Publications

• Reaction mechanism of chalcone isomerase. pH dependence, diffusion control, and product binding differences.

  Jez J.M., Noel J.P.

  Chalcone isomerase (CHI) catalyzes the intramolecular cyclization of bicyclic chalcones into tricyclic (S)-flavanones. The activity of CHI is essential for the biosynthesis of flavanone precursors of floral pigments and phenylpropanoid plant defense compounds. We have examined the spontaneous and ... >> More

  Chalcone isomerase (CHI) catalyzes the intramolecular cyclization of bicyclic chalcones into tricyclic (S)-flavanones. The activity of CHI is essential for the biosynthesis of flavanone precursors of floral pigments and phenylpropanoid plant defense compounds. We have examined the spontaneous and CHI-catalyzed cyclization reactions of 4,2',4',6'-tetrahydroxychalcone, 4,2',4'-trihydroxychalcone, 2',4'-dihydroxychalcone, and 4,2'-dihydroxychalcone into the corresponding flavanones. The pH dependence of flavanone formation indicates that both the non-enzymatic and enzymatic reactions first require the bulk phase ionization of the substrate 2'-hydroxyl group and subsequently on the reactivity of the newly formed 2'-oxyanion during C-ring formation. Solvent viscosity experiments demonstrate that at pH 7.5 the CHI-catalyzed cyclization reactions of 4,2',4',6'-tetrahydroxychalcone, 4,2',4'-trihydroxychalcone, and 2',4'-dihydroxychalcone are approximately 90% diffusion-controlled, whereas cyclization of 4,2'-dihydroxychalcone is limited by a chemical step that likely reflects the higher pK(a) of the 2'-hydroxyl group. At pH 6.0, the reactions with 4,2',4',6'-tetrahydroxychalcone and 4,2',4'-trihydroxychalcone are approximately 50% diffusion-limited, whereas the reactions of both dihydroxychalcones are limited by chemical steps. Comparisons of the 2.1-2.3 A resolution crystal structures of CHI complexed with the products 7,4'-dihydroxyflavanone, 7-hydroxyflavanone, and 4'-hydroxyflavanone show that the 7-hydroxyflavanones all share a common binding mode, whereas 4'-hydroxyflavanone binds in an altered orientation at the active site. Our functional and structural studies support the proposal that CHI accelerates the stereochemically defined intramolecular cyclization of chalcones into biologically active (2S)-flavanones by selectively binding an ionized chalcone in a conformation conducive to ring closure in a diffusion-controlled reaction. << Less

  J. Biol. Chem. 277:1361-1369(2002) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Use of 3-Deoxy-D-arabino-heptulosonic acid 7-phosphate Synthase (DAHP Synthase) to Enhance the Heterologous Biosynthesis of Diosmetin and Chrysoeriol in an Engineered Strain of <i>Streptomyces albidoflavus</i>.

  Perez-Valero A., Serna-Diestro J., Villar C.J., Lombo F.

  Flavonoids are a large family of polyphenolic compounds with important agro-industrial, nutraceutical, and pharmaceutical applications. Among the structural diversity found in the flavonoid family, methylated flavonoids show interesting characteristics such as greater stability and improved oral b ... >> More

  Flavonoids are a large family of polyphenolic compounds with important agro-industrial, nutraceutical, and pharmaceutical applications. Among the structural diversity found in the flavonoid family, methylated flavonoids show interesting characteristics such as greater stability and improved oral bioavailability. This work is focused on the reconstruction of the entire biosynthetic pathway of the methylated flavones diosmetin and chrysoeriol in <i>Streptomyces albidoflavus</i>. A total of eight different genes (TAL, 4CL, CHS, CHI, FNS1, F3'H/CPR, 3'-OMT, 4'-OMT) are necessary for the heterologous biosynthesis of these two flavonoids, and all of them have been integrated along the chromosome of the bacterial host. The biosynthesis of diosmetin and chrysoeriol has been achieved, reaching titers of 2.44 mg/L and 2.34 mg/L, respectively. Furthermore, an additional compound, putatively identified as luteolin 3',4'-dimethyl ether, was produced in both diosmetin and chrysoeriol-producing strains. With the purpose of increasing flavonoid titers, a 3-Deoxy-D-arabino-heptulosonic acid 7-phosphate synthase (DAHP synthase) from an antibiotic biosynthetic gene cluster (BGC) from <i>Amycolatopsis balhimycina</i> was heterologously expressed in <i>S. albidoflavus</i>, enhancing diosmetin and chrysoeriol production titers of 4.03 mg/L and 3.13 mg/L, which is an increase of 65% and 34%, respectively. To the best of our knowledge, this is the first report on the de novo biosynthesis of diosmetin and chrysoeriol in a heterologous host. << Less

  Int J Mol Sci 25:2776-2776(2024) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Structure and mechanism of the evolutionarily unique plant enzyme chalcone isomerase.

