Comparison of Nonheme Manganese- and Iron-Containing Flavone Synthase Mimics

Heme and nonheme-type flavone synthase enzymes, FS I and FS II are responsible for the synthesis of flavones, which play an important role in various biological processes, and have a wide range of biomedicinal properties including antitumor, antimalarial, and antioxidant activities. To get more insight into the mechanism of this curious enzyme reaction, nonheme structural and functional models were carried out by the use of mononuclear iron, [FeII(CDA-BPA*)]2+ (6) [CDA-BPA = N,N,N’,N’-tetrakis-(2-pyridylmethyl)-cyclohexanediamine], [FeII(CDA-BQA*)]2+ (5) [CDA-BQA = N,N,N’,N’-tetrakis-(2-quinolilmethyl)-cyclohexanediamine], [FeII(Bn-TPEN)(CH3CN)]2+ (3) [Bn-TPEN = N-benzyl-N,N’,N’-tris(2-pyridylmethyl)-1,2- diaminoethane], [FeIV(O)(Bn-TPEN)]2+ (9), and manganese, [MnII(N4Py*)(CH3CN)]2+ (2) [N4Py* = N,N-bis(2-pyridylmethyl)-1,2-di(2-pyridyl)ethylamine)], [MnII(Bn-TPEN)(CH3CN)]2+ (4) complexes as catalysts, where the possible reactive intermediates, high-valent FeIV(O) and MnIV(O) are known and well characterised. The results of the catalytic and stoichiometric reactions showed that the ligand framework and the nature of the metal cofactor significantly influenced the reactivity of the catalyst and its intermediate. Comparing the reactions of [FeIV(O)(Bn-TPEN)]2+ (9) and [MnIV(O)(Bn-TPEN)]2+ (10) towards flavanone under the same conditions, a 3.5-fold difference in reaction rate was observed in favor of iron, and this value is three orders of magnitude higher than was observed for the previously published [FeIV(O)(N2Py2Q*)]2+ [N,N-bis(2-quinolylmethyl)-1,2-di(2-pyridyl)ethylamine] species.

Microbial Production of Diosmetin from Hesperetin in Engineered Escherichia coli with Flavone Synthase and Flavonol Synthase

In this study, diosmetin was synthesized with recombinant Escherichia coli expressing flavone synthase (FNS) or flavonol synthase (FLS). Forty-four FNS/FLS were selected from 40 different plants and their bioinformatic data, such as isoelectric point, instability index, grand average of hydropathicity, transmembrane structure, secondary structure, and conservative domain were analyzed with computer tools or software. Nine recombinant E. coli strains expressing FNS/FLS were constructed for diosmetin synthesis, and the products were detected through UPLC, LC-MS, and SDS-PAGE. Results showed that FNS/FLS from different sources were different in transmembrane structures, instability coefficients, and conservative regions. Among the nine recombinant E. coli strains, six recombinant E. coli strains were observed expected bands by SDS-PAGE, four recombinant E. coli strains were detected to have diosmetin with a molecular weight of 300.06 confirmed by LC-MS in broth, and the diosmetin concentration of a DE3/pAnFNS fermentation broth was the highest (39.6 mg/L). The enzyme expression and catalytic reaction were accordance with the results of bioinformatics analysis. In addition, the ratio of predicted intermediate product (4'-O-Methyl taxifolin) and final product (diosmetin) was significant different among FNS/FLS from different sources, although they are similar in physicochemical properties and structures. Therefore, the hypothesis that FNS/FLS catalyzed the synthesis of diosmetin from hesperetin by hydroxylation at the C-2 and C-3 positions respectively, 2-hydroxyhesperetin undergoes an elimination reaction and is converted to diosmetin, 3-hydroxyhesperetin (4'-O-Methyl taxifolin) was retained as a byproduct has been proposed.Key points:1) A possible biosynthesis pathway of diosmetin was proposed.2) Diosmetin was biosynthesized by expressing FNS and FLS in recombinant Escherichia coli strains.3) Enzyme selection in biosynthesis was guided with bioinformatic analysis.4) The speculation that simultaneous hydroxylation of the reaction occurs at both C-2 and C-3 positions of flavanones has been proposed.

Flavonoids are indispensable for complete male fertility in rice

Flavonoids are essential for male fertility in some but not all plant species. In rice (Oryza sativa), the chalcone synthase mutant oschs1 produces flavonoid-depleted pollen and is male sterile. The mutant pollen grains are viable with normal structure, but they display reduced germination rate and pollen-tube length. Analysis of oschs1/+ heterozygous lines shows that pollen flavonoid deposition is a paternal effect and fertility is independent of the haploid genotypes (OsCHS1 or oschs1). To understand which classes of flavonoids are involved in male fertility, we conducted detailed analysis of rice mutants for branch-point enzymes of the downstream flavonoid pathways, including flavanone 3-hydroxylase (OsF3H; flavonol pathway entry enzyme), flavone synthase II (CYP93G1; flavone pathway entry enzyme), and flavanone 2-hydroxylase (CYP93G2; flavone C-glycoside pathway entry enzyme). Rice osf3h and cyp93g1 cyp93g2 CRISPR/Cas9 mutants, and cyp93g1 and cyp93g2 T-DNA insertion mutants showed altered flavonoid profiles in anthers, but only the osf3h and cyp93g1 cyp93g2 mutants displayed reduction in seed yield. Our findings indicate that flavonoids are essential for complete male fertility in rice and a combination of different classes (flavanones, flavonols, flavones, and flavone C-glycosides) appears to be important, as opposed to the essential role played primarily by flavonols that has been previously reported in several plant species.

Flavones Produced by Mulberry Flavone Synthase Type I Constitute a Defense Line against the Ultraviolet-B Stress

Flavones, one of the largest classes of flavonoids in plants, have a variety of bioactivities and participate in the resistance response of plants to biotic and abiotic stresses. However, flavone synthase (FNS), the key enzyme for flavone biosynthesis, has not yet been characterized in mulberry. In this study, we report that the leaves of certain mulberry cultivars, namely BJ7, PS2, and G14, are rich in flavones. We identified a Fe2+/2-oxoglutarate-dependent dioxygenase from Morus notabilis (MnFNSI) that shows the typical enzymatic activity of a FNSI-type enzyme, and directly converts eriodictyol and naringenin into their corresponding flavones. Overexpression of MnFNSI in tobacco increased the flavones contents in leaves and enhanced the tolerance of tobacco to ultraviolet-B (UV-B) stress. We found that mulberry cultivars with higher flavones contents exhibit less UV-B induced damage after a UV-B treatment. Accordingly, our findings demonstrate that MnFNSI, a FNSI-type enzyme, is involved in the biosynthesis of flavones, which provide protection against UV-B radiation. These results lay the foundation for obtaining mulberry germplasm resources that are more tolerant to UV-B stress and richer in their nutritional value.