Glycyrrhizin, a major bioactive compound derived from the underground parts of Glycyrrhiza (licorice) plants, is a triterpene saponin that possesses a wide range of pharmacological properties and is used worldwide as a natural sweetener. Because of its economic value, the biosynthesis of glycyrrhizin has received considerable attention. Glycyrrhizin is most likely derived from the triterpene ␤-amyrin, an initial product of the cyclization of 2,3-oxidosqualene. The subsequent steps in glycyrrhizin biosynthesis are believed to involve a series of oxidative reactions at the C-11 and C-30 positions, followed by glycosyl transfers to the C-3 hydroxyl group; however, no genes encoding relevant oxidases or glycosyltransferases have been identified. Here we report the successful identification of CYP88D6, a cytochrome P450 monooxygenase (P450) gene, as a glycyrrhizin-biosynthetic gene, by transcript profilingbased selection from a collection of licorice expressed sequence tags (ESTs). CYP88D6 was characterized by in vitro enzymatic activity assays and shown to catalyze the sequential two-step oxidation of ␤-amyrin at C-11 to produce 11-oxo-␤-amyrin, a possible biosynthetic intermediate between ␤-amyrin and glycyrrhizin. CYP88D6 coexpressed with ␤-amyrin synthase in yeast also catalyzed in vivo oxidation of ␤-amyrin to 11-oxo-␤-amyrin. CYP88D6 expression was detected in the roots and stolons by RT-PCR; however, no amplification was observed in the leaves or stems, which is consistent with the accumulation pattern of glycyrrhizin in planta. These results suggest a role for CYP88D6 as a ␤-amyrin 11-oxidase in the glycyrrhizin pathway.expressed sequence tag ͉ medicinal plant ͉ secondary metabolite ͉ monooxygenase ͉ isoprenoid

Both CD46 and signaling lymphocytic activation molecule (SLAM) have been shown to act as cellular receptors for measles virus (MV). The viruses on throat swabs from nine patients with measles in Japan were titrated on Vero cells stably expressing human SLAM. Samples from all but two patients produced numerous plaques on SLAM-expressing Vero cells, whereas none produced any plaques on Vero cells endogenously expressing CD46. The Edmonston strain of MV, which can use either CD46 or SLAM as a receptor, produced comparable titers on these two types of cells. The results strongly suggest that the viruses in the bodies of measles patients use SLAM but probably not CD46 as a cellular receptor.Measles virus (MV) is an enveloped negative-strand RNA virus of the Morbillivirus genus in the Paramyxoviridae family (4). Measles remains an important cause of childhood mortality, with approximately one million deaths per year worldwide (2), mainly due to secondary infections caused by MV-induced immunosuppression (4). Human CD46 has been shown to be a cellular receptor for vaccine strains of MV, such as the Edmonston strain (3, 10). However, wild-type MV strains that are commonly isolated in marmoset B-cell line B95a or human B-cell lines usually do not use CD46 as a receptor (5-7, 14, 15, 18), although a study has reported that MV strains isolated from and propagated only in human peripheral blood mononuclear cells (PBMCs) use CD46 as a receptor (9). We have recently demonstrated that signaling lymphocytic activation molecule (SLAM; also known as CDw150) is a cellular receptor for MV, including the Edmonston strain, B95a-isolated strains, and PBMC-isolated strains (20). Thus, some MV strains use SLAM but not CD46 as a receptor, and others, such as the Edmonston strain, use either SLAM or CD46. The type of MV strain obtained depends on the cell types used for virus isolation. In this study, we sought to quantitate the proportions of these two types of MV in measles patients.Vero cells are susceptible to the Edmonston strain but not to B95a-isolated MV strains (6, 18). In order to titrate B95a-isolated MV strains on Vero cells, we transfected them with the expression plasmid encoding human SLAM (pCAGhSLAM) (12) and the vector plasmid pCXN2 (11) containing the neomycin resistance (neo) gene; we selected stable clones in the presence of G418. We used the clone expressing the highest level of human SLAM (Vero/hSLAM) in the following experiments.The expression profile of Vero/hSLAM cells stained with anti-human SLAM monoclonal antibody IPO-3 (Kamiya Biomedical) (17) is shown in Fig. 1A. Vero/hSLAM cells were infected with the B95a-isolated KA strain of MV (18-20) at a multiplicity of infection of 0.1. At 24 h after infection, they developed extensive syncytia (Fig. 1B), unlike the parental Vero cells (6, 18). Then, we used Vero and Vero/hSLAM cells for plaque titration of the KA strain. Vero/hSLAM cells developed clear plaques after infection with the KA strain, whereas Vero cells inoculated with the same amount of the virus did no...

