Source of Chemicals
All chemicals and snake venom nucleotide pyrophosphatase were purchased from Sigma-Aldrich Corporation (USA) unless otherwise mentioned. Calf intestinal phosphatase was obtained from New England Biolabs. LB medium was obtained from EMD Biosciences. Kanamycin, ampicillin and IPTG were purchased from LabScientific Inc. NTA resin was the NTA superflow by Qiagen. E. coli
BL21(DE3) competent cells were obtained from Invitrogen. The microcon membrane filters were from Millipore. TALON metal affinity resin was purchased from BD Biosciences. Analytical HPLC (Agilent 1100 instrument) was carried out using a Phenomenex Gemini C18 110A (150×4.6 mm, 5 µm ID) reverse phase column for thiochrome analysis, a Supelco LC-18-T (150×4.6 mm, 3 µm ID) column for thiazole reconstitution analysis and a Phenosphere Strong Anion-Exchange 80A (250×4.6 mm, 5 µm ID) column for the anion exchange chromatography. HPLC purifications were carried out using a semi-prep Supelco LC-18-T (250×10 mm, 5 µm ID) column. HPLC grade solvents were obtained from Fisher Scientific. The Superdex 200 gel-filtration column was obtained from Pharmacia. A previously synthesized stock of [1-13
was used as the substrate of the thiazole reconstitution reactions and previously synthesized cThz-P and cThz6
were used as reference compounds for the TenI catalyzed reaction.
Enzyme overexpression and purification
E. coli BL21(DE3) containing the ThiSG overexpression plasmid (ThiG is co-purified with ThiS for stability) in pET16b was grown in LB medium containing ampicillin (40 µg/mL) with shaking at 37 °C until the OD600 reached 0.6. At this point, protein overexpression was induced with IPTG (final concentration = 2 mM) and cell growth was continued at 15°C for 16 h. The cells were harvested by centrifugation and the resulting cell pellets were stored at −80°C. To purify the protein, the cell pellets from 1L of culture were resuspended in 25 mL lysis buffer (10 mM imidazole, 300 mM NaCl, 50 mM NaH2PO4, pH 8) and lysed by sonication (Heat Systems Ultrasonics model W-385 sonicator, 2 s cycle, 50% duty). The resulting cell lysate was clarified by centrifugation and the ThiSG protein was purified on Ni-NTA resin following the manufacturer’s instructions. After elution, the protein was desalted using a 10-DG column (BioRad) pre-equilibrated with 50 mM Tris-HCl buffer, pH 7.8. The remaining proteins ThiF (pET22), NifS (pET16), ThiO (pET22) ThiE (pQE32 and pREP4), TenI (pET28b), H122Q TenI (pET28b) and H122A TenI (pET28b) were overexpressed and purified in a similar manner. NifS, ThiO and ThiE were stored in aliquots at −80 °C in 20% glycerol. ThiSG, TenI and ThiF were purified immediately before use.
Reconstitution of the thiazole synthase catalyzed reaction on an analytical scale (in the presence and absence of TenI)
All solutions were made with 50 mM Tris buffer, pH 8. Final concentrations of the reactants are given in parentheses. Cysteine (0.35 mM), DTT (0.70 mM), ATP (0.60 mM) and MgCl2 (3.5 mM) were incubated with purified ThiSG (1.25 µM), ThiF (1.24 µM) and 70 µL NifS (1.38 µM) for 1.5 hours. Total volume of this solution was 425 µL. Glycine (6.50 mM), DXP (0.33 mM), MgCl2 (3.5 mM) and ThiO (6.8 µM) were then added to this reaction mixture and the final volume of the reconstitution mixture now was 610 µL. TenI was added in the reconstitution reaction to a final concentration of 10 µM to check for the acceleration of the rate of thiazole formation. In a control reaction set up in exactly the same way, the same volume of buffer was added into the reaction instead of TenI. This mixture was incubated for an additional 2 hours. The reaction mixture was then analyzed for product formation using the thiochrome assay (see below). In this reconstitution, 16% of the DXP was converted to product. This is a 3-fold improvement over our previously reported reconstitution, and corresponds to about 12 turnovers by the thiazole synthase.
