Lothar Britsch

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.

Divergent evolution of flavonoid 2-oxoglutarate-dependent dioxygenases in parsley1

Flavone synthases (FNSs) catalyze the oxidation of flavanones to flavones, i.e. the formation of apigenin from (2S)‐naringenin. While many plants express a microsomal‐type FNS II, the soluble FNS I appears to be confined to a few species of the Apiaceae and was cloned recently from parsley plants. FNS I belongs to the FeII/2‐oxoglutarate‐dependent dioxygenases characterized by short conserved sequence elements for cofactor binding, and its evolutionary context and mode of action are under investigation. Using a homology‐based reverse transcription polymerase chain reaction approach, two additional flavonoid‐specific dioxygenases were cloned from immature parsley leaflets, which were identified as flavanone 3β‐hydroxylase (FHT) and flavonol synthase (FLS) after expression in yeast cells. Sequence alignments revealed marginal differences among the parsley FNS I and FHT polypeptides of only 6%, while much less identity (about 29%) was observed with the parsley FLS. Analogous to FNS I, FLS oxidizes the flavonoid γ‐pyrone by introducing a C2, C3 double bond, and (2R,3S)‐dihydrokaempferol (cis‐dihydrokaempferol) was proposed recently as the most likely intermediate in both FNS I and FLS catalysis. Incubation of either FNS I or FLS with cis‐dihydrokaempferol exclusively produced kaempferol and confirmed the assumption that flavonol formation occurs via hydroxylation at C3 followed by dehydratation. However, the lack of apigenin in these incubations ruled out cis‐dihydrokaempferol as a free intermediate in FNS I catalysis. Furthermore, neither (+)‐trans‐dihydrokaempferol nor unnatural (−)‐trans‐dihydrokaempferol and 2‐hydroxynaringenin served as a substrate for FNS I. Overall, the data suggest that FNS I has evolved uniquely in some Apiaceae as a paraphyletic gene from FHT, irrespective of the fact that FNS I and FLS catalyze equivalent desaturation reactions.

Flavonol synthase from Citrus unshiu is a bifunctional dioxygenase.

Flavonol synthase was classified as a 2-oxoglutarate-dependent dioxygenase converting natural (2R,3R)-dihydroflavonols, i.e. dihydrokaempferol, to the corresponding flavonols (kaempferol). Flavonol synthase from Citrus unshiu (Satsuma mandarin), expressed in Escherichia coli and purified to homogeneity, was shown to accept also (2S)-naringenin as a substrate, producing kaempferol in high yield and assigning sequential flavanone 3beta-hydroxylase and flavonol synthase activities to the enzyme. In contrast, dihydrokaempferol was identified as the predominant product from assays performed with the unnatural (2R)-naringenin as substrate. The product which was not converted any further on repeated incubations was identified by 1H NMR and CD spectroscopies as (-)-trans-dihydrokaempferol. The data demonstrate that Citrus flavonol synthase encompasses an additional non-specific activity trans-hydroxylating the flavanones (2S)-naringenin as well as the unnatural (2R)-naringenin at C-3.

Purification of flavanone 3β-hydroxylase from Petunia hybrida : antibody preparation and characterization of a chemogenetically defined mutant

Flavanone 3 beta-hydroxylase from Petunia hybrida has been purified to apparent homogeneity utilizing an improved purification protocol including chromatography of the partially purified enzyme on hydroxyapatite, chromatofocusing, and hydrophobic interaction chromatography on phenyl-Superose. The specificity of mouse and rabbit polyclonal antisera directed to flavanone 3 beta-hydroxylase was demonstrated by Western blotting and immunotitration with crude extracts from wild-type flowers of P. hybrida and with the purified enzyme. Cross-reactivity was observed with flavanone 3 beta-hydroxylase from extracts of illuminated parsley cells. A Petunia mutant with white flowers, previously shown to lack 3 beta-hydroxylase activity and to accumulate flavanone glycosides, showed complete absence of the enzyme protein.

Size-exclusion chromatography of plasma proteins with high molecular masses

Two different hydrophilic materials with large pores, Superose 6 and Fractogel EMD BioSec (S), which are designed for size-exclusion chromatography (SEC) of plasma proteins with high molecular masses, are tested for their performance on a preparative scale. The model mixtures are preparations of the clotting factors VIII (FVIII) and IX (FIX). A combination of a Fractogel EMD BioSec (S) column and a Superose 6 column has proved to be particularly effective for separations in a wide molecular size range, from several millions down to about 20,000. Superose 6 showed good results on a small scale as well as on a large scale, even in the molecular mass range over 1,000,000. However, recovery of FVIII clotting activity was less than 70% with this material and therefore not satisfactory. Fractogel did not perform well in terms of separation on a small scale. However, in the case of biopolymers with high molecular masses, separation was improved by using larger columns. With Fractogel, recovery of activity of the two clotting factors FVIII and FIX was satisfactory, above 80%. On a large scale, the active fraction in the clotting factor concentrate was successfully separated from the non-active fraction with either size exclusion (SE) material. In the preparation under investigation, the clotting factor VIII is found in a complex with the von Willebrand factor (vWF). The FVIII-vWF complex has a molecular mass of several millions. It dissociates in the presence of high concentrations of Ca2+ ions. Under such conditions FVIII and vWF were successfully separated with both SEC columns.

Size Exclusion Chromatography on Fractogel EMD BioSEC

Publisher Summary Size exclusion chromatography (SEC) is the predominant chromatographic technique for determining the molecular weight distribution of macromolecules. The separation mode is based on the differential penetration of molecules of different size and shape into the pores of the stationary phase. Thus, gels made for SEC should have controlled range of size of the pores. One main advantage of SEC is that: very mild conditions for biological macromolecules separation is utilized, such as the presence of essential ions or cofactors, high or low ionic strength, or other buffer additives (detergents, urea, glycerol). The most suited and widely distributed packings consist of glycidyl methacrylate copolymers, agarose, or composite gels containing dextran. The methacrylate-based copolymer Fractogel EMD BioSEC used for large-scale purification of biological macromolecules is introduced to overcome the drawback derived from the poor pressure stability of soft gels. One main advantage of the new Fractogel EMD BioSEC is its wide application range for proteins and large peptides over a molecular weight range from 5–1000 kDa. Compared to conventional soft gels for SEC, the rigid polymer Fractogel EMD BioSEC is easier to pack into large columns and it gives stable gel packings that allow relatively high linear flow rates. The high selectivity of Fractogel EMD BioSEC allows efficient purification steps, yielding good purification factors.