A synthetic DNA fragment containing the full-length XylA (GenBank accession no: AM747722) which encodes the enzyme Xylose Isomerase from Burkholderia cenocepacia J2315 strain (de Figueiredo et al. 2013) was obtained from Epoch Life Science (TX, USA). The gene was cloned into HindIII and BglII digested pTrcHis-B plasmid (Thermo Scientific, USA) and transformed in E. coli TOP10 [F− mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80lacZ ΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu) 7697 galU galK rpsL (StrR) endA1 nupGλ] cells. Transformant cells were selected in LB broth with 200 μg/mL ampicillin. Protein expression was performed in 1-liter flasks containing 500 mL of LB broth and inoculated at 0.1 OD600nm starting cell density. Cells were incubated at 37 °C under 160 rpm agitation until OD600nm reached 0.6. Then, the culture was refrigerated to 18 °C and 500 μM IPTG was added to the medium for induction of protein expression for 16 h.
Protein purification
IPTG-induced cells were washed with washing buffer (50 mM sodium phosphate, 300 mm sodium chloride, pH 7) and centrifuged at 2900×g for 30 min at 4 °C. Cell pellet was frozen at − 80 °C. In the day of use, thawed samples were disrupted with 10 sonication cycles of 1 min with 1 min intervals on ice-water bath using a 4 mm titanium micro-point (QR 300, Ultronique). The cell lysate suspension was centrifuged at 9100×g for 30 min at 4 °C and supernatant was collected. A Nickel (Ni2+) resin (Novagen®, Merck, USA) was equilibrated with 30 bed volumes of washing buffer. The cell lysate supernatant was incubated with the nickel resin for 30 min under gentle agitation on ice-cold water bath for His-tagged protein trapping. Then, the resin was added into a disposable Bio-Rad column and washed twice with 10 bed volumes of washing buffer. His-tagged proteins were eluted in a stepwise manner, with elution buffer (50 mM sodium phosphate, 300 mm sodium chloride, pH 7.5) supplemented with 100, 200 or 300 mM imidazole.
Polyacrylamide gel electrophoresis (PAGE), protein concentration and determination
Protein expression and purification were initially analyzed in SDS-PAGE (5% stacking, 12% resolving gel). Sample loading dye contained 100 mM Tris–HCl pH 6.8, 8 M urea, 20% glycerol, 4% SDS, 0.2% bromophenol blue and 100 mM β-mercaptoethanol. Electrophoresis was performed at room temperature at a constant current of 0.06 A. Gel content was evaluated with Coomassie Brilliant Blue R-250 staining (BioRad). Eluted protein samples from the Ni2+ -resin were concentrated with Amicon Ultra 30K (Merck) by centrifuging at 4000×g at 4 °C for 10 min. The concentrated sample was fractionated into the Superdex 200 (GE Healthcare) resin with 1 mL/min flux of buffer (100 mM Tris–HCl, 20 mM NaCl, pH 7.5). The protein elution was followed by absorbance at 280 nm (ÄKTA Prime PLUS, GE Healthcare). Protein concentration after purification was assessed with BCA assay (Smith et al. 1985). The standard curve was made using BSA concentrations varying from 25 to 2000 μg/μL.
The molecular size and oligomerization of native His6-XylA was determined with a non-denaturing gradient PAGE (4–20% Mini-PROTEAN® TGX™ Precast Protein Gel, BIO-RAD). Electrophoresis was performed as described above, with SDS-free sample and running buffers. A mixture of 10 recombinant proteins ranging from 10 a 250 kDa was used as standard (Precision Plus Protein™, BIO-RAD).
