A cluster of genes encodes the two types of chalcone isomerase involved in the biosynthesis of general flavonoids and legume-specific 5-deoxy(iso)flavonoids in Lotus japonicus

Leguminous plants produce 5-deoxyflavonoids and 5-deoxyisoflavonoids that play essential roles in legume-microbe interactions. Together with chalcone polyketide reductase and cytochrome P450 2-hydroxyisoflavanone synthase, the chalcone isomerase (CHI) of leguminous plants is fundamental in the construction of these ecophysiologically active flavonoids. Although CHIs of nonleguminous plants isomerize only 6'-hydroxychalcone to 5-hydroxyflavanone (CHIs with this function are referred to as type I), leguminous CHIs convert both 6'-deoxychalcone and 6'-hydroxychalcone to 5-deoxyflavanone and 5-hydroxyflavanone, respectively (referred to as type II). In this study, we isolated multiple CHI cDNAs (cCHI1-cCHI3) from a model legume, Lotus japonicus. In contrast to previous observations, the amino acid sequence of CHI2 was highly homologous to nonleguminous CHIs, whereas CHI1 and CHI3 were the conventional leguminous type. Furthermore, genome sequence analysis revealed that four CHI genes (CHI1-3 and a putative gene, CHI4) form a tandem cluster within 15 kb. Biochemical analysis with recombinant CHIs expressed in Escherichia coli confirmed that CHI1 and CHI3 are type II CHIs and that CHI2 is a type I CHI. The occurrence of both types of CHIs is probably common in leguminous plants, and it was suggested that type II CHIs evolved from an ancestral CHI by gene duplication and began to produce 5-deoxy(iso)flavonoids along with the establishment of the Fabaceae.     

A genomic approach to isoflavone biosynthesis in kudzu (<Emphasis Type="Italic">Pueraria lobata</Emphasis>)

Roots of kudzu (Pueraria lobata) are a rich source of isoflavone O- and C-glycosides. Although O-glycosylation of (iso)flavonoids has been well characterized at the molecular level, no plant isoflavonoid C-glycosyltransferase genes have yet been isolated. To address the biosynthesis of kudzu isoflavonoids, we generated 6,365 high-quality expressed sequence tags (ESTs) from a subtraction cDNA library constructed using RNA from roots that differentially accumulate puerarin. The ESTs were clustered into 722 TCs and 3,913 singletons, from which 15 family I glycosyltransferases (UGTs) were identified. Hierarchical clustering analysis of the expression patterns of these UGTs with isoflavone synthase (IFS) in a range of tissues identified UGTs with potential functions in isoflavone glycosylation. The open reading frames of these UGTs were expressed in E. coli for functional analysis, and one was shown to preferentially glycosylate isoflavones at the 7-O-position. In addition, ESTs corresponding to chalcone synthase, chalcone reductase, chalcone isomerase (CHI) and 2-hydroxyisoflavanone dehydratase were identified. Recombinant CHI proteins had high activities with both 6′-deoxy- and 6′-hydroxy chalcones, typical of Type II CHIs. Establishment of this EST database and identification of genes associated with kudzu isoflavone biosynthesis and glycosylation provide a new resource for metabolic engineering of bioactive kudzu isoflavones.     

Flavone synthase II (CYP93B16) from soybean (Glycine max L.)

Flavonoids are a very diverse group of plant secondary metabolites with a wide array of activities in plants, as well as in nutrition and health. All flavonoids are derived from a limited number of flavanone intermediates, which serve as substrates for a variety of enzyme activities, enabling the generation of diversity in flavonoid structures. Flavonoids can be characteristic metabolites, like isoflavonoids for legumes. Others, like flavones, occur in nearly all plants. Interestingly, there exist two fundamentally different enzymatic systems able to directly generate flavones from flavanones, flavone synthase (FNS) I and II. We describe an inducible flavone synthase activity from soybean (Glycine max) cell cultures, generating 7,4′-dihydroxyflavone (DHF), which we classified as FNS II. The corresponding full-length cDNA (CYP93B16) was isolated using known FNS II sequences from other plants. Functional expression in yeast allowed the detailed biochemical characterization of the catalytic activity of FNS II. A direct conversion of flavanones such as liquiritigenin, naringenin, and eriodictyol into the corresponding flavones DHF, apigenin and luteolin, respectively, was demonstrated. The enzymatic reaction of FNS II was stereoselective, favouring the (S)- over the (R)-enantiomer. Phylogenetic analyses of the subfamily of plant CYP93B enzymes indicate the evolution of a gene encoding a flavone synthase which originally catalyzed the direct conversion of flavanones into flavones, via early gene duplication into a less efficient enzyme with an altered catalytic mechanism. Ultimately, this allowed the evolution of the legume-specific isoflavonoid synthase activity.     