Glycyrrhizin, a triterpenoid saponin derived from the underground parts of Glycyrrhiza plants (licorice), has several pharmacological activities and is also used worldwide as a natural sweetener. The biosynthesis of glycyrrhizin involves the initial cyclization of 2,3-oxidosqualene to the triterpene skeleton b-amyrin, followed by a series of oxidative reactions at positions C-11 and C-30, and glycosyl transfers to the C-3 hydroxyl group. We previously reported the identification of a cytochrome P450 monooxygenase (P450) gene encoding b-amyrin 11-oxidase (CYP88D6) as the initial P450 gene in glycyrrhizin biosynthesis. In this study, a second relevant P450 (CYP72A154) was identified and shown to be responsible for C-30 oxidation in the glycyrrhizin pathway. CYP72A154 expressed in an engineered yeast strain that endogenously produces 11-oxo-b-amyrin (a possible biosynthetic intermediate between b-amyrin and glycyrrhizin) catalyzed three sequential oxidation steps at C-30 of 11-oxo-b-amyrin supplied in situ to produce glycyrrhetinic acid, a glycyrrhizin aglycone. Furthermore, CYP72A63 of Medicago truncatula, which has high sequence similarity to CYP72A154, was able to catalyze C-30 oxidation of b-amyrin. These results reveal a function of CYP72A subfamily proteins as triterpene-oxidizing enzymes and provide a genetic tool for engineering the production of glycyrrhizin.

The fragment pattern analysis of tandem mass spectrometry (MS/MS) has long been used for the structural characterization of metabolites. The construction of a plant-specific MS/MS data resource and database will enable complex phytochemical structures to be narrowed down to candidate structures. Therefore, a web-based database of MS/MS data pertaining to phytochemicals was developed and named ReSpect (RIKEN tandem mass spectral database). Of the 3595 metabolites in ReSpect, 76% were derived from 163 literature reports, whereas the rest was obtained from authentic standards. As a main web application of ReSpect, a fragment search was established based on only the m/z values of query data and records. The confidence levels of the annotations were managed using the MS/MS fragmentation association rule, which is an algorithm for discovering common fragmentations in MS/MS data. Using this data resource and database, a case study was conducted for the annotation of untargeted MS/MS data that were selected after quantitative trait locus analysis of the accessions (Gifu and Miyakojima) of a model legume Lotus japonicus. In the case study, unknown metabolites were successfully narrowed down to putative structures in the website.

Potatoes (Solanum tuberosum) contain a-solanine and a-chaconine, two well-known toxic steroidal glycoalkaloids (SGAs). Sprouts and green tubers accumulate especially high levels of SGAs. Although SGAs were proposed to be biosynthesized from cholesterol, the biosynthetic pathway for plant cholesterol is poorly understood. Here, we identify sterol side chain reductase 2 (SSR2) from potato as a key enzyme in the biosynthesis of cholesterol and related SGAs. Using in vitro enzyme activity assays, we determined that potato SSR2 (St SSR2) reduces desmosterol and cycloartenol to cholesterol and cycloartanol, respectively. These reduction steps are branch points in the biosynthetic pathways between C-24 alkylsterols and cholesterol in potato. Similar enzymatic results were also obtained from tomato SSR2. St SSR2-silenced potatoes or St SSR2-disrupted potato generated by targeted genome editing had significantly lower levels of cholesterol and SGAs without affecting plant growth. Our results suggest that St SSR2 is a promising target gene for breeding potatoes with low SGA levels.