The thiochrome assay involves conversion of the thiazole product of the reconstitution to thiamin phosphate 18 and further to thiochrome phosphate. The product of the thiazole reconstitution is reacted with HMP-PP 17 (0.5 mM) in the presence of thiamin phosphate synthase (ThiE) (1.00 µM). The reaction is allowed to stand at room temperature for 2 hours and then quenched with an equal volume of 10% TCA. Potassium acetate (50 µL of 4M) is added to 100 µL of the quenched reaction followed by oxidative cyclization to thiochrome phosphate using 50 µL of a saturated solution of K3Fe(CN)6 in 7M NaOH. The oxidation reaction is neutralized after 1 minute with 6M HCl and analyzed by reverse phase HPLC with fluorescence detection (excitation at 365 nm, emission at 450 nm). The following linear gradient, at a flow rate of 1 mL/min, was used. Solvent A is water, solvent B is 100 mM K2HPO4, pH 6.6, solvent C is methanol. 0 min: 100% B; 2 min: 10% A, 90%B; 10 min: 25% A, 15% B, 60% C; 12 min: 25% A, 15% B, 60%; 15 min: 100% B; 17 min: 100%B.
25 mL of assay solution containing 4 mM phenol, 100 mM 4-amino-antipyrene and 2 units/mL HRP was made. To 500 uL of the assay solution, 10 mM, 5 mM, 1 mM, 500 µM, 250 µM and 100 µM glycine was added and the volume each time was diluted to 505 uL. In each case the reaction was initiated by the addition of a final concentration of 6.6 µM ThiO and 10 µM TenI. A parallel set of reactions was similarly run in the absence of TenI. The rate of glycine oxidation was measured, at various glycine concentrations, by monitoring the absorbance change at 500 nm for 600sec.
437 µM of Thz-P and 485 µM HMP-PP were mixed with 10 µM ThiE and 10 µM TenI in a final volume of 700uL of 50 mM Tris-HCl buffer, 2 mM MgCl2, pH 7.8. An identical reaction lacking TenI was run under the same conditions. 100 µL aliquots of each reaction mixture were quenched after 0 min, 0.5 min, 1 min, 2 min, 5 min, 10 min and 20 min and oxidized to thiochrome phosphate. Reaction mixtures were analyzed by HPLC with fluorescence detection.
To evaluate the effect of TenI on ThiG, the thiazole reconstitution reaction was run using preformed ThiS-COSH in the presence and absence of TenI. Cysteine (0.35 mM), DTT (0.70 mM), ATP (0.60 mM) and MgCl2 (3.5 mM) were incubated with purified ThiSG (1.25 µM), ThiF (1.24 µM) and NifS (1.38 µM) for 1.5 hours. The total volume of this solution was 425 µL. Desalting through a 10-DG column (BioRad) pre-equilibrated with 50 mM Tris-HCl, pH 7.8 yielded ThiS-COSH. Two sets of the thiazole reconstitution reactions were carried out as described above, one in the presence of TenI and the other in its absence. 100 µL aliquots of these reaction mixtures were quenched at time points of 0 min, 1 min, 2 min, 5 min and 10 min and oxidized to thiochrome phosphate which was detected by reverse phase HPLC with fluorescence detection.
Activity assay for TenI with cThz*-P
cThz*-P was obtained as described in Supplementary Information
( and , Supplementary Information
). 1 µM TenI was added to 100 µM cThz*-P in a final assay volume of 300 µL of 50mM Tris buffer, pH 7.6. The mixture was incubated for 120 min, filtered using a 10 kDa MW cut-off filter, and analyzed by HPLC. Mutants were similarly analyzed except the reaction time was increased to 12 hours.
HPLC conditions for separation of cThz*-P and cThz-P using analytical strong anion exchange chromatography
The following linear gradient, at a flow rate of 1 mL/min, on the Phenosphere Strong Anion-Exchange 80A (250×4.6 mm, 5 µm ID) column was used: solvent A is water, solvent B is 100 mM triethylammonium acetate, pH 7.8. 0 min: 100% A; 1 min: 100% A; 4 min: 100% B; 7 min: 100% A; 10 min: 100%A.
HPLC conditions for separation of cThz* and cThz using analytical reverse chromatography
The following linear gradient, at a flow rate of 1 mL/min, on the Supelcosil LC-18-T column (150×4.6 mm, 3 µm ID) was used: solvent A is water, solvent B is 100 mM KPi, pH 6.6, solvent C is methanol. 0 min: 100% B; 2 min: 100% B; 4min 10% A, 90%B; 9 min: 10% A, 25% B, 65% C; 14 min: 10% A, 25% B, 65% C; 16 min: 100%B; 20min 100%B.
