Functional expression and mutational analysis of flavonol synthase from Citrus unshiu

Abstract

Flavonols are produced by the desaturation of flavanols catalyzed by flavonol synthase. The enzyme belongs to the class of intermolecular dioxygenases which depend on molecular oxygen and Fe II /2-oxoglutarate for activity, and have been in focus of structural studies recently. Flavonol synthase cDNAs were cloned from six plant species, but none of the enzymes had been studied in detail. Therefore, a cDNA from Citrus unshiu (Satsuma mandarin) designated as flavonol synthase was expressed in Escherichia coli, and the purified recombinant enzyme was subjected to kinetic and mutational chacterizations. The integrity of the recombinant synthase was revealed by a molecular ion from MALDI-TOF mass spectrometry at m/z 37888 ± 40 (as compared to 37899 Da calculated for the translated polypeptide), and by partial N-terminal sequencing. Maximal flavonol synthase activity was observed in the range of pH 5-6 with dihydroquercetin as substrate and a temperature optimum at about 37°C. K m values of 272, 11 and 36 lM were determined for dihydroquercetin, Fe II and 2-oxoglutarate, respectively, with a sixfold higher affinity to dihydrokaempferol (K m 45 lM). Flavonol synthase polypeptides share an overall sequence similarity of 85% (47% identity), whereas only 30-60% similarity were apparent with other dioxygenases. Like the other dioxygenases of this class, Citrus flavonol synthase cDNA encodes eight strictly conserved amino-acid residues which include two histidines (His221, His277) and one acidic amino acid (Asp223) residue for Fe II -coordination, an arginine (Arg287) proposed to bind 2-oxoglutarate, and four amino acids (Gly68, His75, Gly261, Pro207) with no obvious functionality. Replacements of Gly68 and Gly261 by alanine reduced the catalytic activity by 95%, while the exchange of these Gly residues for proline completely abolished the enzyme activity. Alternatively, the substitution of Pro207 by glycine hardly affected the activity. The data suggest that Gly68 and Gly261, at least, are required for proper folding of the flavonol synthase polypeptide.

Metabolomic and genetic analyses of flavonol synthesis in Arabidopsis thaliana support the in vivo involvement of leucoanthocyanidin dioxygenase

“…Like other dioxygenases, FLS contain several strictly conserved amino-acid residues (Lukacin and Britsch 1997) which include two histidines (His221, His277) and one acidic amino acid (223) residue for Fe II -binding, an arginine (Arg287) together with a serine (Ser289) proposed to bind 2-oxoglutarate, and four amino acids (Gly68, His75, Gly261, Pro207) with no obvious functionality, presumed to be required for proper folding of the enzyme polypeptide (Wellmann et al 2002). For FLS1 from A. thaliana, His132, Lys202 and Phe293 were identiWed via computational methods and analysed by mutational analyses in spectroscopic assays, showing their importance for dihydroquercetin substrate binding (Chua et al 2008).…”

Evolution of Flavone Synthase I from Parsley Flavanone 3β-Hydroxylase by Site-Directed Mutagenesis

“…However, the extent and the periphery of the jelly rolls may vary and the enzymes show only little sequence similarity. Regardless of these limitations, a-helices and b-sheets appear to be analogously assembled, leading to almost identical circular dichroism spectroscopic profiles for P. hybrida FHT (Lukacin et al, 2000), C. unshiu FLS (Wellmann et al, 2002), or IPNS (Borovok et al, 1996;Durairaj et al, 1996). Alignment of the 2-ODDs that have been examined by x-ray scattering with parsley FHT and FNS I polypeptides revealed the highest sequence similarity of approximately 30% with ANS.…”

Developmental and metabolic plasticity of white-skinned grape berries in response to Botrytis cinerea during noble rot

“…For FLS and ANS activities, the methods of Wellmann et al (2002) and Pang et al (2007), respectively, were used. The assay mixture for FSL activity (total volume, 300 mL) contained 100 mM dihydroquercetin as a substrate, 83 mM 2-oxoglutarate, 42 mM ammonium iron (II) sulfate, 2.5 mM sodium ascorbate, and 2 mg mL 21 bovine catalase.…”