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.

Gene cloning and biochemical characterization of a catalase from Gluconobacter oxydans.

Gluconobacter oxydans has a large number of membrane-bound dehydrogenases linked to the respiratory chain that catalyze incomplete oxidation of a wide range of organic compounds by oxidative fermentation. Because the respiratory chain is a primary site of reactive oxygen species (ROS) production, the bacterium is expected to have a high capacity to detoxify nascent ROS. In the present study, a gene that encodes a catalase of G. oxydans, which might act as a potential scavenger of H(2)O(2), was cloned, and the expression product (termed rGoxCat) was characterized biochemically. rGoxCat is a heme b-containing tetrameric protein (molecular mass, 320 kDa) consisting of identical subunits. The recombinant enzyme displayed a strong catalase activity with a k(cat) of 6.28×10(4) s(-1) and a K(m) for H(2)O(2) of 61 mM; however, rGoxCat exhibited no peroxidase activity. These results, along with the phylogenetic position of the enzyme, provide conclusive evidence that rGoxCat is a monofunctional, large-subunit catalase. The enzyme was most stable in the pH range of 4-9, and greater than 60% of the original activity was retained after treatment at pH 3.0 and 40°C for 1h. Moreover, the enzyme exhibited excellent thermostability for a catalase from a mesophilic organism, retaining full activity after incubation for 30 min at 70°C. The observed catalytic properties of rGoxCat, as well as its stability in a slightly acidic environment, are consistent with its role in the elimination of nascent H(2)O(2) in a bacterium that produces a large amount of organic acid via oxidative fermentation.