ISSN 0362 1197, Human Physiology, 2012, Vol. 38, No. 5, pp. 541–544. © Pleiades Publishing, Inc., 2012. Original Russian Text © V.M. Pokrovskii, O.G. Kompaniets, 2012, published in Fiziologiya Cheloveka, 2012, Vol. 38, No. 5, pp. 102–107. Influence of the Level of Blood Pressure on the Regulatory–Adaptive State V. M. Pokrovskii and O. G. Kompaniets Kuban State Medical Universi
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: email@example.com (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.
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