Pii: s0031-9422(01)00191-

Stefan Martensa, Gert Forkmanna, Ulrich Maternb,*, Richard Lukacˇinb aTechnische Universita¨t Mu¨nchen, Wissenschaftszentrum fu¨r Erna¨hrung, Landnutzung und Umwelt, Department fu¨r Pflanzenwissenschaften, Lehrstuhl fu¨r Zierpflanzenbau, Am Hochanger 4, D-85350 Freising, Germany bInstitut fu¨r Pharmazeutische Biologie, Philipps-Universita¨t Marburg, Deutschhausstrasse 17 A, D-35037 Marburg, Germany Received 19 March 2001; received in revised form 6 April 2001 A cDNA encoding flavone synthase I was amplified by RT-PCR from leaflets of Petroselinum crispum cv. Italian Giant seedlings and functionally expressed in yeast cells. The identity of the recombinant, 2-oxoglutarate-dependent enzyme was verified in assaysconverting (2S)-naringenin to apigenin. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Petroselinum crispum; Apiaceae; Flavonoid biosynthesis; Flavone synthase I cloning; 2-Oxoglutarate-dependent dioxygenase; Hetero-logous expression During the last decade considerable progress has been cephalosporin biosynthesis (Baldwin and Abraham, achieved towards elucidating the mode of action and 1988), as well as in mammalian tissues (Lindstedt et al., molecular architecture of 2-oxoglutarate-dependent diox- 1977; Kivirikko et al., 1989; Stenflo et al., 1989). Fur- ygenases. These enzymes catalyze diverse reactions, such thermore, these enzymes catalyze numerous reactions in as the hydroxylation, desaturation, epoxidation or cycli- plants, e.g. in the formation of hydroxyproline-rich gly- zation of substrates, and the activities depend on ferrous coproteins (Tanaka et al., 1980), of gibberellins (Hed- iron and molecular oxygen which is reduced during cata- den and Graebe, 1982) and the secondary metabolites lysis by two electrons provided by decarboxylation of the scopolamine (Hashimoto and Yamada, 1986), vindoline cosubstrate (Prescott, 1993; DeCarolis and DeLuca, 1994; (DeCarolis et al., 1990) or of various flavonoids (Fork- Prescott and John, 1996). Although 2-oxoglutarate is the mann et al., 1980; Britsch et al., 1981; Lukacˇin and common cosubstrate, some closely related dioxygenases Britsch, 1997; Lukacˇin et al., 2000 a,b,c).
mobilize the electrons from decarboxylation of the sub- Five 2-oxoglutarate-dependent dioxygenases have been strate itself, e.g. isopenicillin N-synthase (Baldwin and identified so far from flavonoid biosynthesis, which Abraham, 1988) and 4-hydroxyphenylpyruvate dioxy- include the widely distributed anthocyanidin synthase genase (Bradley et al., 1986; Ru¨etschi et al., 1992), or by (Menssen et al., 1990), flavanone 3b-hydroxylase (Fork- the oxidation of ascorbate as in ethylene biosynthesis mann et al., 1980; Britsch et al., 1981; Lukacˇin and (Zhang et al., 1995; Lay et al., 1996). These latter Britsch, 1997; Lukacˇin et al., 2000 a,b,c) and flavonol enzymes may nevertheless classify with the 2-oxogluta- synthase (Britsch et al., 1981; Holton et al., 1993).
rate-dependent dioxygenases stricto sensu in one cate- Another dioxygenase, catalyzing the 6-hydroxylation of gory of intermolecular dioxygenases. Intermolecular partially methylated flavonols, was reported recently dioxygenases fulfill a variety of pivotal functions in pri- from Chrysosplenium americanum (Anzelotti and Ibra- mary and secondary metabolism in bacteria (Omura et him, 2000), while flavone synthase I, FNS I, appears to al., 1984; Salowe et al., 1990) and fungi, including the be confined to species of the Apiaceae (Britsch, 1990).
cyclization and ring expansion reactions in penicillin/ FNS I had been characterized in 1981 as a solubleenzyme from parsley, in contrast to the microsomal fla-vone synthase II, FNS II, from other plants (Kochs andGrisebach, 1987; Martens and Forkmann, 1998), and * Corresponding author. Tel.: +49-6421-282-2461; fax: +49-6421- was partially purified through six-steps of fractionation E-mail address: matern@mailer.uni-marburg.de (U. Matern).
