Properties of a Mixed Function Oxygenase Catalyzing Ipomeamarone 15-Hydroxylation in Microsomes from Cut-Injured and <em>Ceratocystis fimbriata</em>-Infected Sweet Potato Root Tissues
Abstract
Ipomeamarone 15-hydroxylase activity was found in a microsomal fraction from cut-injured and Ceratocystis fimbriata-infected sweet potato (Ipomoea batatas Lam. cv. Norin No. 1) root tissues and its optimum pH was 8.0. The enzyme reaction required O2 and NADPH. The Km values calculated for ipomeamarone and NADH were approximately 60 and 2 micromolar, respectively. NADPH alone had little effect on enzyme activity but activated the reaction in the presence of low concentrations of NADPH. Ipomeamarone 15-hydroxylase activity was strongly inhibited by p-chloromercuribenzoic acid and markedly suppressed by cytochrome c and p-benzoquinone. KCN was an activator rather than an inhibitor for the reaction. CO inhibited the activity strongly and its inhibition was partially reversed by light. CO difference spectra of the reduced microsomal fraction showed two absorption maxima at 423 and 453 nm; the latter maximum may be due to a cytochrome P-450. These results suggest that ipomeamarone 15-hydroxylase is a cytochrome P-450-dependent, mixed-function oxygenase.
Ipomeamarone 15-hydroxylase activity was not found in fresh tissue of sweet potato roots. However, the activity appeared and increased markedly in response to cut-injury or infection by Ceratocystis fimbriata, and reached a maximum after 24 to 36 hours of incubation. The increase in activity in the latter case was 3- to 5-fold higher than in the former. The time course patterns of development and successive decline in ipomeamarone hydroxylase activities were similar to those for cinnamic acid 4-hydroxylase activity, which had been described as a cytochrome P-450-dependent, mixed-function oxygenase. However, little substrate competition was found between ipomeamarone 15-hydroxylase and cinnamic acid 4-hydroxylase in our preparations.
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- Coolbaugh RC, Hirano SS, West CA. Studies on the Specificity and Site of Action of alpha-Cyclopropyl-alpha-[p-methoxyphenyl]-5-pyrimidine Methyl Alcohol (Ancymidol), a Plant Growth Regulator. Plant Physiol. 1978 Oct;62(4):571–576.[PMC free article] [PubMed] [Google Scholar]
- Hasson EP, West CA. Properties of the System for the Mixed Function Oxidation of Kaurene and Kaurene Derivatives in Microsomes of the Immature Seed of Marah macrocarpus: Cofactor Requirements. Plant Physiol. 1976 Oct;58(4):473–478.[PMC free article] [PubMed] [Google Scholar]
- HORIE S. ON THE CARBON MONOXIDE-SHIFT OF ABSORPTION SPECTRUM OF CYTOCHROMA A3. J Biochem. 1964 Aug;56:113–121. [PubMed] [Google Scholar]
- Imai Y, Hashimoto-Yutsudo C, Satake H, Girardin A, Sato R. Multiple forms of cytochrome P-450 purified from liver microsomes of phenobarbital- and 3-methylcholanthrene-pretreated rabbits. I. Resolution, purificaton, and molecular properties. J Biochem. 1980 Aug;88(2):489–503. [PubMed] [Google Scholar]
- Kamataki T, Kitada M, Chiba K, Kitagawa H, Imai Y, Sato R. Enhancement by cyanide of aniline p-hydroxylation activity in rat liver microsomes. Biochem Pharmacol. 1980 Apr 15;29(8):1141–1147. [PubMed] [Google Scholar]
- KEILIN D, HARTREE EF. Purification of horse-radish peroxidase and comparison of its properties with those of catalase and methaemoglobin. Biochem J. 1951 Jun;49(1):88–104.[PMC free article] [PubMed] [Google Scholar]
- LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- Madyastha KM, Meehan TD, Coscia CJ. Characterization of a cytochrome P-450 dependent monoterpene hydroxylase from the higher plant Vinca rosea. Biochemistry. 1976 Mar 9;15(5):1097–1102. [PubMed] [Google Scholar]
- Meehan TD, Coscia CJ. Hydroxylation of geraniol and nerol by a monooxygenase from Vinca rosea. Biochem Biophys Res Commun. 1973 Aug 21;53(4):1043–1048. [PubMed] [Google Scholar]
- Murphy PJ, West CA. The role of mixed function oxidases in kaurene metabolism in Echinocystis macrocarpa Greene endosperm. Arch Biochem Biophys. 1969 Sep;133(2):395–407. [PubMed] [Google Scholar]
- Oguni I. Dehydroipomeamarone as an Intermediate in the Biosynthesis of Ipomeamarone, a Phytoalexin from Sweet Potato Root Infected with Ceratocystis fimbriata. Plant Physiol. 1974 Apr;53(4):649–652.[PMC free article] [PubMed] [Google Scholar]
- Rich PR, Bendall DS. Cytochrome components of plant microsomes. Eur J Biochem. 1975 Jul 1;55(2):333–341. [PubMed] [Google Scholar]
- Potts JR, Weklych R, Conn EE, Rowell J. The 4-hydroxylation of cinnamic acid by sorghum microsomes and the requirement for cytochrome P-450. J Biol Chem. 1974 Aug 25;249(16):5019–5026. [PubMed] [Google Scholar]
- Reichhart D, Salaün JP, Benveniste I, Durst F. Induction by manganese, ethanol, phenobarbital, and herbicides of microsomal cytochrome P-450 in higher plant tissues. Arch Biochem Biophys. 1979 Aug;196(1):301–303. [PubMed] [Google Scholar]
- Russell DW. The metabolism of aromatic compounds in higer plants. X. Properties of the cinnamic acid 4-hydroxylase of pea seedlings and some aspects of its metabolic and developmental control. J Biol Chem. 1971 Jun 25;246(12):3870–3878. [PubMed] [Google Scholar]