Characterization Of The Functional Domains Among Class II EPSP Synthases And Microarray Analysis Of The Glyphosate Stress Response In Escherichia Coli

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is an attractive target for drugs and herbicides. Here we identified a novel RPMXR motif that is strictly conserved among class II EPSP synthases. Site-directed mutational analysis of this motif showed that substitutions of the four strictly conserved amino acid residues, Arg127, Pro128, Met129, and Arg131, resulted in complete loss of enzymatic activity, whereas changes in the non-conserved Asn130 residue strongly influenced glyphosate resistance (all numbering according to Pseudomonas stutzeri A1501 EPSP synthase). These experimental results, combined with 3D structure modeling of the location and interaction of the RPMXR motif with phosphoenolpyruvate (PEP) and shikimate-3-phosphate (S3P), demonstrate that the novel motif is required for enzymatic activity and glyphosate resistance of class II EPSP synthases.Glyphosate is one of the most widely used herbicides in cereal-growing regions worldwide. Noted for its broad effectiveness on competing vegetation, mild effect on conifers, rapid inactivation in soil, and low mammalian toxicity. Benefits of herbicide use must be viewed cautiously, however, environmental risks in effects of glyphosate shock on soil microbes has remained obscure. Firstly, there is a lack of knowledge on the class II EPSP synthase expression patterns in Escherichia coli. Here, we investigate the transcriptome using Affymetrix GeneChip. Expression profling revealed 19 up-regulated and 142 down-regulated genes. Class II aroA, a functional gene, which is essential for the synthesis of aromatic amino acids and many secondary metabolites, did not invasive expression changing. The microarray results, combined with free amino acid assay and BioLog results, suggested that a number of Serine involved in synthesis and transportor were significantly regulated by the expression of class II aroA gene. Secondly, a need exists to analyze the glyphosate-induced expression profiling changes in Escherichia coli, in order to gain a better understanding of the positive responses of glyphosate-responsive expression for future studies. A total of 127 differentially expressed genes were identified from the glyphosate shock sample, wherein 58 were up-regulated and 69 were down-regulated. Among the differentially expressed genes of known function, those encoding amino acid metabolism and transportor accounted for the largest proportion, consistent with observation that glyphosate blocks the shikimate pathway.In this work, we found a new conservative motif, and identified the functional motif, enzyme activity and glyphosate resistance which related to develop the commercial application of this enzyme . The gene expression profiling of E. coli exposed to glyphosate was analyzed to investigate the alternative effects of glyphosate on E. coli genome. The response mechanisms of glyphosate provide an important basis for effects of glyphosate application on soil microbes.