Identification of α-Tomatine 23-Hydroxylase Involved in the Detoxification of a Bitter Glycoalkaloid

Tomato plants (Solanum lycopersicum) contain steroidal glycoalkaloid α-tomatine, which functions as a chemical barrier to pathogens and predators. α-Tomatine accumulates in all tissues and at particularly high levels in leaves and immature green fruits. The compound is toxic and causes a bitter taste, but its presence decreases through metabolic conversion to nontoxic esculeoside A during fruit ripening. This study identifies the gene encoding a 23-hydroxylase of α-tomatine, which is a key to this process. Some 2-oxoglutarate-dependent dioxygenases were selected as candidates for the metabolic enzyme, and Solyc02g062460, designated Sl23DOX, was found to encode α-tomatine 23-hydroxylase. Biochemical analysis of the recombinant Sl23DOX protein demonstrated that it catalyzes the 23-hydroxylation of α-tomatine and the product spontaneously isomerizes to neorickiioside B, which is an intermediate in α-tomatine metabolism that appears during ripening. Leaves of transgenic tomato plants overexpressing Sl23DOX accumulated not only neorickiioside B but also another intermediate, lycoperoside C (23-O-acetylated neorickiioside B). Furthermore, the ripe fruits of Sl23DOX-silenced transgenic tomato plants contained lower levels of esculeoside A but substantially accumulated α-tomatine. Thus, Sl23DOX functions as α-tomatine 23-hydroxylase during the metabolic processing of toxic α-tomatine in tomato fruit ripening and is a key enzyme in the domestication of cultivated tomatoes.

Pathways to defense metabolites and evading fruit bitterness in genus Solanum evolved through 2-oxoglutarate-dependent dioxygenases

The genus Solanum comprises three food crops (potato, tomato, and eggplant), which are consumed on daily basis worldwide and also producers of notorious anti-nutritional steroidal glycoalkaloids (SGAs). Hydroxylated SGAs (i.e. leptinines) serve as precursors for leptines that act as defenses against Colorado Potato Beetle (Leptinotarsa decemlineata Say), an important pest of potato worldwide. However, SGA hydroxylating enzymes remain unknown. Here, we discover that 2-OXOGLUTARATE-DEPENDENT-DIOXYGENASE (2-ODD) enzymes catalyze SGA-hydroxylation across various Solanum species. In contrast to cultivated potato, Solanum chacoense, a widespread wild potato species, has evolved a 2-ODD enzyme leading to the formation of leptinines. Furthermore, we find a related 2-ODD in tomato that catalyzes the hydroxylation of the bitter α-tomatine to hydroxytomatine, the first committed step in the chemical shift towards downstream ripening-associated non-bitter SGAs (e.g. esculeoside A). This 2-ODD enzyme prevents bitterness in ripe tomato fruit consumed today which otherwise would remain unpleasant in taste and more toxic.

Competitive Inhibition of Mammalian Hyaluronidase by Tomato Saponin, Esculeoside A

Esculeoside A, a glycoside of spirosolane-type, is identified as a major component in ripe tomato fruits. Our previous study showed that esculeoside A inhibited hyaluronidase activity in vitro and ameliorated experimental dermatitis in vivo. The aim of this present study is to investigate the inhibition mode on mammalian hyaluronidase by esculeoside A. Measured by a modified Morgan-Elson method, the present kinetic analysis of the hydrolysis reaction using hyaluronic acid revealed that no significant difference was observed in velocity maximum Vmax, and Michaelis-Menten constant Km was shown as 0.74 mM in the absence of esculeoside A, was increased as 1.32 and 1.98 mM with 3 and 30 μM of esculeoside A, respectively. Thus suggested that the inhibition mode on hyaluronidase by esculeoside A was competitive. This competitive inhibition on hyaluronidase activity may become valuable in the amelioration of mice experimental dermatitis by esculeoside A.

Quantification and Enrichment of Esculeoside a from Fresh Tomato Fruits

Esculeoside A, the tomato saponins from Lycopersicon esculentum MILL, may be metabolized into various steroidal hormones such as pregnane derivatives that are expected to exhibit various bioactivities in the body, such as anti-osteoporosis, antimenopausal disorder and anti-tumor actions. In order to enrich large amount of esculeoside A for further scientific research, we firstly developed a high performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD) method for its quantification, and used it to examined the content of esculeoside A in different cultivated varieties of tomato fruits and tomato extract. Statistical analysis of the obtained data demonstrated that our method was simple, and has achieved desired linearity, precision and accuracy. Then we have explored the feasibility of using pectinase as a means for assisting esculeoside A extraction from fresh tomato fruits, and several factors such as the enzyme concentration, pH value of enzyme solution, temperature, incubation time which influenced on extraction were investigated. Moreover, in our previous study, the adsorption characteristics on several types of macroporous resins have been compared. Among them, D-101 resin showed the best separation behaviors on esculeoside A. So in the present paper, in order to get the best parameters for D101 adsorption, the adsorption kinetic curve, the appropriate ethanol concentration for elution and breakthrough volume were analyzed．