Cytochrome P450-Dependent Hydroxylation of Prosulfuron (CGA 152005) by Wheat Seedling Microsomes

Microsomes isolated from shoot tissues of etiolated wheat seedlings (Triticum aestivum L. var. Olaf) oxidized the sulfonylurea herbicide prosulfuron (CGA 152005). One major and two minor enzymatic oxidation products were isolated and identified by negative ion FAB/MS and cochromatography (TLC and HPLC) with reference standards. Identification of the major oxidation product (I) as 1-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)-3-[2-(3,3,3-trifluoropropyl)-5-hydroxyphenylsulfonyl]urea was confirmed by proton NMR spectroscopy. Minor oxidation products were tentatively identified as 1-[4-(hydroxymethyl)-6-methoxy-1,3,5-triazin-2-yl]-3-[2-(3,3,3-trifluoropropyl)phenylsulfonyl]urea (II) and an intermediate oxidation product 1-[4-[(hydroxymethyl)oxy]-6-methyl]-1,3,5-triazin-2-yl]-3-[2-(3,3,3-trifluoropropyl)phenylsulfonyl]urea (III) that degraded to 1-(4-hydroxy-6-methyl-1,3,5-triazin-2-yl)-3-[2-(3,3,3-trifluoropropyl)phenylsulfonyl]urea (IV). Microsomal oxidation of prosulfuron required NADPH and molecular oxygen. Constitutive enzyme activity was increased 5−28-fold by induction with ethanol and with the herbicide safeners naphthalic anhydride or cloquintocet-mexyl (CGA 185072) and by combinations of naphthalic anhydride or cloquintocet-mexyl with ethanol. Inhibition of enzyme activity by CO in the dark was reversible by light. Other inhibitors of prosulfuron oxidation included tetcyclacis, piperonyl butoxide, cytochrome c, polyclonal antibodies raised against wheat cytochrome c (P450) reductase, and the herbicides bifenox and linuron. Kinetic studies established that the apparent K m for prosulfuron was 12.6 ± 1.1 μM and that bifenox and linuron were noncompetitive and mixed-type inhibitors of prosulfuron oxidation, respectively, with apparent K i values of 210 and 59 μM. Keywords: Wheat; microsomes; prosulfuron oxidation; cytochrome P450