Molecular Cloning and Analysis of Strictosidine β-d-Glucosidase, an Enzyme in Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus

Abstract

Strictosidine ␤-D-glucosidase (SGD) is an enzyme involved in the biosynthesis of terpenoid indole alkaloids (TIAs) by converting strictosidine to cathenamine. The biosynthetic pathway toward strictosidine is thought to be similar in all TIA-producing plants. Somewhere downstream of strictosidine formation, however, the biosynthesis diverges to give rise to the different TIAs found. SGD may play a role in creating this biosynthetic diversity. We have studied SGD at both the molecular and enzymatic levels. Based on the homology between different plant ␤-glucosidases, degenerate polymerase chain reaction primers were designed and used to isolate a cDNA clone from a Catharanthus roseus cDNA library. A full-length clone gave rise to SGD activity when expressed in Saccharomyces cerevisiae. SGD shows ϳ60% homology at the amino acid level to other ␤-glucosidases from plants and is encoded by a singlecopy gene. Sgd expression is induced by methyl jasmonate with kinetics similar to those of two other genes acting prior to Sgd in TIA biosynthesis. These results show that coordinate induction of the biosynthetic genes forms at least part of the mechanism for the methyl jasmonate-induced increase in TIA production. Using a novel in vivo staining method, subcellular localization studies of SGD were performed. This showed that SGD is most likely associated with the endoplasmic reticulum, which is in accordance with the presence of a putative signal sequence, but in contrast to previous localization studies. This new insight in SGD localization has significant implications for our understanding of the complex intracellular trafficking of metabolic intermediates during TIA biosynthesis.

Geraniol 10‐hydroxylase1, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis

“…These include enzymes acting in the vindoline-speci¢c branch of the pathway, such as the P450 enzyme tabersonine 16-hydroxylase [27], desacetoxyvindoline-4-hydroxylase [28] and acetyl-CoA:4-O-deacetylvindoline 4-O-acetyltransferase [29], as well as genes acting earlier in the pathway, such as Tdc [30], Str [15], Cpr [22], Sls [9] and Sgd [21]. The availability of the G10H cDNA now allows overexpression of combinations of G10h with other genes in plants and cell cultures.…”

“…RNA extractions and Northern blot analysis were performed as described [20]. DNA probes for tryptophan decarboxylase (Tdc), strictosidine synthase (Str), strictosidine L-D-glucosidase (Sgd), 4OS ribosomal protein S9 (Rps9) [21] and Cpr [22] were described. …”

“…It does so by coordinately switching on the expression of multiple genes in the TIA biosynthetic pathway [5,20,21] and precursor pathways [5]. Therefore it was of interest to study the e¡ect of MeJA on G10h expression.…”

Opium poppy and Madagascar periwinkle: model non‐model systems to investigate alkaloid biosynthesis in plants

“…C. roseus accumulates at least 130 MIAs, which are detected in small quantities in various organs and in specialized cell cultures (van der Heijden et al, 2004). Strictosidine b-glucosidase (SGD) is a remarkable strictosidine-specific enzyme that yields a highly reactive ringopened dialdehyde intermediate that is involved in the biosynthesis of MIAs with corynantheine, iboga and aspidosperma chemical backbones (Geerlings et al, 2000). However, little is known about the mechanisms or enzymes involved in directing the formation of each type.…”

Mutational Analysis of the Medicago Glycosyltransferase UGT71G1 Reveals Residues That Control Regioselectivity for (Iso)flavonoid Glycosylation

“…It involves transfer of a nucleotide diphosphateactivated sugar moiety to an acceptor molecule and is catalyzed by a diverse group of uridine diphosphate glycosyltransferases (UGTs). 2 All major classes of plant secondary metabolites, including flavonoids (1, 2), terpenoids (3)(4)(5), and alkaloids (6) can be modified by glycosylation, and more than 300 different flavonoid glycosides have been identified from plants (7).…”