Purification and Characterization of a Cytochrome P450 (trans-Cinnamic Acid 4-Hydroxylase) from Etiolated Mung Bean Seedlings

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A Cyt P450 (P450_<C4H>) possessing trans-cinnamate 4-hydroxylase (C4H) activity was purified to apparent homogeneity from microsomes of etiolated mung bean seedlings. Upon SDS-poly-acrylamide gel electrophoresis, the purified preparation gave a single protein band with a molecular mass of 58-kDa. Its specific P450 content was 12.6 nmol (mg protein)^<-1>. Using NADPH as electron donor, purified P450_<C4H> aerobically converted trans-cinnamic acid to p-coumaric acid with a specific activity of 68 nmol min^<-1> nmol^<-1> P450 in a reconstituted system containing NADPH-Cyt P450 reductase purified from the seedlings or rabbit liver microsomes, dilauroyl phosphatidylcholine, and cholate. This specific activity is by far the highest for reconstituted C4H systems so far reported and provides direct evidence that C4H activity is actually associated with a P450 protein. In the oxidized state P450_<C4H> Showed a typical low-spin type absorption spectrum with a Soret peak at 419 nm. A partial spectral shift to the high spin state was observed when trans-cinnamic acid was added to oxidized P450_<C4H>' By spectral titration, the dissociation con-stant of the cinnamic acid-P450_<C4H> complex was determined to be 2.8 μM. This value is similar to the K_m value (1.8 μM) for trans-cinnamic acid determined in the reconstituted system.
日本植物生理学会の論文