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Iridoid-specific Glucosyltransferase from Gardenia jasminoides*

Open AccessPublished:July 28, 2011DOI:https://doi.org/10.1074/jbc.M111.242586
      Iridoids are one of the most widely distributed secondary metabolites in higher plants. They are pharmacologically active principles in various medicinal plants and key intermediates in the biosynthesis of monoterpenoid indole alkaloids as well as quinoline alkaloids. Although most iridoids are present as 1-O-glucosides, the glucosylation step in the biosynthetic pathway has remained obscure. We isolated a cDNA coding for UDP-glucose:iridoid glucosyltransferase (UGT85A24) from Gardenia jasminoides. UGT85A24 preferentially glucosylated the 1-O-hydroxyl group of 7-deoxyloganetin and genipin but exhibited only weak activity toward loganetin and no activity toward 7-deoxyloganetic acid. This suggests that, in the biosynthetic pathway of geniposide, a major iridoid compound in G. jasminoides, glucosylation occurs after methylation of 7-deoxyloganetic acid. UGT85A24 showed negligible activity toward any acceptor substrates other than iridoid aglycones. Thus, UGT85A24 has a remarkable specificity for iridoid aglycones. The mRNA level of UGT85A24 overlaps with the marked increase in genipin glucosylation activity in the methyl jasmonate-treated cell cultures of G. jasminoides and is related to iridoid accumulation in G. jasminoides fruits.

