ScienceAsia 33 (2007): 153-160 |doi: 10.2306/scienceasia1513-1874.2007.33.153

Isolation and Purification of Recombinant EPSP Synthase from the Pathogenic Bacterium Pseudomonas aeruginosa

Pilanee Vaithanomsat^a* and Katherine A. Brown^b

 
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.

KEYWORDS: EPSP synthase; P. aeruginosa; Isolation; Purification.

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^a Kasetsart Agricultural and Agro-Industrial Product Improvement Institute, Kasetsart University, Bangkok, Thailand.
^b Department of Biochemistry, Imperial College of Science Technology and Medicine, Exhibition Road, London SW7 2AY, United Kingdom.

* Corresponding author, E-mail: p_vaithanomsat@yahoo.com

Received 24 Apr 2006, Accepted 23 Aug 2006