Equilibration of cThz-P/CThz*-P in the presence of TenI
540 µM TenI in 50mM KPi pH 7.6 in 50% D2O: H2O was added to 540 µM cThz-P (TenI:cThz-P ratio approximately 1:1) in a total volume of 650 µL. The reaction mixture was analyzed by NMR after 12 hours.
Expression and Purification of Bacillis subtilis TenI for crystallography
The TenI overexpression strain was grown at 37 °C with vigorous agitation (200 rpm) LB medium containing 30 µg/mL kanamycin to an OD600 of 0.7, at which point the cells were induced with 500 µM IPTG and allowed to incubate overnight at 22 °C under conditions of mild mixing (180 rpm). The cells were harvested by centrifugation (6,000 g) for 15 min at 4 °C and stored at −80 °C for later use. All purification steps were carried out at 4 °C. The cell pellet was suspended in 50 mL binding buffer (50 mM sodium phosphate, pH 7.0 and 300 mM NaCl), and lysed by sonication. The crude extract was centrifuged at 4 °C for 30 min at 50,000 g and the resulting supernatant was augmented with 5 mM imidazole and loaded onto a column containing 2 mL of TALON metal affinity resin equilibrated with 50 mL binding buffer. The column was washed with 20 column volumes of binding buffer plus 5 mM imidazole, followed by 5 column volumes of binding buffer plus 10 mM imidazole. The His6-tagged TenI was eluted from the column with elution buffer (50 mM sodium phosphate, pH 7.0, 300 mM NaCl and 300 mM imidazole). The recombinant TenI was further purified on a Superdex 200 gel-filtration column and eluted in the storage buffer (25 mM Tris-HCl, pH 8.5, 150 mM NaCl, and 1 mM thiamin-phosphate). The fractions containing pure TenI were combined and concentrated to 12 mg/mL using a 10 kDa cutoff concentrator (Vivaspin) and stored at −80 °C. Protein concentration was determined by the Bradford method with bovine serum albumin as the standard. The purity of TenI was determined by SDS-PAGE analysis and found to be greater than 99%.
Crystallization of TenI with the cThz-P 16 bound in the active site
Crystals of ligand free TenI were grown from 1.65 – 1.75 M ammonium sulfate, 100 mM bicine, pH 8.7 – 9.6, 2% PEG400 (w/v), and 8 mM L-cysteine using the hanging drop vapor diffusion method.12
To obtain the product complex, the crystals were first dialyzed into 2.38 M sodium malonate (pH 7.0) (Hampton Research) to remove the sulfate ions, by gradually increasing the sodium malonate concentration and decreasing the ammonium sulfate concentration in 30 steps with 5 min incubation for each step. Subsequently the crystals were soaked overnight in 2.38 M sodium malonate, 21.5 mM bicine (pH 9.0), 0.01% PEG400 (w/v), 0.5 mM L-cysteine, 4% glycerol and 11.5 mM cThz-P, followed by flash freezing in liquid nitrogen.
X-ray Data Collection and Processing
The X-ray intensity data from the TenI-cThz-P complex were measured at the A1 beamline of the Cornell High Energy Synchrotron Source (CHESS) using a Quantum 210 CCD detector (Area Detector Systems Corp.). Data were collected over 180° using a 10 s exposure time and 1° oscillation per frame with a crystal to detector distance of 200 mm. The data were integrated and scaled using HKL2000.23
Structure Determination and Refinement
The structure of TenI-cThz-P complex was determined by Fourier synthesis using the previously reported TenI structure (PDB: 1YAD) as the starting model. The first round of rigid-body refinement with the starting model by REFMAC524
reduced the R
-factor to 0.286 (Rfree
0.294). cThz-P was modeled in based on clear electron density. The model was refined through iterative cycles of restrained refinement by REFMAC5 and PHENIX25
, and manual rebuilding in COOT.26
Refinement statistics are shown in Table 1, Supplementary Information
Modeling of cThz*-P in the active site
The initial coordinates of proposed intermediate cThz*-P were 26
generated using program SKETCHER of CCP4 suite.27
Using the crystal structure of TenI complexed with Thz-P as the template, the intermediate cThz*-P was docked into the active site and the key interacting residues were adjusted manually in COOT.26