Abstract
GDP-mannose-epimerase (GME) catalyzes the inversion of configuration at carbon-3 and carbon-5 of D-mannose moiety, first committed step for L-ascorbate (Asc) biosynthesis branched off other metabolic pathways. The product, GDP-L-galactose is converted to Asc with four steps via L-galactose. The pathway is specific for plants and some algae species. Therefore, GME reaction is thought to be an important step.
We have isolated a cDNA clone encoding full coding sequence of GME from peach fruit. Transgenic tobacco plants those overexpress GME under the control of 35S promoter were generated. Overexpression of GME transcript was confirmed in 10 transgenic lines. In order to evaluate GME activity, [14C]-GDP-mannose was reacted with ammonium sulfate fractionated proteins, acid hydrolyzed and separated by silica TLC. Separation of radioactive spots were not uniform among preparations, resulting uncertainty in GME activity evaluation. Recombinant GME with HAT tag expressed in E. coli were similarly treated, and generation of [14C]-galactose was confirmed but the ratio of substrate:product was about 6:1. Asc contents in leaf tissue were similar to that in original line SR1.