Physical interactions among flavonoid enzymes in snapdragon and torenia reveal the diversity in the flavonoid metabolon organization of different plant species

Flavonoid metabolons (weakly‐bound multi‐enzyme complexes of flavonoid enzymes) are believed to occur in diverse plant species. However, how flavonoid enzymes are organized to form a metabolon is unknown for most plant species. We analyzed the physical interaction partnerships of the flavonoid enzymes from two lamiales plants (snapdragon and torenia) that produce flavones and anthocyanins. In snapdragon, protein–protein interaction assays using yeast and plant systems revealed the following binary interactions: flavone synthase II (FNSII)/chalcone synthase (CHS); FNSII/chalcone isomerase (CHI); FNSII/dihydroflavonol 4‐reductase (DFR); CHS/CHI; CHI/DFR; and flavonoid 3′‐hydroxylase/CHI. These results along with the subcellular localizations and membrane associations of snapdragon flavonoid enzymes suggested that FNSII serves as a component of the flavonoid metabolon tethered to the endoplasmic reticulum (ER). The observed interaction partnerships and temporal gene expression patterns of flavonoid enzymes in red snapdragon petal cells suggested the flower stage‐dependent formation of the flavonoid metabolon, which accounted for the sequential flavone and anthocyanin accumulation patterns therein. We also identified interactions between FNSII and other flavonoid enzymes in torenia, in which the co‐suppression of FNSII expression was previously reported to diminish petal anthocyanin contents. The observed physical interactions among flavonoid enzymes of these plant species provided further evidence supporting the long‐suspected organization of flavonoid metabolons as enzyme complexes tethered to the ER via cytochrome P450, and illustrated how flavonoid metabolons mediate flower coloration. Moreover, the observed interaction partnerships were distinct from those previously identified in other plant species (Arabidopsis thaliana and soybean), suggesting that the organization of flavonoid metabolons may differ among plant species.     

Engineering isoflavone metabolism with an artificial bifunctional enzyme

Plant secondary metabolism has been a focus of research in recent years due to its significant roles in plant defense and in human medicine and nutrition. A protein engineering strategy was designed to more effectively manipulate plant secondary metabolite (isoflavonoid) biosynthesis. A bifunctional isoflavone synthase/chalcone isomerase (IFS/CHI) enzyme was constructed by in-frame gene fusion, and expressed in yeast and tobacco. The fusion protein was targeted to the endoplasmic reticulum (ER) membrane and the individual enzymatic functions of its component fragments were retained when assayed in yeast. Petals and young leaves of IFS/CHI transgenic tobacco plants produced higher levels of the isoflavone genistein and genistein glycosides as a ratio of total flavonoids produced than did plants transformed with IFS alone. Thus, through a combined molecular modeling, in vitro protein engineering and in planta metabolic engineering approach, it was possible to increase the potential for accumulation of isoflavonoid compounds in non-legume plants. Construction of bifunctional enzymes will simplify the transformation of plants with multiple pathway genes, and such enzymes may find broad uses for enzyme (e.g., cytochrome P450 family) and biochemical pathway engineering.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

大豆异黄酮生物合成关键酶及其代谢工程研究进展

异黄酮是一类具有C-6/C-3/C-6骨架的二次代谢产物,具有抗氧化和抗肿瘤活性。异黄酮与黄酮类物质具有相似的苯丙烷生物合成途径。天然的绝大部分异黄酮分布在豆科植物中,目前在大豆中已经发现了超过12个异黄酮(苷)。大豆异黄酮的生物合成主要涉及三个关键的酶查尔酮合酶(CHS)、查尔酮异构酶(CHI)和异黄酮合酶(IFS)。总结了大豆异黄酮的提取分离方法和生物合成途径,着重综述了CHI、CHS、IFS生物学特征和功能及异黄酮的代谢工程研究。     

