Conversion of 1'-deoxy-2H-ABA to 2H-ABA in cell-free extracts from Cercospora rosicola
1991
Al-Nimri, L.F. | Coolbaugh, R.C.
Abscisic acid (ABA) biosynthesis in the fungus Cercospora rosicola has been studied previously by feeding potential intermediates to growing cultures and examining the products. The present work describes the isolation and partial characterization of an enzyme system which catalyzes the last step in this pathway, the conversion of 1'-deoxy-2H-ABA to 2H-ABA. Enzyme extracts were prepared from cold-pressed mycelia of actively growing C. rosicola in tricine buffer containing sucrose and beta-mercaptoethanol. Low-speed supernatants and resuspended microsomal preparations were active in converting 1'-deoxy-2H-ABA to 2H-ABA. Optimum conditions for the reactions were established at 10 micromole substrate, 300 microliter enzyme extract in a total volume of 1 ml. Reaction products were chromatographed by reverse-phase high-performance liquid chromatography (HPLC). The presumptive ABA fractions were collected and the 2H-ABA was measured by combined gas chromatography-mass spectrometry (GC-MS) with an 2H-ABA standard. Most of ABA standard. Most of the enzyme activity was found in the microsomal fraction. Typical reaction rates were on the order of 1.5 pmol 2H-ABA formed/min/mg protein. The reaction required NADPH and was enhanced by FAD. Triarimol, a substituted pyrimidine, which inhibits ABA synthesis in C. rosicola, had no effect on the conversion of 1'-deoxy-2H-ABA to 2H-ABA.
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