Mass spectrometry
Protein bands were extracted from either native or SDS-PAGE and unstained on a solution of 30% methanol (v/v) and 10% acetic acid (v/v). Then, the sliced gel was incubated on a washing solution containing 50% acetonitrile (v/v) and 25 mM ammonium bicarbonate at pH 8.0 for 15 min. Protein samples were reduced on 8 M urea, 10 mM DTT and 25 mM ammonium bicarbonate solution at room temperature for 30 min. Then, the samples were incubated in a dark chamber in a 55 mM iodoacetamide and 25 mM ammonium bicarbonate solution for 30 min at room temperature. Trypsin (Promega) was added to the solution for proteolysis at 37 °C overnight. The digestion reaction was stopped with the addition of 1% formic acid (v/v). The peptides were cleaned using homemade C-18 stage tips (Rappsilber et al. 2007), dried, and resuspended in 0.1% formic acid (v/v) solution.
Peptides were fractionated using a nanoHPLC system Easy-nLC II (Proxeon) system on an in-house packed 2 cm × 150 μm i.d. pre-column (Reprosil-Pur C18-AQ, 5 μm, 120 Å, Dr. Maisch), and 15 cm × 75 μm i.d. analytical column (Reprosil-Pur C18-AQ, 3 μm, 120 Å, Dr. Maisch) coupled to an LTQ Velos Orbitrap (Thermo Fisher Scientific). Chromatography was performed at 300 nL/min. flow rate with 95% water, 5% Acetonitrile (ACN) and 0.1% formic acid (FA) as mobile phase A and 85% ACN, 15% water, and 0.1% FA as phase B. Two technical replicates of each sample were performed in optimized gradient of 55 min. Mass spectra were acquired by Tune and Xcalibur software operating in data-dependent acquisition (DDA) mode, switching between full scan MS1 (60,000 resolution at 200 m/z, 100 ms accumulation time, AGC 1 × 106 ions, range 375–1800 m/z) and MS2 (15,000 resolution at 200 m/z, 200 ms accumulation time, AGC 105 ions, range 100–2000 m/z). MS2 spectra were obtained by HCD fragmentation of the 10 most intense ions using 30 normalized collision energy.
Peptide spectrum match (PSM) search was performed using Comet search engine from PatternLab for Proteomics v4.0 (Carvalho et al. 2016) against forward and reverse protein sequences from E. coli and B cenocepacia, downloaded from UniProtKB on January 31, 2017, and containing 34,214 entries. Carboxamidomethylation of cysteines was set as a fixed modification and oxidation of methionine was set as a variable modification. Semi-tryptic peptides were considered with a maximum of 3 missed cleavages and mass tolerance for MS1 was set at 40 ppm. Resultant peptides were processed and evaluated by Search Engine Processor (SEPro) from using the following filter parameters: 10 ppm deviation from theoretical peptide precursor, peptides longer than six amino acid residues, minimum number of peptides per protein 3, Delta CN of 0.001, and a 1% estimated protein-level FDR.
Enzymatic assays
XI activity was measured according to Brat et al. (2009). A solution containing 195 μg/mL of purified XI was incubated at the desired working temperature until the moment of use. Assays were performed in a quartz cuvette with 1 cm optical path. A reaction mixture containing 0.23 mM NADH, 10 mM MgCl2, 2 U/mL sorbitol dehydrogenase and 29.25 μg of XI in 100 mM Tris–HCl (pH 7.2) was fresh made for a final volume of 1 mL and incubated for 5 min at temperatures varying from 25 to 37 °C. The assay was performed in a quartz cuvette with 1 cm optical path. The reaction started with the addition of d-xylose and then followed for 30 min by measuring the oxidation of NADH at 340 nm until its complete oxidation to NAD+. Variations of d-xylose concentration from 10 to 500 mM were used to determine kinetic parameters. The protein theoretical isoelectric point (pI) was calculated using ProtParam Tool (http://web.expasy.org/protparam/) in order to determine the pH interval for activity measurement. From that, optimal pH was found by varying the buffer pH from 5.8 to 7.6. For pH values between 5.8 and 7.0, it was used a 100 mM Bis–Tris + 70 mM NaCl buffer. The ionic strength of each buffer was calculated for each condition to remain constant at 87 mM. Xylitol inhibition assay was done by measuring enzyme activity in the presence of 125 mM xylose and 10–50 mM xylitol at pH 7.2, 37 °C.
