The New β-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␣(S)-epimer) is considered to be a part of the reactions for emetine biosynthesis, whereas its 1␤(R)-epimer N-deacetylipecoside is converted to ipecoside in P. ipecacuanha. Here, we isolated a cDNA clone Ipeglu1 encoding Ipecac alkaloid ␤-D-glucosidase from P. ipecacuanha. The deduced protein showed 54 and 48% identities to raucaffricine ␤-glucosidase and strictosidine ␤-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-Omethyl 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 ␤-glucosidase that stereospecifically hydrolyzes 3␣(S)-epimer in terpenoid-indole alkaloid biosynthesis, IpeGlu1 lacked stereospecificity for its substrates where 1␤(R)-epimers were preferred to 1␣(S)-epimers, although ipecoside (1␤(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␣(S)-and 1␤(R)-epimers and for the involvement of IpeGlu1 in the metabolic fate of both of them.

Functional Characterization of CsBGlu12, a β-Glucosidase from Crocus sativus, Provides Insights into Its Role in Abiotic Stress through Accumulation of Antioxidant Flavonols

“…However, EDTA (1 mM), the nonionic detergent Triton X-100 (1%), and reducing agent 2-mercaptoethanol (1%) had little or no effect on the enzyme activity. These observations are also in agreement with the studies carried out previously (23).…”

“…The kinetic constants K m and V max values were calculated with non-linear regression analysis using GraphPad Prism 6 software. Effect of additives on enzyme activity was evaluated using a standard protocol (23).…”

Characterization of a Novel β-Glucosidase from a Compost Microbial Metagenome with Strong Transglycosylation Activity

“…The eluent was water with a linear gradient of CH 3 CN from 80 to 74% over 30 min. The column was kept at 40°C and eluted at 1.0 ml min…”

“…terpenols and flavonoids) from glycoside precursors (3,4), and cyanide-based biological defense mechanisms (the release of cyanide from cyanoglucosides) in plants (5). Recent interest in ␤-glucosidases arose from their involvement in the biological conversion of cellulose.…”

Molecular Cloning of anO-Methyltransferase from Adventitious Roots ofCarapichea ipecacuanha

“…4,5) Ipecac alkaloids are synthesized from the condensation of dopamine derived from tyrosine, for isoquinoline moieties, and from secologanin, as a monoterpenoid molecule (Fig. 1).…”