Detoxification of the phytoalexin pisatin by a fungal cytochrome P-450

The fungus Nectria haematococca, a pathogen of garden pea (Pisum sativum), can demethylate pisatin, an antimicrobial compound synthesized by infected pea tissue. The phenolic product is less toxic than pisatin to many microorganisms. Cell extracts catalyzing pisatin demethylation were obtained from N. haematococca, and the properties of the reaction were examined. The enzyme activity was greatest in the high-speed pellet fraction, in which rates up to 20 nmol/min/mg protein were observed. The K_m for pisatin was relatively low, less than 5 μm. The reaction was dependent on NADPH, which could not be replaced by any other cofactor tested. However, in the presence of NADPH, NADH increased the rate of demethylation. Oxygen uptake by the enzyme was stimulated by addition of pisatin, the increment of oxygen utilization being approximately equimolar with pisatin added. Formaldehyde was a product of the reaction. The effects of various inhibitors were tested to determine whether this reaction is mediated by cytochrome P-450. The respiratory inhibitors KCN (1 mm) and antimycin A strongly inhibited the demethylation of pisatin by intact cells of the fungus, but not by the NADPH-supplemented enzyme. The cytochrome P-450 inhibitors SKF 525-A and 1-(2-isopropylphenyl)imidazole inhibited demethylation both in whole cells and in the enzyme preparation, though the latter compound was effective only at high concentrations. Most other cytochrome P-450 inhibitors tested had little effect. However the reaction was quite sensitive to CO, and this inhibition was readily reversed by light at wavelengths near 450 nm. It is concluded that pisatin demethylase is a cytochrome P-450 monooxygenase.