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 (1α(S)-epimer) is considered to be a part of the reactions for emetine biosynthesis, whereas its 1β(R)-epimer N-deacetylipecoside is converted to ipecoside in P. ipecacuanha. Here, we isolated a cDNA clone Ipeglu1 encoding Ipecac alkaloid β-D-glucosidase from P. ipecacuanha. The deduced protein showed 54 and 48% identities to raucaffricine β-glucosidase and strictosidine β-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 β-glucosidase that stereospecifically hydrolyzes 3α(S)-epimer in terpenoid-indole alkaloid biosynthesis, IpeGlu1 lacked stereospecificity for its substrates where 1β(R)-epimers were preferred to 1α(S)-epimers, although ipecoside (β(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 1α(S)- and 1β(R)-epimers and for the involvement of IpeGlu1 in the metabolic fate of both of them. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.