Regulation of Flavonoid Biosynthetic Genes in Germinating Arabidopsis Seedlings

Many higher plants, including Arabidopsis, transiently display purple anthocyanin pigments just after seed germination. We observed that steady state levels of mRNAs encoded by four flavonoid biosynthetic genes, PAL1 (encoding phenylalanine ammonia-lyase 1), CHS (encoding chalcone synthase), CHI (encoding chalcone isomerase), and DFR (encoding dihydroflavonol reductase), were temporally regulated, peaking in 3-day-old seedlings grown in continuous white light. Except for the case of PAL1 mRNA, mRNA levels for these flavonoid genes were very low in seedlings grown in darkness. Light induction studies using seedlings grown in darkness showed that PAL1 mRNA began to accumulate before CHS and CHI mRNAs, which, in turn, began to accumulate before DFR mRNA. This order of induction is the same as the order of the biosynthetic steps in flavonoid biosynthesis. Our results suggest that the flavonoid biosynthetic pathway is coordinately regulated by a developmental timing mechanism during germination. Blue light and UVB light induction experiments using red light- and dark-grown seedlings showed that the flavonoid biosynthetic genes are induced most effectively by UVB light and that blue light induction is mediated by a specific blue light receptor.     

Chalcone synthesis with enzyme extracts from tulip anther tapetum using a biphasic enzyme assay

The in vitro synthesis of chalcones has been demonstrated using a special biphasic enzyme assay. The highly viscous lower phase in this assay stems from a tapetum fraction of anthers of Tulipa cv. “Apeldoorn” which has been used an enzyme source. The upper phase of this system consists of a reaction mixture of the normal “flavanone synthase” assay. It is suggested that chalcone synthesis occurs at the boundary layer between the two phases. To prevent spontaneous as well as enzymatic cyclization of the chalcones formed (phloroglucinyl type), the pH of the upper phase must not be allowed to exceed pH 4.0. Under these pH conditions, chalcone formation by a reverse reaction of chalcone-flavanone isomerase can be excluded. The measured substrate specificity of the “chalcone synthase” corresponds to the conditions of chalcone formation in the natural system. Using p-coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA, respectively, as substrates, the enzyme system forms the correspondingly substituted chalcones which are also accumulated in the loculus of tulip anthers. It is suggested that this chalcone synthase is identical to the previously described “flavanone synthase”. The results can be further explained as follows. (i) Not flavanones, but rather chalcones are the first C₁₅ intermediates of flavonoid biosynthesis in tulip anthers. (ii) In this Tulipa system, the substitution pattern of three different hydroxycinnamic acids can be transferred unchanged into the flavonoid C₁₅ stage. (iii) The role of chalcone-flavanone isomerase is to cyclize chalcones to flavanones on the direct biosynthetic pathway to the further accumulated flavonol glycosides. (iv) The sensitivity of the reaction with regard to chalcone production points to the localization of chalcone synthase in a most unstable and, up to now, unknown tapetal compartment. Since purification of the enzyme results in exclusive production of flavanones, it is suggested that certain “chalcone stabilizing factors” must occur in the natural system. (v) The phenomenon of chalcone accumulation in tulip anthers, however, must be caused by a complex system, distinguished by cooperation of certain biochemical and physiological conditions, and, finally, by special compartmentation of the enzymes which are responsible for the biosynthesis of flavonoids.     

Responses of the flavonoid pathway to UV-B radiation stress and the correlation with the lipid antioxidant characteristics in the desert plant Caryopteris mongolica

