Computational protocol: Phosphorylation of bacterial-type phosphoenolpyruvate carboxylase at Ser425 provides a further tier of enzyme control in developing castor oil seeds

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Protocol publication

[…] Full-length cDNAs for AtPpc3 and RcPpc4 were cloned into a pET28b His-tag vector (Novagen) and transformed into Escherichia coli [BL21-CodonPlus (DE3)-RIL] (Stratagene) and recombinantly expressed as previously described []. The QuikChange® II site-directed mutagenesis kit (Stratagene) was used to generate desired mutations in RcPpc4 (S425D, P426A and R760A) and AtPpc3 (R644A). Oligonucleotide primer pairs used to introduce the mutations were designed using the SiteFind online software [] and obtained through Eurofins MWG Operon in HPLC purified form (primers are described in Supplementary Table S1 at http://www.BiochemJ.org/bj/433/bj4330065add.htm). A typical PCR amplification consisted of denaturing RcPpc4 or AtPpc3 in pET28b vector at 95 °C for 30 s followed by 16–18 cycles of 95 °C for 30 s, 55 °C for 30 s and 68 °C for 8.5 min []. After the prescribed DpnI treatment of PCR products, 0.5–2 μl was used to transform E. coli using electroporation. The positive clones selected on LB (Luria–Bertani) plates containing 50 μg/ml kanamycin were further screened for the desired mutation by restriction digestion and sequencing []. The PEPC coding portion in each plasmid from the confirmed clones was sequence verified. [...] PEPC activity was assayed at 25 °C by following NADH oxidation at 340 nm using a kinetics microplate reader (Molecular Devices) and the following optimized 200-μl assay mixture: 50 mm Hepes/KOH, pH 8.0, containing 10% (v/v) glycerol, 10 mm PEP, 5 mm KHCO3, 10 mm MgCl2, 2 mm dithiothreitol, 0.15 mm NADH and 5 units/ml of porcine muscle L-malate dehydrogenase (Roche). One unit of PEPC is defined as the amount of PEPC resulting in the production of 1 μmol of oxaloacetate per min. PEP saturation kinetic data for Class-2 PEPCs were fitted to both single and two active site models using non-linear regression analysis software (SigmaPlot Version 10.0; SPSS) as described previously [,]. Apparent I50 values (inhibitor concentration producing 50% inhibition of PEPC activity) were calculated using a non-linear least-square regression computer program []. All kinetic parameters represent means of at least four independent experiments and are reproducible to within ±15% S.E.M. of the mean value. Metabolite stock solutions were made equimolar with MgCl2 and adjusted to pH 7.0. Protein concentrations were determined by Coomassie Blue G-250 dye-binding using bovine γ-globulin as the protein standard []. […]

Pipeline specifications

Software tools SiteFind, SigmaPlot
Applications Miscellaneous, Genome annotation
Chemicals Phosphoenolpyruvate