Protein tertiary structure prediction
Templates were chosen over the Protein Databank (PDB) based on their % identities and coverage to sequences using the BLAST tool (NCBI). For homology modeling, a multiple sequence alignment between target and templates were generated using MUSCLE (Edgar 2004) and MODELLER (Martí-Renom et al. 2000; Webb and Sali 2014) salign script. Alignments were manually checked for overall quality and edited when necessary. Modeling of the tertiary structures was performed by MODELLER v9.17 with 500 models generated. In order to choose the best model, Modeller’s objective function, DOPE score and Z-score (normalized DOPE) were used to pick the best models from the pool. Each model quality was assessed with PROCHECK for overall structure geometry, ERRAT for non-bonded interactions statistics and VERIFY 3D for compatibility of the atomic model of tertiary structure with its primary structure (Bowie et al. 1991; Lüthy et al. 1992; Laskowski et al. 1993). Identified anomalies in loop regions were fixed by running MODELLER’s Loop refining protocol. RMSD was calculated with PyMol v1.8.4.
Molecular dynamics and energy minimization
Structural models for B. cenocepacia, C. phytofermentans and Piromyces sp. were positioned to 1a0c, 1a0d, 1s5 m and 1s5n crystallographic structures. Cyclical d-xylose structure was obtained by modifying the glucose (removal of the methyl group and addition of H) from 1s5 m and transferring the coordinates to the constructed models. Water molecules from 1a0c crystal structure were preserved, except for HOH 494, 495, 496, 639 (chain A), 527, 528, 529, 671 (chain B), 525, 526, 527, 670 (chain C), 558, 559, 560, 702 (chain D) that were overlapping xylose atoms at the active site.
Amino acid side chain protonation state was determined according to PROPKA algorithm (Olsson et al. 2011) for pH 7.0. Structures were prepared with AMBER 14 program package (Simmerling 2015). Topology parameters for protein construction were obtained from AMBER 14SB force field. Xylose topology parameters were obtained from GLYCAM 06j-1 force field (Kirschner et al. 2008). The system was involved in an octahedral water box with 15 Å distance from sides and neutralized with Na+ or Cl− when necessary.
The model structures were relaxed before molecular dynamics simulation with the following protocol: (i) structural minimization with position restrictions at 100 kcal/mol for heavy atoms present on the protein and xylose; (ii) structural minimization with position restrictions at 10 kcal/mol for protein backbone and xylose heavy atoms; (iii) whole system minimization with weak positioned restrictions at 1 kcal/mol for the complex heavy atoms. Then, the system temperature was increased from 0 to 100 K with the canonical ensemble (NVT) for 20 ps. Next, it was used the isothermal-isobaric (NPT) ensemble from 100 to 29,815 K for 20 ps. Both were controlled with Berendsen temperature protocol (Berendsen et al. 1984). After the system heating, the system went through 250 ps at 1 kcal/mol for restriction density equilibration. Finally, the system went through another cycle of 250 ps for heavy atoms restriction equilibration at 0.1 kcal/mol. All production cycles were performed by approximately 20 ns.
Electrostatic surface and electric field vector \(\left( {\mathop {\text{E}}\limits^{ \to } } \right)\)
The file preparation for electric field calculations was done using the online software PDB2PQR (Dolinsky et al. 2004). The protein topology parameters were calculated using PARSE force field. Amino acid side chain protonation state was designated with PROPKA software at the following pH: 7.2 (B. cenocepacia) and 7.5 (C. phytofermentans and Piromyces sp.). Electrostatic field calculation and visualization were done on VMD v1.9.3 with APBS v1.4.1 plugin. Grid boxes were automatically calculated and kept at 298.15 K for C. phytofermentans and Piromyces sp. and 310.15 K for B. cenocepacia.
Statistical analysis
All values obtained during enzymatic assays were analyzed on GraphPad Prism 6 and were considered statistically different on a 95% confidence interval using ANOVA test. All assays were performed as two independent replicates with three technical replicates. Results presented are the mean values of the assay replicates with standard error values.