PURIFICATION AND CHARACTERIZATION OF 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE FROM MAIZE

The proposed target enzyme for benzoylcyclohexanedione herbicides, 4-h ydroxyphenylpyruvate dioxygenase (HPPD) was purified from etiolated ma ize seedlings with a purification factor of 105. Enzyme activity was m easured by detection of carbon dioxide formed from radiolabelled subst rate. The enzyme has a pH optimum of 7 . 3 and an apparent molecular m ass of 43 kDa, similar to that of the mammalian liver enzyme. Activity needs the presence of a reducing system glutathione/dichlorophenol in dophenol or ascorbate and catalase. Surprisingly, a commercial catalas e preparation of low specific activity-generally used for the enzyme a ssay-showed HPPD activity which was separable from the catalase activi ty on a gel filtration column. According to kinetic studies with purif ied maize HPPD, experimental herbicides from the family mentioned were strong competitive inhibitors of the plant enzyme in nanomolar range with K-i values of 5 and 15 nM for 2-(2-nitro-4-chlorobenzoyl)-5-(2-me thoxyethyl) cyclohexane- 1,3-dione and 2-(2-chloro-4-methanesulfonylbe nzoyl)- cyclohexane-1,3-dione (SC-0051; sulcotrione), respectively.