title: Identification of strictly conserved histidine and arginine residues as part of the active site in Petunia hybrida flavanone 3beta-hydroxylase.
abstract: Flavanone 3beta-hydroxylase, involved in the biosynthesis of flavonoids, catechins, and anthocyanidins, is a non-heme iron enzyme, dependent on Fe2+, 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, [Gln121]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 Km value with respect to iron binding, as compared to the wild-type enzyme. The most drastic effect on the reaction rate of flavanone 3beta-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 delta-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 3beta-hydroxylase.
authors.affiliation: Institut für Pharmazeutische Biologie, Universität Marburg, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Lukacin
authors.forename: L
authors.initials: L
authors.lastname: Britsch
mesh_terms: D000272:Adipates / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis; D019657:Solanaceae / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D000272:Adipates; D007656:Ketoglutaric Acids; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C001918:alpha-ketoadipic acid; D006899:Mixed Function Oxygenases; C048797:flavanone 3-dioxygenase; D004047:Diethyl Pyrocarbonate