tailieunhanh - Báo cáo Y học: Heterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids

Strictosidine glucosidase (SG) is an enzyme that catalyses the second step in the biosynthesis of various classes of monoterpenoid indole alkaloids. Based on the comparison of cDNA sequences of SG from Catharanthus roseus and raucaffricine glucosidase (RG) from Rauvolfia serpentina, primers for RT-PCR were designed and the cDNA encoding SG was cloned from R. serpentina cell suspension cultures. The active enzyme was expressed in Escherichia coli and purified to homogeneity. Analysis of its deduced amino-acid sequence assigned the SG from R. serpentina to family 1 of glycosyl hydrolases. In contrast to the SG from C. roseus, the enzyme from R | Eur. J. Biochem. 269 2204-2213 2002 FEBS 2002 doi Heterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase a biosynthetic key to over 2000 monoterpenoid indole alkaloids Irina Gerasimenko Yuri Sheludko Xueyan Ma and Joachim Stockigt Lehrstuhl fur Pharmazeutische Biologie Institut fur Pharmazie Johannes Gutenberg-Universitat Mainz Germany Strictosidine glucosidase SG is an enzyme that catalyses the second step in the biosynthesis of various classes of monoterpenoid indole alkaloids. Based on the comparison of cDNA seu lienees of SG from Catharanthus roseus and raucaffricine glucosidase RG from Rauvolfia serpentina primers for RT-PCR were designed and the cDNA encoding SG was cloned from R. serpentina cell suspension cultures. The active enzyme was expressed in Escherichia coli and purified to homogeneity. Analysis of its deduced amino-acid sequence assigned the SG from R. serpentina to family 1 of glycosyl hydrolases. In contrast to the SG from C. roseus the enzyme from R. serpentina is predicted to lack an uncleavable N-terminal signal sequence which is believed to direct proteins to the endoplasmic reticulum. The temperature and pH optimum enzyme kinetic parameters and substrate specificity of the heterologously expressed SG were studied and compared to those of the C. roseus enzyme revealing some differences between the two glucosidases. In vitro deglucosylation of strictosidine by R. serpentina SG proceeds by the same mechanism as has been shown for the C. roseus enzyme preparation. The reaction gives rise to the end product cathenamine and involves 4 21-dehydrocory-nantheine aldehyde as an intermediate. The enzymatic hydrolysis of dolichantoside Nb-methylstrictosidine leads to several products. One of them was identified as a new compound 3-isocorreantine A. From the data it can be concluded that the divergence of the biosynthetic pathways leading to different classes of indole alkaloids formed in R. .