Caryopteris mongolica is a dwarf shrub mainly found in grassland and desert areas of north-west China, and which can survive severe environmental stress. This study aimed to assess the responses of the flavonoid pathway to UV-B radiation treatments and its correlation to the lipid peroxide and antioxidant systems in C. mongolica. In UV-B radiation experiments, plants were exposed to UV-B radiation treatments with a intensity of 30 J/s for 1, 4 and 24 h, respectively. A control group without UV-B radiation treatment was also used. The chlorophyll fluorescence parameters, contents of chlorophyll and carotenoid, levels of lipid peroxidation, activities of antioxidant system enzymes, accumulations of total flavonoids and anthocyanins, and activities of phenylalanine ammonialyase (PAL) and chalcone isomerase (CHI) under different UV-B radiation treatments were investigated. The correlations between products and key enzymes in the flavonoid pathway and the lipid peroxide and antioxidant systems were also analyzed. The results showed that chlorophyll fluorescence parameters decreased within 24 h of treatment. The chlorophyll contents decreased within 4 h and remained stable after 24 h. Carotenoid content significantly increased. The level of MDA, the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and peroxidase (POD) and the contents of total flavonoids and anthocyanidins increased, while catalase (CAT) activity decreased under UV-B stress. The activities of PAL and CHI also increased with the increased content of total flavonoids. The flavonoid products anthocyanidins had a significant positive correlation with MDA level, as well as the activities of antioxidant enzyme SOD. In conclusion, UV-B radiation induced the degradation of photosynthetic pigments and decreased photochemical efficiency of Photosystem II; increased the contents of MDA, total flavonoids and anthocyanidins; and also enhanced activities of antioxidant enzymes (SOD, APX and POD) and key enzymes (PAL and CHI) in the flavonoid pathway in C. mongolica. Thus, we speculate that the flavonoid pathway were involved in the regulation of stress resistance in C. mongolica.     

Flavonoid genes of pear (<Emphasis Type="Italic">Pyrus communis</Emphasis>)

Pear (Pyrus sp.) is a major fruit crop of temperate regions with increasing extent of cultivation. Pear flavonoids contribute to its fruit color, pathogen defense, and are health beneficial ingredients of the fruits. Comparative Southern analyses with apple (Malus x domestica) cDNAs showed comparable genomic organization of flavonoid genes of both related genera. A homology-based cloning approach was used to obtain the cDNAs of most enzymes of the main flavonoid pathway of Pyrus: phenylalanine ammonia lyase, chalcone synthase, chalcone isomerase, flavanone 3β-hydroxylase, flavonol synthase, dihydroflavonol 4-reductase, leucoanthocyanidin reductase 1 and 2, anthocyanidin synthase, anthocyanidin reductase, and UDP-glucose : flavonoid 7-O-glucosyltransferase. The substrate specificities of the recombinant enzymes expressed in yeast were determined for physiological and non-physiological substrates and found to be in general agreement with the characteristic pear flavonoid metabolite pattern of mainly B-ring dihydroxylated anthocyanins, flavonols, catechins, and flavanones. Furthermore, significant differences in substrate specificities and gene copy numbers in comparison to Malus were identified. Cloning of the cDNAs and studying the enzymes of the Pyrus flavonoid pathway is an essential task toward a comprehensive knowledge of Pyrus polyphenol metabolism. It also elucidates evolutionary patterns of flavonoid/polyphenol pathways in the Rosaceae, which allocate several important crop plants.     

Flavonoid 6-hydroxylase from soybean (Glycine max L.), a novel plant P-450 monooxygenase

Cytochrome P-450-dependent hydroxylases are typical enzymes for the modification of basic flavonoid skeletons. We show in this study that CYP71D9 cDNA, previously isolated from elicitor-induced soybean (Glycine max L.) cells, codes for a protein with a novel hydroxylase activity. When heterologously expressed in yeast, this protein bound various flavonoids with high affinity (1.6 to 52 microm) and showed typical type I absorption spectra. These flavonoids were hydroxylated at position 6 of both resorcinol- and phloroglucinol-based A-rings. Flavonoid 6-hydroxylase (CYP71D9) catalyzed the conversion of flavanones more efficiently than flavones. Isoflavones were hardly hydroxylated. As soybean produces isoflavonoid constituents possessing 6,7-dihydroxy substitution patterns on ring A, the biosynthetic relationship of flavonoid 6-hydroxylase to isoflavonoid biosynthesis was investigated. Recombinant 2-hydroxyisoflavanone synthase (CYP93C1v2) efficiently used 6,7,4'-trihydroxyflavanone as substrate. For its structural identification, the chemically labile reaction product was converted to 6,7,4'-trihydroxyisoflavone by acid treatment. The structures of the final reaction products for both enzymes were confirmed by NMR and mass spectrometry. Our results strongly support the conclusion that, in soybean, the 6-hydroxylation of the A-ring occurs before the 1,2-aryl migration of the flavonoid B-ring during isoflavanone formation. This is the first identification of a flavonoid 6-hydroxylase cDNA from any plant species.     

