Molecular characterization of flayanone 3β‐hydroxylases

Abstract

A heterologous cDNA probe from Petunia hybrida was used to isolate flavanone-3P-hydroxylase-encoding cDNA clones from carnation (Dianthus caryophyllus), china aster (Callisteplzus chinensis) and stock (Matthiola incana). The deduced protein sequences together with the known sequences of the enzyme from l ? hybrida, barley (Hordeum vulgare) and snapdragon (Antirrhinum majus) enabled the determination of a consensus sequence which revealed an overall 84% similarity (53 % identity) of flavanone 3P-hydroxylases from the different sources. Alignment with the sequences of other known enzymes of the same class and to related non-heme iron-(11) enzymes demonstrated the strict genetic conservation of 14 anuno acids, in particular, of three histidines and an aspartic acid. The conservation of the histidine motifs provides strong support for the possible conservation of structurally similar iron-binding sites in these enzymes. The putative role of histidines as chelators of ferrous ions in the active site of tlavanone 3P-hydroxylases was corroborated by diethyl-pyrocarbonate modification of the partially purified recombinant Petunia enzyme.Flavanone 3P-hydroxylase (FHT) catalyzes 3P-hydroxylation of 2S-flavanones to 2R,3R-dihydroflavonols [( +)-dihydroflavonols] which are intermediates in the biosynthesis of flavonols, anthocyanidins, catechins and proanthocyanidins in plants (Heller and Forkmann, 1988;Forkmann, 1991). The enzyme from Petunia hybrida has been purified to homogeneity and characterized as a typical 2-oxoglutarate-dependent dioxygenase (Britsch and Grisebach, 1986;Britsch, 1990). Recently, we have isolated a near-full-length FHTencoding cDNA from a Petunia library (Britsch et al., 1992). Heterologous expression of the cDNA in Escherichia coli led to the formation of highly active recombinant FHT, thereby verifying the identity of the cDNA clone.Antibodies against FHT from Petunia readily crossreact with FHT from a number of other sources and were used to characterize specific FHT mutants (Britsch, 1990; Koch, 1992). Therefore, structural similarities of the FHT protein from different plants can be assumed. A comparison of the deduced amino acid sequence of the Petunia FHT with the sequence of FHT from barley (Meldgaard, 1992) indeed showed a high degree of identity (75%; Britsch et al., 1992).Consequently, the FHT cDNA from Petunia should serve as a useful heterologous probe to clone this enzyme from other plant sources. Utilizing the 945-bp EcoRI fragment from Petunia FHT as a hybridization probe, we were able to clone the corresponding cDNAs from carnation, china aster and stock. In this study, we report the cloning and sequence analysis of these particular FHT enzymes, the analysis of the consensus sequence derived from six individual FHT sequences and the modification of purified recombinant enzyme with diethyl pyrocarbonate, performed to elucidate the possible function of strictly conserved histidines. Furthermore, the sequences were also compared with other known 2-oxoglutarate-dependent dioxygenases f...

The monomeric polypeptide comprises the functional flavanone 3β‐hydroxylase from Petunia hybrida

“…The FHT polypeptide contains five cysteine residues all of which could account for the observed cross‐linking. These residues are clustered with Cys‐60 and Cys‐67 in one region and Cys‐172, Cys‐193 and Cys‐208 as part of a conserved amphipathic α‐helix spanning the amino acids 165 to 210 [32].…”

Expression cloning of a gibberellin 20-oxidase, a multifunctional enzyme involved in gibberellin biosynthesis.

“…Higher amino acid-sequence homology might be expected between the different GA,2-oxoglutarate dioxygenases, although at present no other sequences are available for comparison. The derived sequence contains three conserved histidines, which may serve to bind Fe at the enzyme active center (17,20,21). The sequence contains no recognizable N-terminal targeting signals for particular cell compartments.…”

Expression quantitative trait loci infer the regulation of isoflavone accumulation in soybean (Glycine max L. Merr.) seed

“…These two products are the precursors of DZ and GT, which are formed after the catalysis of the precursors by the key enzyme 2-hydroxyisoflavanone synthase (IFS; EC 1.14.13.136) [30, 31]. The enzyme F3H, that competes with IFS in utilizing naringenin, catalyzes the conversion of flavanones to dihydroflavonols, which are intermediates in the biosynthesis of flavonols, anthocyanidins, catechins and proanthocyanidins [32, 33]. For the synthesis of GC, isoliquiritigenin is likely a precursor to form GC after several biochemical steps, which are not entirely known yet [34].…”