Purification and Characterization of 4-Hydroxyphenylpyruvate Dioxygenase from Maize

✍ Scribed by Barta, Istvan Cs.; Böger, Peter

• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 794 KB
• Volume: 48
• Category: Article
• ISSN: 1526-498X
• DOI: 10.1002/(sici)1096-9063(199610)48:2<109::aid-ps447>3.0.co;2-7

✦ Synopsis

The proposed target enzyme for benzoylcyclohexanedione herbicides, 4-hydroxyphenylpyruvate dioxygenase (HPPD) was purified from etiolated maize seedlings with a purification factor of 105. Enzyme activity was measured by detection of carbon dioxide formed from radiolabelled substrate. The enzyme has a pH optimum of 7.3 and an apparent molecular mass of 43 kDa, similar to that of the mammalian liver enzyme. Activity needs the presence of a reducing system glutathione/dichlorophenol indophenol or ascorbate and catalase. Surprisingly, a commercial catalase preparation of low specific activity-generally used for the enzyme assay-showed HPPD activity which was separable from the catalase activity on a gel filtration column. According to kinetic studies with purified maize HPPD, experimental herbicides from the family mentioned were strong competitive inhibitors of the plant enzyme in nanomolar range with Ki values of 5 and 15 nM for 2-(2-nitro-4-chlorobenzoyl)-5-(2-methoxyethyl) cyclohexane-1,3-dione and 2-(2-chloro-4-methanesulfonylbenzoyl)-cyclohexane-1,3-dione (SC-0051 ; sulcotrione), respectively.