Land plants produce diverse flavonoids for growth, survival, and reproduction. Chalcone synthase is the first committed enzyme of the flavonoid biosynthetic pathway and catalyzes the production of 2′,4,4′,6′-tetrahydroxychalcone (THC). However, it also produces other polyketides, including p-coumaroyltriacetic acid lactone (CTAL), because of the derailment of the chalcone-producing pathway. This promiscuity of CHS catalysis adversely affects the efficiency of flavonoid biosynthesis, although it is also believed to have led to the evolution of stilbene synthase and p-coumaroyltriacetic acid synthase. In this study, we establish that chalcone isomerase-like proteins (CHILs), which are encoded by genes that are ubiquitous in land plant genomes, bind to CHS to enhance THC production and decrease CTAL formation, thereby rectifying the promiscuous CHS catalysis. This CHIL function has been confirmed in diverse land plant species, and represents a conserved strategy facilitating the efficient influx of substrates from the phenylpropanoid pathway to the flavonoid pathway.

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.

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Background: Carboxylesterases typically have an alkaline pH optima, which limits their industrial application. Results: An acidophilic carboxylesterase was crystallized and changed into an alkaliphilic enzyme via quadruple mutations. Conclusion:The extended hydrogen bonds in the active site were important for adaptation to a low pH. Significance: The first structure of an acidophilic carboxylesterase revealed a novel strategy for the low pH adaptation of carboxylesterase.

cDNAs coding for the chalcone isomerase-fold proteins (CIFP) of the snapdragon (Antirrhinum majus L.) were cloned and characterized. One of these CIFPs was a Cluster-1 member of the CIFP family, which was a catalytically active chalcone isomerase and thus termed AmCHI1. The recombinant AmCHI1 could act on 2′,4,4′,6′-tetrahydroxychalcone (THC) and 2′,3,4,4′,6′-pentahydroxychalcone (PHC) to produce naringenin and eriodictyol with k cat /K m values of 0.25 s −1 µM −1 and 0.071 s, respectively, at pH 7.5 and 4°C. The enzyme could not act on 4′-O-glucosides of THC and PHC. In the yellow snapdragon petals (cv. Yellow Butterfly), the temporal expression patterns of AmCHI1 were consistent with the observed temporal accumulation patterns of flavones. Thus, regulation of the AmCHI1 transcription and substrate specificity of the expressed AmCHI1 should serve as the key mechanisms that allows for partitioning of the flavonoid biosynthetic pathways into the aurones and the non-aurone flavonoids in snapdragon petal cells. The other CIFP cDNA, AmCIFP4, was a Cluster-4 member of the family and was similar in its primary structure to enhancers of the flavonoid production of torenia (Torenia×hybrida) and petunia (Petunia×hybrida). AmCIFP4 was more abundantly expressed than AmCHI1 irrespective of flower color.