A chalcone isomerase‐like protein enhances flavonoid production and flower pigmentation

Flavonoids are major pigments in plants, and their biosynthetic pathway is one of the best‐studied metabolic pathways. Here we have identified three mutations within a gene that result in pale‐colored flowers in the Japanese morning glory (Ipomoea nil). As the mutations lead to a reduction of the colorless flavonoid compound flavonol as well as of anthocyanins in the flower petal, the identified gene was designated enhancer of flavonoid production (EFP). EFP encodes a chalcone isomerase (CHI)‐related protein classified as a type IV CHI protein. CHI is the second committed enzyme of the flavonoid biosynthetic pathway, but type IV CHI proteins are thought to lack CHI enzymatic activity, and their functions remain unknown. The spatio‐temporal expression of EFP and structural genes encoding enzymes that produce flavonoids is very similar. Expression of both EFP and the structural genes is coordinately promoted by genes encoding R2R3‐MYB and WD40 family proteins. The EFP gene is widely distributed in land plants, and RNAi knockdown mutants of the EFP homologs in petunia (Petunia hybrida) and torenia (Torenia hybrida) had pale‐colored flowers and low amounts of anthocyanins. The flavonol and flavone contents in the knockdown petunia and torenia flowers, respectively, were also significantly decreased, suggesting that the EFP protein contributes in early step(s) of the flavonoid biosynthetic pathway to ensure production of flavonoid compounds. From these results, we conclude that EFP is an enhancer of flavonoid production and flower pigmentation, and its function is conserved among diverse land plant species.     

Modular Optimization of Heterologous Pathways for De Novo Synthesis of (2S)-Naringenin in Escherichia coli

Due to increasing concerns about food safety and environmental issues, bio-based production of flavonoids from safe, inexpensive, and renewable substrates is increasingly attracting attention. Here, the complete biosynthetic pathway, consisting of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS), chorismate mutase/prephenate dehydrogenase (CM/PDH), tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), malonate synthetase, and malonate carrier protein, was constructed using pre-made modules to overproduce (2S)-naringenin from D-glucose. Modular pathway engineering strategies were applied to the production of the flavonoid precursor (2S)-naringenin from L-tyrosine to investigate the metabolic space for efficient conversion. Modular expression was combinatorially tuned by modifying plasmid gene copy numbers and promoter strengths to identify an optimally balanced pathway. Furthermore, a new modular pathway from D-glucose to L-tyrosine was assembled and re-optimized with the identified optimal modules to enable de novo synthesis of (2S)-naringenin. Once this metabolic balance was achieved, the optimum strain was capable of producing 100.64 mg/L (2S)-naringenin directly from D-glucose, which is the highest production titer from D-glucose in Escherichia coli. The fermentation system described here paves the way for the development of an economical process for microbial production of flavonoids.     

Metabolic engineering of isoflavone genistein in Brassica napus with soybean isoflavone synthase

Genistein, 4′,5,7-trihydroxyisoflavone, is an isoflavonoid compound predominantly restricted to legumes and known to possess phyto-oestrogenic and antioxidative activities. The key enzyme that redirects phenylpropanoid pathway intermediates from flavonoids to isoflavonoids is the isoflavone synthase (IFS). Brassica napus is a non-legume oilseed crop with vegetative tissues producing phenylpropanoids and flavonoids, but does not naturally accumulate isoflavones due to the absence of IFS. To demonstrate whether exogenous IFS is able to use endogenous substrate to produce isoflavone genistein in oilseed crop, the soybean IFS gene (GmIFS2) was incorporated into B. napus plants. The presence of GmIFS2 in B. napus was shown to direct the synthesis and accumulation of genistein derivatives in leaves up to 0.72 mg g⁻¹ DW. In addition, expression levels for most B. napus genes in the phenylpropanoid pathway were altered. These results suggest that the heterologous GmIFS2 enzyme is functionally active at using the B. napus naringenin as a substrate to produce genistein in oilseed rape.     

