Catalytic properties of the plant cytochrome P450 CYP73 expressed in yeast. Substrate specificity of a cinnamate hydroxylase.

The catalytic properties of CYP73, a cinnamate 4-hydroxylase isolated from Helianthus tuberosus tuber [Teutsch, H. G., Hasenfratz, M. P., Lesot, A., Stoltz, C., Garnier, J. M., Jeltsch, J. M., Durst, F. & Werck-Reichhart, D. (1993) Proc. Natl Acad. Sci. USA 90, 4102-4106] and expressed in an optimised yeast system [Urban, P., Werck-Reichart, D., Teutsch, G. H., Durst, F., Regnier, S., Kazmaier, M. & Pompon, D. (1994) Eur. J. Biochem. 222, 843-850] have been investigated. Microsomes from transformed yeast catalysed trans-cinnamate hydroxylation with high efficiency. CYP73 was highly specific for its natural substrate, and did not catalyse oxygenation of p-coumarate, benzoate, ferulate, naringenin or furanocoumarins. No metabolism of terpenoids or fatty acids, known substrates of plant P450s, was observed. CYP73 however demethylated the natural coumarin herniarin into umbelliferone. In addition, it was shown to oxygenate five xenobiotics and mechanism-based inactivators, including the herbicide chlorotoluron. All substrates of CYP73 were small planar aromatic molecules. Comparison of the kinetic parameters of CYP73 for its various substrates showed that, as expected, cinnamate was by far the best substrate of this P450. The physiological and toxicological significance of these observations are discussed.

Mini-Mu transduction: cis-inhibition of the insertion of Mud transposons.

Mud (mini-Mu) transposons are defective phage Mu genomes that conserve the Mu ends. The transduction of Mud transposons is strictly dependent on Mu complementation, inefficient, and affected by modifications in the Mud internal sequences. The transduction of Mud transposons depends on transposition, which appears to be low, relative to wild-type Mu. Insertions of Mud into a plasmid can be frequently recovered among transductants; new Mud insertions into plasmids that already have both Mu ends, or just one, are rarely found. This suggests that the presence of Mu ends "immunizes" the plasmid against further insertion. This phenomenon may be similar to the transposition immunity of Tn3.