Identification of Strictly Conserved Histidine and Arginine Residues as Part of the Active Site in Petunia hybrida Flavanone 3β‐Hydroxylase

Abstract

Flavanone 3P-hydroxylase, involved in the biosynthesis of flavonoids, catechins, and anthocyanidins, is a non-heme iron enzyme, dependent on Fez+, molecular oxygen, 2-oxoglutarate, and ascorbate, the typical cofactors of the class of 2-oxoglutarate-dependent dioxygenases.Sequence alignment analysis of various 2-oxoglutarate-dependent dioxygenases and related enzymes revealed eight amino acid residues that seem to be strictly conserved within this group of enzymes. Among these residues, two histidines (His220 and His278) and one aspartic acid (Asp222) were identified as part of the putative iron-binding site and an arginine residue (Arg288) as part of the 2-oxoglutarate binding site, by site-directed mutagenesis and functional analysis of the mutated recombinant enzyme.The mutant genes were expressed in Escherichia coli to give soluble proteins whose molecular masses were in excellent agreement with the wild-type enzyme. Four out of nine mutant enzymes, [Gln78]FHT, [Glnl21]FHT, [Gln264]FHT and [Gln266]FHT, were enzymatically active with activities reduced to 26-57%, implying that the mutated amino acid residues are not essential for catalysis. Replacement of His220 by glutamine and Asp222 by asparagine remarkably reduced the catalytic activity to about 0.15% and 0.4%, respectively. The [Gln220]FHT and [Asn222]FHT enzymes showed a slightly increased K,,, value with respect to iron binding, as compared to the wild-type enzyme. The most drastic effect on the reaction rate of flavanone 3P-hydroxylase was achieved by mutating His278 to glutamine. The mutant had no detectable enzyme activity, indicating that His278 was essential for the catalytic reaction. The observed protection of purified enzyme from inactivation by diethylpyrocarbonate after the addition of cofactors provided further independent confirmation for the involvement of histidine residues in the active site.The substitution of Arg288 by lysine or glutamine induced a precipitous decrease in catalytic activity and a fivefold and 160-fold increase in the Michaelis constants for 2-oxoglutarate, respectively. In addition, the enzymatic activities of the latter two mutant enzymes showed a strong pH dependence in the weakly acidic as well as in the neutral pH range, unlike the wild-type enzyme.These results clearly indicate that Arg288 probably contributes to the specific binding of 2-oxoglutarate at the active site of the enzyme, most likely by providing a positive charge, properly located in order to interact with the S-carboxyl function of 2-oxoglutarate. Furthermore, we conclude that His220, His278 and Asp222 constitute three of the possible ligands for iron binding in the active site of flavanone 38-hydroxylase.

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

“…Although the FLS2 gene is transcribed, it is fairly unlikely that the resulting protein has FLS activity. The deduced polypeptide from the FLS2 transcript is missing the C-terminal protein part (Supplementary Figure 1), including the last histidine residue of the HxD…H motiv involved in 2-oxoglutarate binding (Britsch et al 1993;Roach et al 1995;Lukacin and Britsch 1997) as well as the amino acid residues involved in iron binding (Myllylä et al 1992;Britsch et al 1993;Lukacin and Britsch 1997;Clifton et al 2006). Taken together, these results indicate that only FLS1, FLS3 and FLS5 encode full-length proteins.…”

“…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).…”

A sensitive microextraction by packed sorbent-based methodology combined with ultra-high pressure liquid chromatography as a powerful technique for analysis of biologically active flavonols in wines

“…1) in grapes has been reported to occur through the phenylpropanoid metabolism [1]. Specifically, naringenin is transformed into quercetin and kaempferol by means of the enzyme flavanone 3␤-hydroxylase (FHT), which leads to the production of dihydroflavonols [1][2][3]. Regarding myricetin, although it is also produced through the phenylpropanoid metabolism, its synthesis occurs in general terms further in the pathway [4][5][6].…”

A flavonol synthase gene GtFLS defines anther-specific flavonol accumulation in gentian

“…FLS belongs to the 2-oxoglutarate-dependent dioxygenase family, along with anthocyanidin synthase (ANS) and F3H. GtFLS contained characteristic conserved motifs (data not shown), including two histidines and one aspartic acid as part of the putative iron-binding site, and an arginine residue as a part of the 2-oxoglutarate binding site (Lukacin and Britsch 1997). Therefore, the isolated GtFLS gene was concluded to be a FLS ortholog, although the enzymatic properties of GtFLS remain to be determined.…”