Iridoid-specific glucosyltransferase from Gardenia jasminoides

Abstract

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.

FIGURE 1.

A, 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.

FIGURE 2.

HPLC profiles of the biotransformation products of genipin by cell suspension cultures of G. jasminoides. Genipin was added to cell suspension cultures of G. jasminoides and incubated for 4 h (A) and 72 h (B). The methanol extracts of the collected cells were subjected to HPLC analysis. C, HPLC profile of a mixture comprising standards for genipin, geniposide, and gardenoside.

FIGURE 3.

Genipin glucosylation by cell suspension cultures of G. jasminoides. A, time course changes in the accumulation of glucosylation products in cultured G. jasminoides cells. G. jasminoides cell cultures were supplemented with genipin (25 μmol/flask). The cells were collected at 4, 8, 24, 48, and 72 h after the addition of genipin, and the amounts of the glucosides formed from genipin were estimated by HPLC. B, glucosylation activity of G. jasminoides cell cultures. Genipin (25 μmol/flask) was added to the cell cultures at 3, 6, 9, 12, and 15 days after cell inoculation, and glucoside formation was estimated after the cells were cultured for 1 additional day. ●, biomass fresh weight per flask at 3, 6, 9, 12, and 15 days after cell inoculation. Each circle and bar represent an average value with S.D. from triplicate measurements.

FIGURE 4.

Non-rooted molecular phylogenetic tree of family 1 PSPGs. The PSPG clones isolated in this investigation are shown in boldface, and GjUGT2 (UGT84A24) is shown in red. The tree was generated by the neighbor-joining method following multiple alignments by the ClustalW algorithm. The bar indicates 0.1 amino acid substitution/site. The nucleotide sequences of GjUGT1–GjUGT13 have been submitted to the DDBJ/GenBank^TM/EBI DNA Database under accession numbers AB555731–AB555743, respectively. For the names and DDBJ/GenBank^TM/EBI accession numbers of other PSPGs, see supplemental Table S4.

FIGURE 5.

Analysis of the glucosyltransferase activity of recombinant UGT85A24 expressed in E. coli. A, results from SDS-PAGE analysis of the crude protein from E. coli JM109 harboring pQE-30-GjUGT2 (left lane: C) and the recombinant enzyme (UGT85A24) purified using a nickel-nitrilotriacetic acid resin column (right: P). B, time course changes in genipin glucosylation by incubation with recombinant UGT85A24.

FIGURE 6.

A and B, glucosylation of 7-deoxyloganetin and loganetin, respectively, by UGT85A24. Each iridoid aglycone was incubated with UGT85A24 for 60 min, and the assay mixture was subjected to HPLC analysis. C, HPLC profile of a mixture comprising standards. 3, 7-deoxyloganin; 4, 7-deoxyloganetin; 5, loganin; 6, loganetin.

FIGURE 7.

MJ-induced activity of genipin glucosylation in G. jasminoides. A, the suspension cultures of G. jasminoides were supplemented with methyl jasmonate at a final concentration of 250 μm 3 days after cell inoculation, and genipin was exogenously added to the cells 24 h after methyl jasmonate addition. The geniposide contents of the cells were estimated 1 and 2 days after genipin addition. Open bars, geniposide formation in mock (Me₂SO)-treated cells; closed bars, geniposide formation in methyl jasmonate-treated cells. B, genipin glucosylation activity in the crude enzyme from methyl jasmonate (MJ)-treated cells. Each bar represents an average value with S.D. from triplicate measurements. C, Northern blot analysis of mRNA levels of UGT85A24. Methyl jasmonate was added to the cells as described above, and the crude enzyme and total RNA were prepared 0, 4, 8, 24, and 48 h after methyl jasmonate addition.

FIGURE 8.

Temporal and tissue distribution of UGT85A24 mRNA (A) and iridoid glucosides (B) in G. jasminoides. A, fruits, leaves, and stems were collected from a single G. jasminoides plant on the same day, powdered in liquid nitrogen, and stored until used. The fruits were classified into five maturation stages based on the red coloration. Total RNA and methanol extract were prepared from the tissues and used for RT-PCR analysis and HPLC analysis, respectively. B, closed and open bars represent contents of geniposide and gardenoside, respectively, and represent average values with S.D. from five replicates.