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.

Identification of protein-protein interactions of isoflavonoid biosynthetic enzymes with 2-hydroxyisoflavanone synthase in soybean (Glycine max (L.) Merr.).

Metabolic enzymes, including those involved in flavonoid biosynthesis, are proposed to form weakly bound, ordered protein complexes, called "metabolons". Some hypothetical models of flavonoid biosynthetic metabolons have been proposed, in which metabolic enzymes are believed to anchor to the cytoplasmic surface of the endoplasmic reticulum (ER) via ER-bound cytochrome P450 isozymes (P450s). However, no convincing evidence for the interaction of flavonoid biosynthetic enzymes with P450s has been reported previously. Here, we analyzed binary protein-protein interactions of 2-hydroxyisoflavanone synthase 1 (GmIFS1), a P450 (CYP93C), with cytoplasmic enzymes involved in isoflavone biosynthesis in soybean. We identified binary interactions between GmIFS1 and chalcone synthase 1 (GmCHS1) and between GmIFS1 and chalcone isomerases (GmCHIs) by using a split-ubiquitin membrane yeast two-hybrid system. These binary interactions were confirmed in planta by means of bimolecular fluorescence complementation (BiFC) using tobacco leaf cells. In these BiFC analyses, fluorescence signals that arose from the interaction of these cytoplasmic enzymes with GmIFS1 generated sharp, network-like intracellular patterns, which was very similar to the ER-localized fluorescence patterns of GmIFS1 labeled with a fluorescent protein. These observations provide strong evidence that, in planta, interaction of GmCHS1 and GmCHIs with GmIFS1 takes place on ER on which GmIFS1 is located, and also provide important clues to understand how enzymes and proteins form metabolons to establish efficient metabolic flux of (iso)flavonoid biosynthesis.

Structural insights into the low pH adaptation of a unique carboxylesterase from Ferroplasma: altering the pH optima of two carboxylesterases.

To investigate the mechanism for low pH adaptation by a carboxylesterase, structural and biochemical analyses of EstFa_R (a recombinant, slightly acidophilic carboxylesterase from Ferroplasma acidiphilum) and SshEstI (an alkaliphilic carboxylesterase from Sulfolobus shibatae DSM5389) were performed. Although a previous proteomics study by another group showed that the enzyme purified from F. acidiphilum contained an iron atom, EstFa_R did not bind to iron as analyzed by inductively coupled plasma MS and isothermal titration calorimetry. The crystal structures of EstFa_R and SshEstI were determined at 1.6- and 1.5-Å resolutions, respectively. EstFa_R had a catalytic triad with an extended hydrogen bond network that was not observed in SshEstI. Quadruple mutants of both proteins were created to remove or introduce the extended hydrogen bond network. The mutation on EstFa_R enhanced its catalytic efficiency and gave it an alkaline pH optimum, whereas the mutation on SshEstI resulted in opposite effects (i.e. a decrease in the catalytic efficiency and a downward shift in the optimum pH). Our experimental results suggest that the low pH optimum of EstFa_R activity was a result of the unique extended hydrogen bond network in the catalytic triad and the highly negatively charged surface around the active site. The change in the pH optimum of EstFa_R happened simultaneously with a change in the catalytic efficiency, suggesting that the local flexibility of the active site in EstFa_R could be modified by quadruple mutation. These observations may provide a novel strategy to elucidate the low pH adaptation of serine hydrolases.

Xylanase from Marine Filamentous Fungus Pestalotiopsis sp. AN-7 Was Activated with Diluted Salt Solution Like Brackish Water.

The genus Pestalotiopsis are endophytic fungi that have recently been identified as cellulolytic system producers. We herein cloned a gene coding for a xylanase belonging to glycoside hydrolase (GH) family 10 (PesXyn10A) from Pestalotiopsis sp. AN-7, which was isolated from the soil of a mangrove forest. This protein was heterologously expressed by Pichia pastoris as a host, and its enzymatic properties were characterized. PesXyn10A was produced as a glycosylated protein and coincident to theoretical molecular weight (35.3 kDa) after deglycosylation by peptide-NfF-glycosidase F. Purified recombinant PesXyn10A exhibited maximal activity at pH 6.0 and 50 °C, and activity was maintained at 90 % at pH 5.0 and temperatures lower than 30 °C for 24 h. The substrate specificity of PesXyn10A was limited and it hydrolyzed glucuronoxylan and arabinoxylan, but not ß-glucan. The final hydrolysis products from birchwood xylan were xylose, xylobiose, and 1,23-α-D-(4-O-methyl-glucuronyl)-1,4-ß-D-xylotriose. The addition of metallic salts (NaCl, KCl, MgCl2, and CaCl2) activated PesXyn10A for xylan degradation, and maximal activation by these divalent cations was approximately 160 % at a concentration of 5 mM. The thermostability of PesXyn10A significantly increased in the presence of 50 mM NaCl or 5 mM MgCl2. The present results suggest that the presence of metallic salts at a low concentration, similar to brackish water, exerts positive effects on the enzyme activity and thermal stability of PesXyn10A.

A conserved strategy of chalcone isomerase-like protein to rectify promiscuous chalcone synthase specificity.

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.

Characterization of Acetylxylan Esterase from White-Rot Fungus Irpex lacteus.

The carbohydrate esterase family 1 (CE1) in CAZy contains acetylxylan esterases (AXEs) and feruloyl esterases (FAEs). Here we cloned a gene coding for an AXE belonging to CE1 from Irpex lacteus (IlAXE1). IlAXE1 was heterologously expressed in Pichia pastoris, and the recombinant enzyme was purified and characterized. IlAXE1 hydrolyzed p-nitrophenyl acetate, α-naphthyl acetate and 4-methylumbelliferyl acetate, however, it did not show any activity on ethyl ferulate and methyl p-coumarate. We also examined the activity on partially acetylated and feruloylated xylan extracted from corncob by hydrothermal reaction. Similarly, ferulic and p-coumaric acids were not liberated, and acetic acid was only detected in the reaction mixture. The results indicated that IlAXE1 is an acetylxylan esterase actually reacted to acetyl xylan. However, since IlAXE1 was unable to completely release acetic acid esterifying xylopyranosyl residues, it is assumed that acetyl groups exhibiting resistance to deacetylation by IlAXE1 are present in corn cob xylan.