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 biosynt hesis of terpenoid indole alkaloids (TLAs) by converting strictosidine to c athenamine, 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. W e 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 ac tivity when expressed in Saccharomyces cerevisiae, SGD shows similar to 60% homology at the amino acid level to other beta-glucosidases from plants an d is encoded by a single-copy gene. Sgd expression is induced by methyl jas monate with kinetics similar to those of two other genes acting prior to Sg d in TLA biosynthesis. These results show that coordinate induction of the biosynthetic genes forms at least part of the mechanism for the methyl jasm onate-induced increase in TIA production. Using a novel in vivo staining me thod, subcellular localization studies of SGD were performed, This showed t hat SGD is most likely associated with the endoplasmic reticulum, which is in accordance with the presence of a putative signal sequence, but in contr ast to previous localization studies. This new insight in SGD localization has significant implications for our understanding of the complex intracell ular trafficking of metabolic intermediates during TIA biosynthesis.