Molecular cloning and analysis of strictosidine beta-D-glucosidase, an enzyme in terpenoid indole alkaloid biosynthesis in Catharanthus roseus.
Strictosidine beta-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 beta-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 approximately 60% homology at the amino acid level to other beta-glucosidases from plants and is encoded by a single-copy 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.
Geerlings
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Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Leiden University, Gorlaeus Laboratories, 2300 RA Leiden, The Netherlands.
Ibañez
M M
MM
Memelink
J
J
van Der Heijden
R
R
Verpoorte
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R
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017343:Genes, Plant; D026121:Indole Alkaloids; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D013729:Terpenes / Q000378:metabolism; D014748:Vinca Alkaloids / Q000378:metabolism*; D001617:beta-Glucosidase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism
D026121:Indole Alkaloids; D010940:Plant Proteins; D013729:Terpenes; D014748:Vinca Alkaloids; C021093:strictosidine; D001617:beta-Glucosidase