Characterization of a class II 5-enopyruvylshikimate-3-phosphate synthase with high tolerance to glyphosate from Sinorhizobium fredii.

Abstract

5-Enopyruvylshikimate-3-phosphate synthase (EPSP synthase) is an important enzyme in the shikimate pathway mediating the biosynthesis of aromatic compounds in plants and microorganisms. A novel class II EPSP synthase AroA S. fredii from Sinorhizobium fredii NGR234 was overexpressed in Escherichia coli BL21. It was purified to homogeneity and its catalytic properties were studied. The enzyme exhibited optimum catalytic activity at pH 8.0 and 50 °C. It was stable below 40 °C, and over a broad range of pH 5.0-9.0. The EPSP synthase was increasingly activated by 100 mM of the chlorides of NH4 (+), K(+), Na(+) and Li(+). Kinetic analysis of AroA S. fredii suggested that the enzyme exhibited a high glyphosate tolerance and high level of affinity for phosphoenolpyruvate, which indicates the enzyme with a high potential for structural and functional studies and its potential usage for the generation of transgenic crops resistant to the herbicide.