| The regulation of plant cell growth and defense involves the insolubilization of hydroxyproline-rich glycoproteins (HRGPs), such as extensin, in the primary cell wall. In tomato (Solanum lycopersicum ), insolublization occurs by the formation of tyrosylcrosslinks catalyzed specifically by the pI 4.6 extensin peroxidase (EP). To date, neither the gene encoding EP nor the protein itself has been identified.;Here, tomato EP candidates were identified using both proteomic and bioinformatic approaches. Bioinformatic screening of the tomato genome yielded eight EP gene candidates, which encoded a putative signal sequence and a protein with a predicted pI near 4.6. Biochemical fractionation of tomato culture media followed by proteomic detection further refined our list of EP candidates to three, with the lead candidate designated a "CG5". The CG5 open-reading frame from a tomato cDNA was then cloned into a bacterial expression vector to test for EP crosslinking activity.;CG5 was expressed in E. coli, fractionated from inclusion bodies, and folded in vitro. Peroxidase activity of CG5 was assayed and quantified by the ABTS (2,2'- zinobis(3-ethylbenzothiazoline-6-sulphonic acid)) assay. Subsequent extensin crosslinking assays showed that CG5 can covalently crosslink authentic tomato P1 extensin and P3-type extensin analogs in vitro supporting the hypothesis that CG5 encodes a tomato EP. Recombinant CG5 was then purified for enzyme kinetics study which gave out a Km of 0.105 mM. A computational 3D model was generated for CG5 with highlighted active site and predicted heme group binding. |
