Microsomal Flavonoid 3'-Monooxygenase from Maize

Identification of flavonoid 3'-monooxygenase establishes another reaction in the biosynthesis of flavonoid compounds in maize (Zea mays L.). The flavonoid 3'-hydroxylase was obtained as a microsomal enzyme preparation by buffer extraction of 5 day old maize seedlings and ultracentrifugation. Seedlings were exposed to light 24 hours prior to enzyme extraction. The extraction buffer required the addition of sucrose or glycerin and dithiothreitol to obtain an active hydroxylase that retained its activity on storage at -70°C. Enzymic activity required 02 and NADPH, was optimum at pH 8.5 and 30°C, and could be inhibited 79% by carbon monoxide. Carbon monoxide inhibition could be reduced to 21% by irradiation of the samples with 450 nanometer light during incubation. Kaempferol, a flavonol; naringenin, a flavanone; and apigenin, a flavone, all served as substrates for the hydroxylase. Treatment of the microsomal enzyme preparation, previously reduced with sodium dithionite, with carbon monoxide gave a 455 nanometer absorption peak which disappeared on oxidation of the preparation with the formation of a 420 nanometer peak. These results suggest a cytochrome P450 type monooxygenase enzyme. The concentration of cytochrome P450 was 0.21 nanomoles per milligram protein. Identification of the monooxygenase provides further biochemical information about a biosynthetic sequence for which the genetics have been studied intensely. The principal anthocyanins found in maize (Zea mays L.) are Pg-gl2 and Cy-gl (3). These compounds differ structurally in that an additional hydroxyl group is found at the 3'-position in the B-ring of Cy-gl. Structures for these and the other substrates and products considered in this paper are given in Table I. The timing of the addition of this hydroxyl group remains a point of discussion since there is evidence for this to occur either at the 9or 15-carbon stage of synthesis. Hess (12) observed the incorporation of substituted cinnamic acids, without further modification, into anthocyanin and proposed the 'cinnamic acid starter hypothesis', which suggested hydroxylation occurred at the 9-carbon level. Subsequently, Fritsch and Grisebach (9) observed 3'hydroxylation of naringenin and eriodictyol by a microsomal preparation obtained from Haplopappus gracilis. Similar 3'hydroxylation was observed in microsomal preparations ob'Cooperative investigations, Agricultural Research Service, United States Department of Agriculture, and Missouri Agricultural Experiment Station, Columbia, MO 6521 1. Journal Series No. 9825. 2Abbreviations: Pg-gl, pelargonidin 3-O-glucoside; Cy-gl, cyanidin 3O-glucoside; UFGT, uridinediphosphoglucose:flavonoid glucosyltransferase; Ches, 2(N-cyclohexylamino)ethane sulfonic acid; NEM, N-ethylmaleimide; PCMB. p-chloromercuribenzoate. tained from Matthiola incana (7), snapdragon (Antirrhinum majus) (8), and cell cultures of parsley (Petroselinum hortense Hoffm.) (2, 11). Although the enzyme preparation had been suggested to be a heme-containing monooxygenase (7), Hagmann et al. (1 1) identified the activity as a Cyt P-450 containing enzyme. The 3'-hydroxylases referred to here all required 02 and NADPH as the electron donor. The only report of hydroxylase activity at the 9-carbon level in any of the preparations was in that from H. gracilis (9). Although the genetics of anthocyanin biosynthesis in maize has been studied extensively, the only enzyme activities to be demonstrated have been chalcone synthase (5) and UFGT (13). The subject of this report is the extraction and partial characterization of flavonoid 3'-hydroxylase, a microsomal enzyme involved in anthocyanin biosynthesis in maize. MATERIALS AND METHODS Plant Materials. Seeds were geminated in the dark at 30°C, on filter paper saturated with 1 mm CaSO4, and grown for a period of 4 d after which they were kept under cool-white fluorescent light for 24 h prior to enzyme extraction. The seedlings were washed in distilled H20 and separated into roots and the shoots, which included mesocotyl minus roots and any further growth beyond the first internode. Unless otherwise indicated the seed was of a uniform, defined genetic background which included all the genetic factors necessary for anthocyanin synthesis. Chemicals. Chemicals used in this study were readily available commercially with the exception of eriodictyol which was obtained from Sarsynthese3, Avenue du President J.-F. Kennedy, Table I. Chemical Structures ofthe Flavonoid Compounds Involved in the Hydroxylation Reaction Compound Hydroxylation Basic Structure