Purification and Partial Characterization of the Streptomyces viridochromogenes TÜ494 Phosphinothricin-N-acetyltransferase Mediating Resistance to the Herbicide Phosphinothricin in Transgenic Plants

A purification scheme for the enzyme phosphinothricin-N-acetyltransferase (PAT) origi­ nating from Streptomyces viridochromogenes {pat-gene product from Streptomyces virido­ chromogenes) and mediating herbicide resistance to transgenic plants was developed. The enzyme was isolated from a transformed and overproducing Escherichia coli strain. With a combination of ammonium sulfate fractionation, chromatography on DEAE-Sephadex A50-, Phenylsepharose-, Hydroxylapatiteand FPLC-Superose 12-columns it was possible to obtain PAT which was at least 90 % homogeneous on the basis of SDS-PAGE. The properties of the isolated PAT were compared with the properties of PAT from S. hygroscopicus (bargene product from S. hygroscopicus) previously isolated and characterisized by Botterman, J., Gossele, V., Thoen, C., Lauwereys, M. (1991), Gene 102, 33-37. Differences were observed in the molecular masses of the two native enzymes (PAT from S. viridochromogenes being a dimer of 40 kD and PAT from S. hygroscopicus being a monomer of 21 kD), and in the temperature sensitivity of the two enzymes (the PAT from S. viridochromogenes being slightly more temperature stable than PAT from S. hygroscopicus). However, since the pat and the bar-gene are to 85 % homologous, substantial similarities exist between the two enzymes especially in the kinetic values and the substrate specificity. The isolated S. virido­ chromogenes PAT did not acetylate putative substrates present in the plant cell. Antibodies were raised against the isolated protein. This antiserum was able to detect PAT in transgenic plants and therefore is suitable to analyse the fate of the protein in such plants under various stress conditions.