from irradiated cell cultures (Britsch, 1990). This 0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
P I I : S 0 0 3 1 - 9 4 2 2 ( 0 1 ) 0 0 1 9 1 - 1 S. Martens et al. / Phytochemistry 58 (2001) 43–46 enzyme was then employed in kinetic studies aiming at 1992), and the similarity of prolyl 4-hydroxylase with the reaction mechanism, which revealed that synthetic lysyl hydroxylase from chicken (Myllyla¨ et al., 1991) or 2-hydroxynaringenin did not compete with flavanone of fungal isopenicillin N-synthase with desacetoxy- substrates. Accordingly, the 2,3-desaturation of flava- cephalosporin C synthase ranged only at approx. 20% nones by FNS I was postulated to proceed by direct (Britsch et al., 1993). Nevertheless, superimposing the abstraction of the vicinal hydrogen atoms (Britsch, structural models of the penicillin and cephalosporin 1990), which would assign FNS I to a distinct desatur- synthases revealed an almost identical architecture for ase subgroup among the 2-oxoglutarate-dependent these two enzymes (Lloyd et al., 1999), and comparison of the CD spectra of Petunia flavanone 3b-hydroxylase exluding the successive hydroxylation and dehydrata- and isopenicillin N-synthase suggested the same pattern tion, was proposed for the desaturation of alkanes to of helical, non-helical and b-sheet motifs for the Petunia olefines suggesting a reaction via radical intermediates dioxygenase (Lukacˇin et al., 2000b). Flavanone 3b- (Mansuy, 1998). The exact course of FNS I catalysis hydroxylase and FNS I both use 2-oxoglutarate as the requires further experimental support, but appears to cosubstrate and depend on the same flavanone sub- proceed analogous to that of the cytochrome P450- strates (Fig. 1). Accordingly, a thorough examination of dependent FNS II expressed in many plants except for the sequential and spatial differences of these two the Apiaceae. The first full size FNS II cDNAs were enzymes, together with in vitro mutagenesis studies, recently cloned from Gerbera hybrida (Martens and might be rather helpful to pinpoint the putative sub- Forkmann, 1999), Antirrhinum majus and Torenia strate binding sites and to explain the formation of fla- hybrida (Akashi et al., 1999) by differential display PCR and expressed in yeast cells. As anticipated for a P450- Based on alignments of fourteen intermolecular diox- dependent monoxygenase, this FNS II converted label- ygenase polypeptides from public data bases two con- led flavanones to the corresponding flavones apparently served sequence motifs were chosen, and, similar to the without any intermediate (Martens and Forkmann, previous cloning of flavonol synthase (Fig. 1) from Pet- unia hybrida (Holton et al., 1993), degenerate oligonu- The common mode of oxygen activation by inter- cleotide primers were designed for the cloning of FNS I.
molecular dioxygenases, particularly among the 2-oxo- In combination with oligo(dT), the primers were glutarate-dependent enzymes, seems to predict a high employed for RT-PCR amplification of cDNAs from degree of homology at the DNA and polypeptide levels.
total RNA that had been extracted from young leaflets However, only 30% similarity was observed in the at four stages of development of flavone-producing polypeptide sequences of, for example, flavanone 3b- Petroselinum crispum cv. Italian Giant plants (Martens hydroxylase from Petunia hybrida and hyoscyamine 6b- and Forkmann, 1999). A whole set of intermolecular hydroxylase from Hyoscyamus niger (Britsch et al., dioxygenase cDNAs was amplified, and the full-size Fig. 1. Reaction catalyzed by flavone synthase I (FNS I), converting (2S)-naringenin to apigenin, in comparison to the activities of flavanone 3b-hydroxylase (FHT) and flavonol synthase (FLS), which sequentially convert (2S)-naringenin to dihydrokaempferol and kaempferol.
S. Martens et al. / Phytochemistry 58 (2001) 43–46 unpublished). Moreover, the yeast strain transfectedwith the pYES2 vector hosting the FNS I cDNA in theinverse orientation did not express flavone synthaseactivity (Fig. 2 B). Recombinant FNS I lacked flavonolsynthase activity (Fig. 1), and the sequences of these twoenzymes differ considerably. Thus, soluble FNS I pre-vailing in the Apiaceae was cloned for the first time andhas become available in quantity for mechanistic studiesas well as for the convenient preparative synthesis ofradiolabeled flavones which enable further biosyntheticand biotechnological studies. The evolutionary contextfor the expression of the soluble synthase exclusively inthe Apiaceae remains to be established. In addition, therecombinant enzyme may be of value for the productionof flavone-nutraceuticals due to their antioxidant andanticancer potentials (Harborne and Williams, 2000).
We are indebted to Dr. L. Britsch, Dr. R. Zimmer- mann and Dr. H. Mu¨ller (Merck KGaA, Darmstadt)for the ESI–MS analysis of apigenin as the product ofthe recombinant parsley FNS I.
Akashi, T., Fukuchi-Mizutani, M., Aoki, T., Ueyama, Y., Yonekura- Fig. 2. Catalytic activity of parsley FNS I expressed in yeast cells.
Sakakibara, K., Tanaka, Y., Kusumi, T., Ayabe, S., 1999. Mole- Crude extracts from yeast cells expressing the FNS I from the cDNA cular cloning and biochemical characterization of novel cytochrome inserted in the pYES2 expression vector (A) or harbouring the FNS I P450, flavone synthase II, that catalyzes direct conversion of flava- cDNA in the inverse orientation (B) were incubated with (2S)-[4a,6- nones to flavones. Plant Cell Physiology 40, 1182–1186.
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