Structure, function, and engineering of enzymes in isoflavonoid biosynthesis

Isoflavonoids are a large group of plant natural products and play important roles in plant defense. They also possess valuable health-promoting activities with significant health benefits for animals and humans. The isoflavonoids are identified primarily in leguminous plants and are synthesized through the central phenylpropanoid pathway and the specific isoflavonoid branch pathways in legumes. Structural studies of some key enzymes in the central phenylpropanoid pathway shed light on the early stages of the (iso)flavonoid biosynthetic process. Significant impact has also been made on structural studies of enzymes in the isoflavonoid branch pathways. Structures of isoflavonoid-specific NADPH-dependent reductases revealed how the (iso)flavonoid backbones are modified by reduction reactions and how enzymes specifically recognize isoflavonoids and catalyze stereo-specific reductions. Structural studies of isoflavonoid methyltransferases and glycosyltransferases revealed how isoflavonoids are further decorated with methyl group and sugars in different methylation and glycosylation patterns that determine their bioactivities and functions. In combination with mutagenesis and biochemical studies, the detailed structural information of these enzymes provides a basis for understanding the complex biosynthetic process, enzyme catalytic mechanisms, and substrate specificities. Structure-based homology modeling facilitates the functional characterization of these large groups of biosynthetic enzymes and their homologs. Structure-based enzyme engineering is becoming a new strategy for synthesis of bioactive isoflavonoids and also facilitates plant metabolic engineering towards improvement of quality and production of crop plants.     

Regulation of gene expression involved in flavonol and anthocyanin biosynthesis during petal development in lisianthus (Eustoma grandiflorum)

To elucidate gene regulation of flower colour formation, the gene expressions of the enzymes involved in flavonoid biosynthesis were investigated in correlation with their product during floral development in lisianthus. Full-length cDNA clones of major responsible genes in the central flavonoid biosynthetic pathway, including chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3',5'-hydroxylase (F3'5'H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and flavonol synthase (FLS), were isolated and characterized. In lisianthus, the stage of the accumulation of flavonols and anthocyanins was shown to be divided clearly. The flavonol content increased prior to anthocyanin accumulation during floral development and declined when anthocyanin began to accumulate. CHS, CHI, and F3H were necessary for both flavonol and anthocyanin biosynthesis and were coordinately expressed throughout all stages of floral development; their expressions were activated independently at the stages corresponding to flavonol accumulation and anthocyanin accumulation, respectively. Consistent with flavonol and anthocyanin accumulation patterns, FLS, a key enzyme in flavonol biosynthesis, was expressed prior to the expression of the genes involved in anthocyanin biosynthesis. The genes encoding F3'5'H, DFR, and ANS were expressed at later stages, just before pigmentation. The genes responsible for the flavonoid pathways branching to anthocyanins and flavonols were strictly regulated and were coordinated temporally to correspond to the biosynthetic order of their respective enzymes in the pathways, as well as in specific organs. In lisianthus, FLS and DFR, at the position of branching to flavonols and anthocyanins, were supposed to play a critical role in regulation of each biosynthesis.     

Stereoselectivity of chalcone isomerase with chalcone derivatives: a computational study

Chalcone isomerase (CHI) catalyzes the intramolecular cyclization of chalcones into flavonoids. The activity of CHI is essential for the biosynthesis of flavonoids precursors of floral pigments and phenylpropanoid plant defense compounds. In the present study, we explored the detailed binding structures and binding free energies for two different active site conformations of CHI with s-cis/s-trans conformers of three chalcone compounds by performing molecular dynamics (MD) simulations and binding free energy calculations. The computational results indicate that s-cis/s-trans conformers of chalcone compounds are orientated in the similar binding position in the active site of CHI and stabilized by the different first hydrogen bond network and the same second hydrogen bond network. The first hydrogen bond network results in much lower binding affinity of s-trans conformer of chalcone compound with CHI than that of s-cis conformer. The conformational change of the active site residue T48 from indirectly interacting with the substrate via the second hydrogen bond network to directly forming the hydrogen bond with the substrates cannot affect the binding mode of both conformers of chalcone compounds, but remarkably improves the binding affinity. These results show that CHI has a strong stereoselectivity. The calculated binding free energies for three chalcone compounds with CHI are consistent with the experimental activity data. In addition, several valuable insights are suggested for future rational design and discovery of high-efficiency mutants of CHI.

Characterization and expression analysis of chalcone synthase and chalcone isomerase genes in Phyllanthus emblica (L.)

Journal of Plant Biochemistry and Biotechnology - The two decisive enzymes in flavonoid biosynthetic pathway are chalcone synthase (CHS) and chalcone isomerase (CHI), wherein the former carries the...