant, which was removed. The resin was then washed by the addition of washing buffer [Tris⋅HCl (50 mm, pH 7.4), imidazole (20 mm), NaCl (0.5 m)]. After thorough mixing the resin was centrifuged at (4000 g, 7 min) and the supernatant removed, and this process was repeated a further three times. Elution buffer [20–30 mL, Tris⋅HCl (50 mm, pH 7.4), imidazole (0.5 m), NaCl (0.5 m)] was then added to the resin, after a 1 h incubation period (with agitation at 4 °C) this was separated from the resin by centrifugation (4000 g, 7 min), yielding the purified protein in the supernatant. The eluted protein was then dialysed against Tris⋅HCl buffer (20 mm, pH 7.4). Further purification was achieved by size-exclusion chromatography using an S-200 gel-filtration column (GE Healthcare Life Sciences) in the same buffer before concentration and storage. Protein purity was assessed by SDS-PAGE, and molecular weight was confirmed with ESI-MS. The purified enzymes were usually stored as lyophilised powders before chemical modification.Kinetic analyses: Kinetic parameters of the aldol cleavage reaction were determined at 30 °C using a standard coupled assay, with lactate dehydrogenase (LDH) and NADH. The reaction (1 mL final volume) contained varying volumes (2–300 μL) of substrate [Neu5Ac, 100 mm in Tris⋅HCl (50 mm, pH 7.4)], LDH (0.5 units), NADH (0.2 mm) and Tris⋅HCl (50 mm, pH 7.4). NAL samples, in Tris⋅HCl (50 mm, pH 7.4), were added in volumes of 10–200 μL. The decrease in absorbance at 340 nm was recorded on a Uvikon 930 spectrophotometer as the measure of enzyme activity. The rate of substrate cleavage was calculated using a molar extinction coefficient of NADH of 6220 m−1 cm−1. Kinetic parameters were estimated by fitting the data to the Michaelis–Menten equation.The pH-activity profiles of the wild-type and K165-γ-thialysine S. aureus NAL were obtained at 30 °C using the same assay but with an acetic acid/MES/Tris three-component buffering system covering the pH range 4–9 whilst maintaining a constant ionic strength. Steady-state parameters were measured by fitting the data to the Michealis–Menten equation. The values of kcat/Km against pH were then fitted to a standard bell-shaped curve by using the equation:Mass spectrometry: Samples were prepared using PD10 desalting columns and then analysed in acetonitrile/1 % aq. formic acid (50:50 v/v) by nano-electrospray ionisation MS using a quadrupole-ion mobility spectrometry-orthogonal time-of-flight spectrometer (Synapt HDMS, Waters, Manchester, UK). The MS was operated in positive TOF “V” mode using a capillary voltage of 1.2 kV, cone voltage of 50 V, nano-electrospray nitrogen gas pressure of 0.1 bar and backing pressure of 1.78 mbar. The source and desolvation temperatures were set at 80 °C and 150 °C, respectively. Nitrogen was used as buffer gas at a pressure of 8.0×10−3 mbar in the trap and transfer regions and 3.6×10−4 mbar in the ion mobility cell. Mass calibration was performed by a separate injection of sodium iodide at a concentration of 2 μg μL−1 in acetonitrile/water (50:50 v/v). Data processing was performed using the MassLynx v4.1 suite of software supplied with the mass spectrometer.Chemical modification of K165C to yield γ-thialysine: Lyophilised K165C S. aureus NAL (2.5 mg) was dissolved in of prewarmed (37 °C) sodium phosphate buffer, (1.25 mL, 50 mm, pH 8.0) containing urea (6 m). A solution of 2,5-dibromo-1,6-hexadiamide (diBr, synthesised as described; 0.13 mg μL−1, 97 μL) in DMF was added to the protein, and was mixed immediately using a vortex mixer for 30 s. The protein/diBr solution was incubated for 1.5 h at 37 °C, with agitation at 200 rpm. An aliquot (70 μL) was then desalted into ammonium acetate buffer (20 mm, pH 7.0), and analysed by ESI-MS to ensure complete conversion of K165C into K165Dha. The conjugate addition of 2-aminoethanethiol onto the K165Dha protein was then carried out on the unfolded K165Dha NAL. A solution of 2-aminoethanethiol (0.1 mg mL−1, 40 μL) in Tris⋅HCl buffer (1.5 m, pH 8.8) was added to K165Dha NAL (2 mg mL−1, 1 mL) and was incubated for 2 h at 37 °C, with agitation at 200 rpm. ESI-MS was used to check for complete conversion of Dha to the γ-thialysine. For determining the pH-activity profile for the thialysine-containing NAL, the above procedure was scaled up so that 50 mg of enzyme was modified.Protein refolding: Refolding of the modified enzyme was carried out in two stages. The modified protein was first dialysed into Tris⋅HCl buffer (50 mm, pH 7.4) containing urea (6 m) to remove excess modification reagents, followed by dialysis into the same buffer in the absence of urea to refold the enzyme. Size-exclusion chromatography was performed using an ÄKTA Prime purification system (GE Healthcare Life Sciences) with a Superdex S200 column. Protein (8 mg mL−1, 5 mL) was injected onto the column which was run at 2 mL min−1.Circular dichroism: CD spectra were obtained at room temperature using a Chirascan CD spectrometer (Applied Photophysics, Leatherhead, UK). All spectra were recorded in a quartz cuvette with path length 1.0 mm. Protein samples were prepared in sodium phosphate buffer (50 mm, pH 8.0) with or without urea (6 m).Protein crystallisation: Crystallisation conditions for the E. coli NAL had been previously established and these were used to crystallise the S. aureus NAL. Crystals were grown at 18 °C by hanging drop vapour diffusion. A ratio of 2 μL of protein (8 mg mL−1) to 2 μL mother liquor was used. The crystallisation conditions were Tris⋅HCl (100 mm, pH 7.0–8.5), NaCl (200 mm) and 18–28 % (w/v) poly(ethylene glycol) (PEG) 3350.To form the pyruvate complexes of the wild-type and K165-γ-thialysine S. aureus NALs crystals were soaked in the mother liquor containing sodium pyruvate (100 mm) and 15 % (v/v) PEG 400 for 1 min before being sequentially transferred to mother liquor with 5 % increments in PEG 400 concentration. The final soak contained the mother liquor containing sodium pyruvate (100 mm) and 25 % (v/v) PEG 400. Crystals were then flash-cooled in liquid nitrogen prior to data collection.Data collection and refinement: Diffraction data for all structures were collected from single crystals at the Diamond Light Source macromolecular crystallography beam lines I02 and I04-1. The temperature for data collection was 100 K. Integration and scaling of data was carried out by MOSFLM and SCALA. The structure of the wild-type S. aureus enzyme was solved by molecular replacement in Phaser using the structure of H. influenza NAL (PDB ID: 1F74) as the search model and subsequent structures were solved by direct Fourier methods. REFMAC5 was used for refinement of the data and after each refinement cycle model building was performed in COOT. Coordinates and restraint library files for the lysine residue covalently bound to a pyruvoyl moiety (HET code: KPI) were as previously described. Those for the thialysine side chain (HET code: SLZ) and for the thialysine covalently bound to pyruvate (HET code: KPY) were generated using the PRODRG server and were manually edited. The models were validated using the PDB validation server. […]

Pipeline specifications

Software tools iMosflm, REFMAC5, Coot, PRODRG
Applications Drug design, Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Staphylococcus aureus, Escherichia coli, Epipremnum aureum
Chemicals Cysteine, N-Acetylneuraminic Acid, Pyruvic Acid