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Abstract

ABSTRACT:The Pseudomonas aeruginosa aroA gene encodes an enzyme called 5-enol-pyruvylshikimate-3-phosphate (EPSP) synthase, which is the primary target of the herbicide glyphosate. We have amplified and cloned the aroA gene from the P. aeruginosa genomic DNA and subcloned it into a vector suitable for high level expression of a recombinant form of this enzyme in Escherichia coli. The E. coli transformed with the resulting plasmid, pTrcPA, produced the EPSP synthase in large quantities, which allowed it to be purified to homogeneity. Furthermore, site-directed mutants of P. aeruginosa ESPS synthase have been constructed in order to compare in vitro glyphosate sensitivity between the wild-type and the mutant enzymes. The k cat and K m values for substrates in both forward and reverse reactions were obtained from both wild-type and mutant EPSP synthases.Abbreviations: IPTG, isopropyl-b-D-thiogalactopyranoside; SDS, sodium dodecyl sulfate; S3P, shikimate-3-phosphate; PEP, phosphoenolpyruvate.