PURIFICATION AND CHARACTERIZATION OF CINNAMOYL-COENZYME A-NADP OXIDOREDUCTASE IN EUCALYPTUS-GUNNII

Cinnamoyl-coenzyme A:NADP oxidoreductase (CCR, EC 1.2.1.44), the entry -point enzyme into the monolignol biosynthetic pathway, was purified t o apparent electrophoretic homogeneity from differentiating xylem of E ucalyptus gunnii Hook. The purified protein is a monomer of 38 kD and has an isoelectric point of 7. Although Eucalyptus gunnii CCR has appr oximately equal affinities for all possible substrates (p-coumaroyl-co enzyme A, feruloyl-coenzyme A, and sinapoyl-coenzyme A), it is approxi mately three times more effective at converting feruloyl-coenzyme A th an the other substrates. To gain a better understanding of the catalyt ic regulation of Eucalyptus CCR, a variety of compounds were tested to determine their effect on CCR activity. CCR activity is inhibited by NADP and coenzyme A. Effecters that bind lysine and cysteine residues also inhibit CCR activity. As a prerequisite to the study of the regul ation of CCR at the molecular level, polyclonal antibodies were obtain ed.