      Introduction

      Iridoids are cyclopenta[c]pyran monoterpenoids distributed in numerous plant families, usually as glucosides (
      • Dinda B.
      • Debnath S.
      • Harigaya Y.
      ). In plants, iridoids function as important defense compounds in plant-herbivore interactions (
      • Harvey J.A.
      • van Nouhuys S.
      • Biere A.
      ). They play an important biogenetic role linking terpenes and alkaloids (
      • El-Sayed M.
      • Verpoorte R.
      ). Iridoids exhibit a wide range of pharmacological activities, such as cardiovascular, anti-hepatotoxic, choleretic, anti-inflammatory, immunomodulatory, and purgative activities, and are present in various folk medicinal plants as bioactive principles (
      • Ghisalberti E.L.
      ,
      • Dinda B.
      • Debnath S.
      • Harigaya Y.
      ).
      In vivo tracer studies have shown that the iridane skeleton is formed by the cyclization of 10-oxogeranial (10-oxoneral) to yield iridodial, which is subsequently oxidized to iridotrial. Iridotrial is converted to a series of iridoid compounds through various reactions, including oxidation, methylation, and glycosylation as shown in Fig. 1 (
      • Meijer A.H.
      • Verpoorte R.
      • Hoge J.H.
      ,
      • Oudin A.
      • Courtois M.
      • Rideau M
      • Clastre M.
      ). However, few studies have investigated the biochemical and molecular biological characterization of the iridoid biosynthetic pathway. In particular, the 1-O-glucosylation step in the iridoid biosynthetic pathway has been poorly characterized, although most iridoids are present as 1-O-glucosides in nature. Glucosyltransferase activity toward loganetin, 7-deoxyloganetin, and 7-deoxyloganetic acid was detected in the crude extract of Lonicera japonica (
      • Yamamoto H.
      • Sha M.
      • Kitamura K.
      • Yamaguchi Y.
      • Katano N.
      • Inoue K.
      ). However, genes for enzymes relevant to the glucosylation step in the iridoid biosynthetic pathway have yet to be characterized.
      Figure thumbnail gr1
      FIGURE 1A, iridoid biosynthetic pathway. B, proposed biosynthetic pathway from iridotrial to geniposide based on the present results. G10H, geraniol 10-hydroxylase; ADH, acyclic monoterpene primary alcohol dehydrogenase; MC, monoterpene cyclase; MIAs, monoterpenoid indole alkaloids.
      Glucosylation steps in plant secondary metabolism are catalyzed by family 1 plant secondary product glycosyltransferases (PSPGs)
      The abbreviations used are: PSPG
      plant secondary product glycosyltransferase
      RACE
      rapid amplification of cDNA ends
      Gj
      G. jasminoides.
      that attach a sugar molecule from a UDP-sugar to a low-molecular-weight acceptor substrate (
      • Vogt T.
      • Jones P.
      ,
      • Lim E.K.
      • Bowles D.J.
      ,
      • Gachon C.M.
      • Langlois-Meurinne M.
      • Saindrenan P.
      ). PSPGs are defined by the presence of a 44-amino acid C-terminal signature motif designated the PSPG box (
      • Hughes J.
      • Hughes M.A.
      ). Recent investigations on the three-dimensional structures of PSPGs by x-ray crystallography revealed that the PSPG box functions as a sugar donor-binding pocket (
      • Offen W.
      • Martinez-Fleites C.
      • Yang M.
      • Kiat-Lim E.
      • Davis B.G.
      • Tarling C.A.
      • Ford C.M.
      • Bowles D.J.
      • Davies G.J.
      ,
      • Li L.
      • Modolo L.V.
      • Escamilla-Trevino L.L.
      • Achnine L.
      • Dixon R.A.
      • Wang X.
      ,
      • Brazier-Hicks M.
      • Offen W.A.
      • Gershater M.C.
      • Revett T.J.
      • Lim E.K.
      • Bowles D.J.
      • Davies G.J.
      • Edwards R.
      ). Homology-based cloning using the conserved amino acid sequence within the PSPG box has been shown to be an efficient tool for isolating cDNAs encoding PSPGs (
      • Noguchi A.
      • Fukui Y.
      • Iuchi-Okada A.
      • Kakutani S.
      • Satake H.
      • Iwashita T.
      • Nakao M.
      • Umezawa T.
      • Ono E.
      ,
      • Masada S.
      • Terasaka K.
      • Oguchi Y.
      • Okazaki S.
      • Mizushima T.
      • Mizukami H.
      ).
      Gardenia jasminoides (Rubiaceae) fruits accumulate iridoid compounds, such as geniposide and gardenoside (
      • Yu Y.
      • Xie Z.L.
      • Gao H.
      • Ma W.W.
      • Dai Y.
      • Wang Y.
      • Zhong Y.
      • Yao X.S.
      ), and the dried fruits have been used as a crude drug in traditional Chinese medicine. Cultured G. jasminoides cells have been used to investigate iridoid biosynthesis because they produce small amounts of iridoids even after dedifferentiation (
      • Ueda S.
      • Kobayashi K.
      • Muramatsu T.
      • Inouye H.
      ,
      • Uesato S.
      • Ueda S.
      • Kobayashi K.
      • Inouye H.
      ,
      • Uesato S.
      • Ueda S.
      • Kobayashi K.
      • Miyauchi M.
      • Inouye H.
      ). Thus, suspension cultures of G. jasminoides are suitable as sources for cDNA isolation and the molecular characterization of glucosyltransferases that catalyze the 1-O-glucosylation step in the iridoid biosynthetic pathway. Geniposide is a major iridoid compound present in G. jasminoides fruits, and genipin, an aglycone of geniposide, is the only iridoid aglycone commercially available in relatively large amounts. Homology-based cloning using the conserved amino acid sequence within the PSPG box combined with screening of the glucosylation activity of recombinant enzymes from candidate cDNA clones with genipin as a glucose-accepting substrate, instead of the intrinsic substrates such as 7-deoxyloganetic acid and 7-deoxyloganetin, which are difficult to obtain, led to the isolation of a cDNA encoding an iridoid-specific glucosyltransferase for the first time. Here, we describe the molecular cloning and characterization of a UDP-glucose:iridoid glucosyltransferase (UGT85A24) from G. jasminoides in culture.

