CHARACTERIZATION OF A MORPHINONE REDUCTASE HOMOLOGUE IN PSEUDOMONAS SYRINGAE PV. GLYCINEA
B ROHDE1, R SCHMID2 and M ULLRICH1
1Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, Germany; 2FB5-Mikrobiologie, Universität Osnabrück, Germany
Background and objectives
Results and conclusions
A PCR screening with different Pseudomonas strains was carried out using primers generated from the morB sequence of Pseudomonas syringae pv. glycinea. To confirm positive strains, the PCR products were blotted on nitrocellulose membranes and subjected to Southern blot analysis. It revealed that the morB gene is widespread among P. syringae pv. glycinea strains, but not in other pathovars of P. syringae. The morB gene was cloned into pMal-C2 and overexpressed in E. coli cells. The MBP-MorB fusion protein was purified by affinity chromatography on amylose resin. The purified fusion protein was then cleaved by Factor Xa protease, separated by SDS-PAGE, and the 40-kDa MorB protein was used to immunize rabbits. So far, a substrate for the MorB-homologue could not be identified. As shown for morphinone reductase of P. putida, enzymatic activity was measured with morphinone, codeinone, neopinone and 2-cyclohexen-1-one as substrates . Morphinone belongs to the alkaloids. Several alkaloids and phytoalexins are produced by plants and possess antimicrobial activity. P. syringae might have developed a defence mechanism, in which MorB plays an important role in the detoxification of antimicrobial agents. Other possible groups of compounds which might act as substrates are structurally related phytoalexins, chalcones and flavones. The enzymatic characterization is currently investigated.