The new beta-D-glucosidase in terpenoid-isoquinoline alkaloid biosynthesis in Psychotria ipecacuanha

Abstract

Ipecac alkaloids produced in the medicinal plant Psychotria ipecacuanha such as emetine and cephaeline possess a monoterpenoid-tetrahydroisoquinoline skeleton, which is formed by condensation of dopamine and secologanin. Deglucosylation of one of the condensed products N-deacetylisoipecoside (1 alpha(S)-epimer) is considered to be a part of the reactions for emetine biosynthesis, whereas its 1 beta(R)-epimer N-deacetylipecoside is converted to ipecoside in P. ipecacuanha. Here, we isolated a cDNA clone Ipeglu1 encoding Ipecac alkaloid beta-D-glucosidase from P. ipecacuanha. The deduced protein showed 54 and 48% identities to raucaffricine beta-glucosidase and strictosidine beta-glucosidase, respectively. Recombinant IpeGlu1 enzyme preferentially hydrolyzed glucosidic Ipecac alkaloids except for their lactams, but showed poor or no activity toward other substrates, including terpenoid-indole alkaloid glucosides. Liquid chromatography-tandem mass spectrometry analysis of deglucosylated products of N-deacetylisoipecoside revealed spontaneous transitions of the highly reactive aglycons, one of which was supposed to be the intermediate for emetine biosynthesis. IpeGlu1 activity was extremely poor toward 7-O-methyl and 6,7-O,O-dimethyl derivatives. However, 6-O-methyl derivatives were hydrolyzed as efficiently as non-methylated substrates, suggesting the possibility of 6-O-methylation prior to deglucosylation by IpeGlu1. In contrast to the strictosidine beta-glucosidase that stereospecifically hydrolyzes 3 alpha(S)-epimer in terpenoid-indole alkaloid biosynthesis, IpeGlu1 lacked stereospecificity for its substrates where 1 beta(R)-epimers were preferred to 1 alpha(S)-epimers, although ipecoside (1 beta(R)) is a major alkaloidal glucoside in P. ipecacuanha, suggesting the compartmentalization of IpeGlu1 from ipecoside. These facts have significant implications for distinct physiological roles of 1 alpha(S)- and 1 beta(R)-epimers and for the involvement of IpeGlu1 in the metabolic fate of both of them.

FIGURE 1.

Schematic representation of the biosynthetic pathway of emetine, cephaeline, and related alkaloidal glucosides found in P. ipecacuanha (*) and A. lamarckii (†).

FIGURE 2.

Phylogenetic tree of the Ipecac alkaloid β-glucosidases (IpeGlu1 and IpeGlu9) with plant family 1 glycosyl hydrolases. Full-length amino acid sequences were initially aligned using the ClustalW version 1.83 (clustalw.ddbj.nig.ac.jp/top-j.html). The highly divergent N-terminal region where significant alignment was not constructed was removed, and truncated sequences were aligned again. Calculation was done based on the neighbor-joining method (42), and the tree was visualized with Treeview version 1.66. The amino acid sequences used were IpeGlu1 (P. ipecacuanha, GenBank™ accession no. AB455576, residues 26–543), IpeGlu9 (P. ipecacuanha, AB455584, 26–540), raucaffricine β-glucosidase (R. serpentina, AF149311, 26–540), strictosidine β-glucosidase (R. serpentina, AJ302044, 47–532), strictosidine β-glucosidase (C. roseus, AF112888, 55–555), isoflavone conjugate β-glucosidase (Glycine max, AB259819, 49–514), dalcochinin β-glucosidase (Dalbegia cochinchinensis, AF163097, 49–547), prunasin hydrolase (Prunus serotina, AF411131, 47–542), amygdalin hydrolase (Prunus serotina, U26025, 49–553), β-primeverosidase (Camellia sinensis, AB088027, 43–507), furcatin hydrolase (Viburnum furcatum, AB122081, 78–538), myrosinase (Sinapis alba, X59879, 49–544), furostanol glycoside glucosidase (Costus speciosus, D83177, 100–562), DIMBOA β-glucosidase (Triticum aestivum, AB236422, 82–569), dhurrinase (Sorghum bicolor, U33817, 80–565), and DIMBOA β-glucosidase (Zea mays, U25157, 82–566). The bar corresponds to 10% change.

FIGURE 3.

SDS-PAGE of the recombinant IpeGlu1 enzyme expressed in E. coli. Proteins were separated on 10% SDS-PAGE and stained with Coomassie Brilliant Blue G-250. Lane M, molecular size marker; lane 1, crude extract from the E. coli-expressing IpeGlu1; lane 2, column run through in metal chelation chromatography; lanes 3–5, stepwise elution of the protein from the metal-resin with buffers containing imidazole at 5 mm (lane 3), 50 mm (lane 4), and 200 mm (lane 5).

FIGURE 4.

Plausible reaction scheme of the aglycon formed by IpeGlu1 from N-deacetylisoipecoside and N-deacetylipecoside under normal (A) and reduced (B) conditions. The molecular ion detectable in LC-MS/MS analysis is shown below each structure.

FIGURE 5.

Overview of the involvement of IpeGlu1 in Ipecac alkaloid biosynthesis on the basis of substrate specificity of the IpeGlu1.