      DISCUSSION

      Cultured G. jasminoides cells efficiently glucosylated exogenously added genipin to geniposide and converted geniposide to gardenoside. We also examined genipin glucosylation using cell suspension cultures of C. roseus, which produces no geniposide in planta. Only ∼3% of the initially supplemented genipin was converted to glucoside in the cell suspension cultures, and no further transformation of geniposide to gardenoside was observed. Therefore, the cellular activity of genipin glucosylation is specific to G. jasminoides, which has biosynthetic activity for geniposide-related iridoid glucosides. The glucosylation activity in the G. jasminoides cells was increased by the addition of methyl jasmonate to the cultures, which is consistent with the possible roles of iridoid glucosides as defense compounds against herbivores (
      • Harvey J.A.
      • van Nouhuys S.
      • Biere A.
      ).
      Homology-based cloning using a characteristic motif conserved in the C-terminal part of glycosyltransferases involved in plant secondary metabolism (PSPGs) led to isolation of 13 cDNA clones (GjUGT1–GjUGT13) encoding novel PSPGs from cultured G. jasminoides cells. Biochemical analysis revealed that GjUGT2 encodes a glucosyltransferase (UGT85A24) that catalyzes 1-O-glucosylation of iridoid aglycones, such as genipin and 7-deoxyloganetin, but does not exhibit glucosyltransferase activity toward any other substrates examined that have unique chemical structures. The deduced amino acid sequence of UGT85A24 showed high identity to that of UGT85A21 from Lycium barbarum (78% identity), UGT85A19 from P. dulcis (66%), and UGT85A1 from Arabidopsis thaliana (62%). Molecular phylogenetic analysis revealed that UGT85A24 belongs to group G of PSPGs with other UGT85 family enzymes. PSPGs of this group exhibit glucosyltransferase activity toward various acceptor substrates. For example, UGT85A19 from P. dulcis is a mandelonitrile glucosyltransferase (
      • Franks T.K.
      • Yadollahi A.
      • Wirthensohn M.G.
      • Guerin J.R.
      • Kaiser B.K.
      • Sedgley M.
      • Ford C.M.
      ), and UGT85A1 from A. thaliana exhibits glucosyltransferase activity toward zeatin (
      • Hou B.
      • Lim E.K.
      • Higgins G.S.
      • Bowles D.J.
      ). UGT85A24 exhibits glucosyltransferase activity toward neither mandelonitrile nor zeatin. Recently, we isolated a cDNA clone encoding an iridoid glucosyltransferase (UGT85A23) from C. roseus. The deduced amino acid sequence of UGT85A23 is 88% identical to that of UGT85A24. UDP-glucose:iridoid glucosyltransferases may form a subgroup within the group G PSPGs as shown in Fig. 4.
      UGT85A24 is apparently responsible for iridoid glucosylation in cultured G. jasminoides cells because 1) marked enhancement of genipin glucosylation activity by adding methyl jasmonate to G. jasminoides cell cultures was consistent with the strong up-regulation of UGT85A24 mRNA expression, and 2) G. jasminoides cells efficiently glucosylated exogenously added 7-deoxyloganetin to glucoside but poorly glucosylated 7-deoxyloganetic acid (data not shown), which is consistent with the substrate preferentiality of UGT85A24. UGT85A24 mRNA expression was also detected in various G. jasminoides organs. The gradual increase in the mRNA level during the early phase of fruit ripening correlates with an increase in iridoid accumulation during the fruit development. Furthermore, the relatively low level of iridoid accumulation (geniposide plus gardenoside) in the vegetative tissues is consistent with the distribution pattern of UGT85A24 mRNA in the plant organs. Thus, UGT85A24 may at least partially limit the iridoid accumulation in planta.
      Iridoid biosynthesis has been extensively studied using cell cultures of C. roseus and L. japonica because the biosynthesis of secologanin, a major iridoid metabolite in these species, has been considered a bottleneck for monoterpenoid indole alkaloid production in C. roseus cell cultures (
