Restriction enzymes were purchased from New England Biolabs. KlenTaq-LA DNA Polymerase Mix was purchased from Sigma. Ni-NTA resin was from Qiagen. MEP was obtained from Echelon Biosciences, Inc. IPTG were from USB. ATP, CTP were obtained from Sigma. γ-[32P] ATP and α-[32P] CTP was from NEN. All other chemicals were purchased from Sigma-Aldrich. TLC plates (Polygram Sil N-HR) were purchased from Macherey & Nagel. ImageQuant 5.2 (Molecular Dynamics) was used to quantify the radioactivity on TLC plates.
PCR amplification of Agrobacterium tumefaciens IspDF from genomic DNA (ATCC33970D) was performed with primers CL0610DFC58sen and CL0610DFC58ant and Klen Taq polymerase according to the following procedure: 94°C (3 min), 61°C (2 min) for 1 cycle, following by 68°C (2 min), 94°C (45 s), 61°C (45 s), for 30 cycles (). The PCR product was purified using the GFX Gel Band Purification Kit and ligated into pGEM-Teasy with T4 ligase (Promega). The resulting plasmid was transformed into JM109 cells, and positive colonies were detected by blue/white screening in presence of IPTG and X-Gal. Constructs possessing the correct insert were chosen based upon restriction digests with NdeI and BamHI and were confirmed by automated DNA sequencing (University of Utah Core Facility). The NdeI/BamH1 insert was subcloned into the NdeI and BamHI sites of pET-15b resulting in an expression clone containing an N-terminal His6-tag sequence. The desired construct, pET15b/AtDF, was used to transform electrocompetent XL1-blue cells. Positive constructs based upon restriction enzyme analysis with NdeI and BamHI were sequenced.
Oligonucleotides used in this study
IspE was obtained by PCR amplification using primers CL0429EC58sen and CL0429EC58ant and Agrobacterium tumefaciens genomic DNA (ATCC33970D) using the following PCR settings: 94°C (2 min), 61°C (2 min) for 1 cycle, following by 68°C (2 min), 94°C (30 s), 61°C (30 s), for 30 cycles. The IspE expression clone was obtained using a procedure like that described above for IspDF.
The IspE(D152A) site directed mutant was obtained using the Quick Change kit from Stratagene. The mutated gene was amplified using primers AMAISPE007 and GC-AMAISPE007 () to give pET15b/AtE(D158A), which was then sequenced to verify the mutation.
Expression of IspDF, IspE and IspE(D152A) and Protein Purification
BL21(DE3)pLysS was transformed with pET15b/AtDF, pET15b/AtE and pET15b/AtE(D152A) and 5 mL LB cultures (100 μg/mL ampicillin) of the transformants were grown overnight at 37° C, with shaking. Four cultures (two for each enzyme), each containing 1 L of LB, 100 μg/mL ampicillin, were inoculated with 2 mL of the overnight culture and were grown at 37° C, with shaking at 250 rpm, until OD600 = 0.6. IPTG was then added to 1 mM (final concentration), incubation was continued for 5.5 h, and cells were harvested by centrifugation and stored at -80°C.
Cell paste was resuspended in 50 mM sodium phosphate, pH = 8.0, 300 mM NaCl, 10 mM imidazole (lysis buffer). Complete Protease Inhibitor Cocktail Tablets (Roche) and lysozyme (1 mg/mL) were added, the cells were lysed by sonication, cellular debris was removed by centrifugation, and the His-tagged enzymes were purified by chromatography on Ni-NTA agarose (Qiagen).
(2.5 mM final concentration) was added to the solutions of His6
-tagged IspDF, IspE
, each containing a thrombin cleavage site. Thrombin (50 units) was added to 6 mg of each protein in elution buffer (50 mM sodium phosphate, pH = 8.0, 300 mM NaCl, 250 mM imidazole) at 4°C. After 12 h, thrombin was removed by adding benzamidine-agarose (Sigma). The samples were filtered and dialyzed with the lysis buffer to reduce the imidazole concentration below 10 mM. Finally, Ni-NTA resin was added, and unproteolyzed proteins and the His6
fragment were removed by Ni-NTA chromatography. The flow through fractions were concentrated with a Centriprep-10 unit (Centricon) and dialyzed against 0.1 mM Tris·HCl pH 7.6 (measured at 37°C) using a 10,000 molecular weight cut-off dialysis cassette (Pierce). The protein concentrations were determined by the Bradford method12
Sedimentation velocity experiments were performed using a Beckman XL-1 analytical ultracentrifugation with absorbance optical detection. Three different concentrations of purified wild-type IspE and IspE(D152A) in 25 mM HEPES, pH 7.5 and 100 mM NaCl were used for sedimentation studies. Protein samples were centrifuged at a rotor speed (An50 Ti) of 42,000 rpm, and absorbance data at 280 nm were collected. Analyses of the sedimentation velocity data were performed using the program SEDFIT13
Enzyme Generated Products
Enzymatic reactions were carried out at 37°C in 0.1 mM Tris·HCl buffer, pH 7.6, containing 5 mM DTT, 10 mM MgCl2, 150 μM CTP, 150 μM ATP, 500 μM racemic MEP, 0.24 μM IspDF, 0.31 μM IspE and varying concentrations (0, 0.3, 0.6, 1.4, 3.1, 6.3, 12.5 μM) of IspE(D152A) in a final volume of 50 μL. [32P]NTPs were diluted from 5 mM stock solutions of 40 μCi/μmol CTP and 320 μCi/μmol ATP. In control experiments, samples were pre-incubated for 5 min and 20 min, respectively, in the presence of all substrates, except for radio-labeled ATP and CTP, or enzyme. Reactions were initiated by addition of CTP. After 15 min, the reactions were quenched with 50 μL of methanol and were put on ice. TLC analysis (Polygram Sil N-HR; Macherey & Nagel) was performed by spotting 3.5 μL of the reaction mixture and developing the plates with n-propanol / ethyl acetate / H2O (6:1:3, v/v/v). Radioactivity was quantified with a Molecular Dynamics Typhoon 8600 Phosphorimager.
Time Course Studies
A solution of 500 μM MEP (racemic), 0.24 μM IspDF and 0.31 μM IspE in 0.1 M Tris·HCl buffer, pH 7.6 (37°C), containing 5 mM DTT, in a final volume of 150 μL was pre-incubated for 10 min at 37°C. ATP (150 μM) and α-[32P]CTP (150 μM, 40 μCi/μmol) were added sequentially to initiate the reaction. At various times, 6 μL portions of the mixture were removed and quenched with 6 μL of methanol. After 61 min, additional 0.5 μg portions of each enzyme were added to the reaction mixture. Identical reactions were run in presence of 3.1 μM IspE or a mixture of 3.1 μM IspE and 31 μM IspE(D152A). After 61 min, 0.5 μg of IspDF and 0.5 μg of IspE were added to the reaction mixtures. The reactions were repeated in 0.1 M Tris·HCl buffer containing 30% (v/v) glycerol at pH 7.6 (37°C) with 3.12 μM IspE or 3.12 μM IspE and 31 μM IspE(D152A). After 61 min, 0.5 μg of IspDF and 0.5 μg of IspE were added to the reaction mixtures.