  Jez J.M., Bowman M.E., Dixon R.A., Noel J.P.

  Chalcone isomerase (CHI) catalyzes the intramolecular cyclization of chalcone synthesized by chalcone synthase (CHS) into (2S)-naringenin, an essential compound in the biosynthesis of anthocyanin pigments, inducers of Rhizobium nodulation genes, and antimicrobial phytoalexins. The 1.85 A resolutio ... >> More

  Chalcone isomerase (CHI) catalyzes the intramolecular cyclization of chalcone synthesized by chalcone synthase (CHS) into (2S)-naringenin, an essential compound in the biosynthesis of anthocyanin pigments, inducers of Rhizobium nodulation genes, and antimicrobial phytoalexins. The 1.85 A resolution crystal structure of alfalfa CHI in complex with (2S)-naringenin reveals a novel open-faced beta-sandwich fold. Currently, proteins with homologous primary sequences are found only in higher plants. The topology of the active site cleft defines the stereochemistry of the cyclization reaction. The structure and mutational analysis suggest a mechanism in which shape complementarity of the binding cleft locks the substrate into a constrained conformation that allows the reaction to proceed with a second-order rate constant approaching the diffusion controlled limit. This structure raises questions about the evolutionary history of this structurally unique plant enzyme. << Less

  Nat. Struct. Biol. 7:786-791(2000) [PubMed] [EuropePMC]

• Role of hydrogen bonds in the reaction mechanism of chalcone isomerase.

  Jez J.M., Bowman M.E., Noel J.P.

  In flavonoid, isoflavonoid, and anthocyanin biosynthesis, chalcone isomerase (CHI) catalyzes the intramolecular cyclization of chalcones into (S)-flavanones with a second-order rate constant that approaches the diffusion-controlled limit. The three-dimensional structures of alfalfa CHI complexed w ... >> More

  In flavonoid, isoflavonoid, and anthocyanin biosynthesis, chalcone isomerase (CHI) catalyzes the intramolecular cyclization of chalcones into (S)-flavanones with a second-order rate constant that approaches the diffusion-controlled limit. The three-dimensional structures of alfalfa CHI complexed with different flavanones indicate that two sets of hydrogen bonds may possess critical roles in catalysis. The first set of interactions includes two conserved amino acids (Thr48 and Tyr106) that mediate a hydrogen bond network with two active site water molecules. The second set of hydrogen bonds occurs between the flavanone 7-hydroxyl group and two active site residues (Asn113 and Thr190). Comparison of the steady-state kinetic parameters of wild-type and mutant CHIs demonstrates that efficient cyclization of various chalcones into their respective flavanones requires both sets of contacts. For example, the T48A, T48S, Y106F, N113A, and T190A mutants exhibit 1550-, 3-, 30-, 7-, and 6-fold reductions in k(cat) and 2-3-fold changes in K(m) with 4,2',4'-trihydroxychalcone as a substrate. Kinetic comparisons of the pH-dependence of the reactions catalyzed by wild-type and mutant enzymes indicate that the active site hydrogen bonds contributed by these four residues do not significantly alter the pK(a) of the intramolecular cyclization reaction. Determinations of solvent kinetic isotope and solvent viscosity effects for wild-type and mutant enzymes reveal a change from a diffusion-controlled reaction to one limited by chemistry in the T48A and Y106F mutants. The X-ray crystal structures of the T48A and Y106F mutants support the assertion that the observed kinetic effects result from the loss of key hydrogen bonds at the CHI active site. Our results are consistent with a reaction mechanism for CHI in which Thr48 polarizes the ketone of the substrate and Tyr106 stabilizes a key catalytic water molecule. Hydrogen bonds contributed by Asn113 and Thr190 provide additional stabilization in the transition state. Conservation of these residues in CHIs from other plant species implies a common reaction mechanism for enzyme-catalyzed flavanone formation in all plants. << Less

  Biochemistry 41:5168-5176(2002) [PubMed] [EuropePMC]