      • Oudin A.
      • Courtois M.
      • Rideau M
      • Clastre M.
      ). Tracer experiments and enzymatic studies revealed that the iridane skeleton is formed by the cyclization of 10-oxogeranial biosynthesized from geraniol through 10-hydroxygeraniol. Conversion of the cyclization product iridodial to loganin and then to secologanin involves several steps, including 1-O-glucosylation of the iridane skeleton (Fig. 1). However, the glucosylation step has remained elusive so far. Kinetic analysis of UGT85A24 indicated that the Km value for 7-deoxyloganetin is ∼13-fold lower than that for genipin, whereas the kcat value for genipin is ∼8 times higher than that for 7-deoxyloganetin. Thus, the catalytic efficiency (kcat/Km) is ∼2-fold higher for 7-deoxyloganetin than for genipin. This result suggests that 7-deoxyloganetin is the preferential sugar acceptor for UGT85A24 and probably an endogenous substrate of the glucosylation step in the biosynthetic pathways of geniposide and gardenoside in G. jasminoides, although genipin is a good substrate because of its structural similarity to 7-deoxyloganetin. In G. jasminoides, 7-deoxyloganetic acid is converted to 7-deoxyloganetin, which is then glucosylated to 7-deoxyloganin. Although the Km value of UGT85A24 for 7-deoxyloganetin (∼0.6 mm) seems to be higher compared with the values of, for example, flavonoid glucosyltransferases (
      • Nakatsuka T.
      • Sato K.
      • Takahashi H.
      • Yamamura S.
      • Nishihara M.
      ,
      • Noguchi A.
      • Horikawa M.
      • Fukui Y.
      • Fukuchi-Mizutani M.
      • Iuchi-Okada A.
      • Ishiguro M.
      • Kiso Y.
      • Nakayama T.
      • Ono E.
      ), it is in the same range of the Km values for terpenoid glucosyltransferases so far reported (
      • Achnine L.
      • Huhman D.V.
      • Farag M.A.
      • Sumner L.W.
      • Blount J.W.
      • Dixon R.A.
      ,
      • Naoumkina M.A.
      • Modolo L.V.
      • Huhman D.V.
      • Urbanczyk-Wochniak E.
      • Tang Y.
      • Sumner L.W.
      • Dixon R.A.
      ).
      Hydroxylation of 7-deoxyloganin at C-10 results in the formation of geniposide as shown in Fig. 1B. Yamamoto and co-workers (
      • Yamamoto H.
      • Sha M.
      • Kitamura K.
      • Yamaguchi Y.
      • Katano N.
      • Inoue K.
      ) examined the kinetic parameters of the partially purified glucosyltransferase preparation from L. japonica cells and noted that the preparation had the highest affinity for 7-deoxyloganetic acid compared with 7-deoxyloganetin and loganetin, although the specific activity was the highest for loganetin. On the basis of their result, they proposed that 7-deoxyloganetic acid is a sugar-accepting substrate in the iridoid biosynthetic pathway leading to loganin. Glucosylation of iridoid precursors may occur in different steps between loganin biosynthesis in L. japonica and geniposide biosynthesis in G. jasminoides. However, the partially purified enzyme preparation from L. japonica cells may be a mixture of glucosyltransferases, which makes the estimated the kinetic parameters difficult to discern.
      In conclusion, we isolated a novel UDP-glucose:7-deoxyloganetin glucosyltransferase, UGT85A24 from G. jasminoides. Our results shed light not only on the glucosylation step in iridoid biosynthesis in G. jasminoides but also on the isolation of various iridoid-specific glucosyltransferases from diverse iridoid- and indole alkaloid-producing plant species, leading to the engineering of biosynthetic pathways producing iridoids and/or monoterpenoid indole alkaloids.

      Acknowledgments

      We thank Professor K. Inoue for the generous gift of 7-deoxyloganin tetraacetate.

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