Purification and characterization of flavone synthase I, a 2-oxoglutarate-dependent desaturase.
Soluble flavone synthase I from illuminated parsley cells was purified to near homogeneity by a six-step procedure. A molecular mass of 48 +/- 2 kDa was determined by gel permeation chromatography and denaturing polyacrylamide gel electrophoresis. A single protein with an isoelectric point at pH 4.8 +/- 0.1 was detected on isoelectric focusing gels, which catalyzed the overall conversion of 2S-flavanones into the corresponding flavones in the presence of molecular oxygen, 2-oxoglutarate, ferrous ion, and ascorbate. Apparent Michaelis constants for 2S-naringenin, 2S-eriodictyol, and 2-oxoglutarate were determined as 5, 8, and 16 microM, respectively. (+)-Dihydrokaempferol and 2R-naringenin were not accepted as substrates. The enzyme was strongly inhibited by Cu2+ and Zn2+. Potent competitive inhibition with respect to 2-oxoglutarate was observed with 2,4-pyridinedicarboxylate (Ki = 1.8 microM). With crude extracts as well as with the purified enzyme neither the hypothetical intermediate 2-hydroxyflavanone nor a dehydratase activity capable of converting the chemically synthesized compound to flavone could be observed. Moreover, the introduction of the double bond into the substrate naringenin was not altered by addition of chemically synthesized 2-hydroxynaringenin into the reaction mixture. Therefore, 2-hydroxyflavanones are apparently not freely dissociable intermediates in the biosynthesis of flavones in parsley and are not capable of entering the active site of the enzyme to compete with the flavanone. It is postulated that flavone synthase I catalyzes double-bond formation by direct abstraction of vicinal hydrogen atoms at C-2 and C-3 of the substrate. Thus, flavone synthase I is a member of a novel subgroup within the 2-oxoglutarate-dependent dioxygenases that can be referred to as 2-oxoglutarate-dependent desaturases.
Britsch
L
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Biologisches Institut II, Lehrstuhl für Biochemie der Pflanzen, Albert-Ludwigs-Universität, Freiburg, Bundesrepublik Deutschland.
D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D005419:Flavonoids / Q000138:chemical synthesis; D007525:Isoelectric Focusing; D008027:Light; D046911:Macromolecular Substances; D006899:Mixed Function Oxygenases / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D013379:Substrate Specificity
D002250:Carbon Radioisotopes; D005419:Flavonoids; D046911:Macromolecular Substances; D006899:Mixed Function Oxygenases; C065998:flavone synthase I