Raymonde Fonne-Pfister

Hydroxylation of primisulfuron by an inducible cytochrome P450-dependent monooxygenase system from maize

Abstract Microsomes were prepared from etiolated maize seedlings and incubated with [14C]primisulfuron (2-[3-(4,6-bis(difluoromethoxy)-pyrimidin-2-yl)-ureidosulfonyl]-benzoic acid methylester). Two enzymatic reaction products were formed in the presence of O2 and NADPH. Comparison on high-performance liquid chromatography with synthetic reference standards and mass spectrometry of the two in vitro metabolites revealed that [14C]primisulfuron was hydroxylated at two different sites, i.e., at the phenyl and at the pyrimidine ring, respectively. Both hydroxylation reactions were inhibited in vitro by tetcyclacis as well as by CO in the presence of O2. CO inhibition was reversed by irradiation of the reaction mixture with white light. Enzyme activities were localized predominantly in the shoots. Apparent Km values for [14C]primisulfuron were estimated to be 137 and 47 μM, and Vmax to be 427 and 261 pmol/hr/mg protein, for the hydroxylation on the pyrimidine and phenyl ring, respectively. Formation of these metabolites from [14C]primisulfuron was barely detectable in microsomes from germinating seedlings. However, seed treatment (0.2% w w ) with the safener, CGA 154281 (4-[dichloroacetyl]-3,4-dihydro-3-methyl-2H-1,4-benzoxazine), increased microsomal cytochrome P450 levels twofold and dramatically stimulated in vitro [14C]primisulfuron phenyl- and pyrimidine-ring hydroxylation. From these data it is concluded that hydroxylation of primisulfuron in maize microsomes is catalyzed by an inducible cytochrome P450 monooxygenase system.

Herbicide-insecticide interaction in maize : malathion inhibits cytochrome P450-dependent primisulfuron metabolism

Abstract Tolerance of maize to sulfonylurea herbicides such as primisulfuron has recently been reported to be impaired by the use of some organophosphorus insecticides. In an effort to elucidate the mechanism of this interaction, the effect of the insecticide, malathion, on the metabolism of primisulfuron was studied in whole plants, in excised leaves, and in a microsomal in vitro system from maize. Foliar application of malathion to 7-day-old plants had no influence on leaf uptake and translocation of primisulfuron, but caused a decrease in the rate of herbicide metabolism. In excised leaves, malathion increased the metabolic half-life of primisulfuron. In microsomal preparations, malathion inhibited cytochrome P450-dependent primisulfuron phenyl- and pyrimidinering hydroxylation. Loss of primisulfuron phenyl-ring hydroxylase activity was time-dependent, saturable with respect to malathion concentration, and attenuated in the absence of NADPH. The kinetic data suggest a mechanism-based cytochrome P450 inactivation by malathion. The oxoanalogue of malathion, malaoxon, did not influence the metabolic half-life of primisulfuron in excised leaves and was a poor inhibitor of microsomal primisulfuron hydroxylation. Neither insecticide had any effect in vitro on total microsomal cytochrome P450 content. From the present results it may be concluded that malathion affects primisulfuron tolerance of maize due to the inhibition of cytochrome P450 monooxygenases involved in herbicide metabolism.

Ring-methyl hydroxylation of chlortoluron by an inducible cytochrome P450-dependent enzyme from maize

Abstract An enzyme catalysing ring-methyl hydroxylation of the substituted phenylurea herbicide chlortoluron was detected in isolated microsomes from germinating maize. Chlortoluron hydroxylation was absolutely dependent on NADPH and molecular oxygen. The enzyme was inhibited by carbon monoxide in the presence of oxygen and this inhibition could be reversed by light. Several known inhibitors of cytochrome P450 enzymes inhibited chlortoluron hydroxylation to varying degrees. It is concluded that the newly detected enzyme is a cytochrome P450-dependent mixed function oxidase. Enzyme activity was stimulated 15-fold by treatment of maize seeds with the herbicide antidote CGA 154281 [4-(dichloroacetyl)-3,4-dihydro-3-methyl-2H-1,4-benzoxazine].

An Enzyme‐Bound Bisubstrate Hybrid Inhibitor of Adenylosuccinate Synthetase

Two relatively weak herbicides, hydantocidin phosphate and hadacidin were linked by a C(3) chain to afford a potent inhibitor (the 2S hybrid is shown) of the enzyme adenylosuccinate synthetase. The crystal structures of the bisubstrate-enzyme complexes were determined.