Search results for "Strictosidine"

Expression, purification, crystallization and preliminary X-ray analysis of strictosidine glucosidase, an enzyme initiating biosynthetic pathways to …

2005

Abstract Strictosidine β- d -glucosidase, a plant enzyme initiating biosynthetic pathways to about 2000 monoterpenoid indole alkaloids with an extremely large number of various carbon skeletons, has been functionally expressed in Escherichia coli and purified to homogeneity in mg scale. Crystals suitable for X-ray analysis were found by robot-mediated screening. Using the hanging-drop technique, optimum conditions were 0.3 M ammonium sulfate, 0.1 M sodium acetate, pH 4.6 and PEG 4000 (10%) as precipitant buffer. The crystals of strictosidine glucosidase belong to the space group P 42 1 2 with unit cell dimensions of a =157.63, c =103.59 A and diffract X-rays to 2.48-A resolution.

Heterologous expression of aRauvolfiacDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids

2002

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. serpentina is predicted to lack an uncleavable N…

Structures of Alkaloid Biosynthetic Glucosidases Decode Substrate Specificity

2011

Two similar enzymes with different biosynthetic function in one species have evolved to catalyze two distinct reactions. X-ray structures of both enzymes help reveal their most important differences. The Rauvolfia alkaloid biosynthetic network harbors two O-glucosidases: raucaffricine glucosidase (RG), which hydrolyses raucaffricine to an intermediate downstream in the ajmaline pathway, and strictosidine glucosidase (SG), which operates upstream. RG converts strictosidine, the substrate of SG, but SG does not accept raucaffricine. Now elucidation of crystal structures of RG, inactive RG-E186Q mutant, and its complexes with ligands dihydro-raucaffricine and secologanin reveals that it is the…

Ligand structures of synthetic deoxa-pyranosylamines with raucaffricine and strictosidine glucosidases provide structural insights into their binding…

2014

Insight into the structure and inhibition mechanism of O-β-d-glucosidases by deoxa-pyranosylamine type inhibitors is provided by X-ray analysis of complexes between raucaffricine and strictosidine glucosidases and N-(cyclohexylmethyl)-, N-(cyclohexyl)- and N-(bromobenzyl)-β-d-gluco-1,5-deoxa-pyranosylamine. All inhibitors anchored exclusively in the catalytic active site by competition with appropriate enzyme substrates. Thus facilitated prospective elucidation of the binding networks with residues located at <3.9 A distance will enable the development of potent inhibitors suitable for the production of valuable alkaloid glucosides, raucaffricine and strictosidine, by means of synthesis in …

Molecular Architecture of Strictosidine Glucosidase: The Gateway to the Biosynthesis of the Monoterpenoid Indole Alkaloid Family[W]

2007

Abstract Strictosidine β-d-glucosidase (SG) follows strictosidine synthase (STR1) in the production of the reactive intermediate required for the formation of the large family of monoterpenoid indole alkaloids in plants. This family is composed of ∼2000 structurally diverse compounds. SG plays an important role in the plant cell by activating the glucoside strictosidine and allowing it to enter the multiple indole alkaloid pathways. Here, we report detailed three-dimensional information describing both native SG and the complex of its inactive mutant Glu207Gln with the substrate strictosidine, thus providing a structural characterization of substrate binding and identifying the amino acids …

The Structure of Rauvolfia serpentina Strictosidine Synthase Is a Novel Six-Bladed β-Propeller Fold in Plant Proteins

2006

Abstract The enzyme strictosidine synthase (STR1) from the Indian medicinal plant Rauvolfia serpentina is of primary importance for the biosynthetic pathway of the indole alkaloid ajmaline. Moreover, STR1 initiates all biosynthetic pathways leading to the entire monoterpenoid indole alkaloid family representing an enormous structural variety of ∼2000 compounds in higher plants. The crystal structures of STR1 in complex with its natural substrates tryptamine and secologanin provide structural understanding of the observed substrate preference and identify residues lining the active site surface that contact the substrates. STR1 catalyzes a Pictet-Spengler–type reaction and represents a novel…

Strictosidine—The Biosynthetic Key to Monoterpenoid Indole Alkaloids

1999

Enzymatic formation of the sarpagan-bridge: a key step in the biosynthesis of sarpagine- and ajmaline-type alkaloids.

1995

The glucoalkaloid strictosidine has been converted under cell-free conditions into 10-deoxysarpagine (= normacusine B) in the presence of a crude soluble enzyme extract and microsomal protein isolated from cell suspensions of Rauwolfia serpentina. The enzymatic formation of this alkaloid bearing the C-5/C-16 bond (sarpagan-bridge), which is characteristic for all sarpagine- and ajmaline-type alkaloids, is dependent on NADPH and oxygen. Inhibition studies indicate that for the synthesis of 10-deoxysarpagine a cytochrome P450 dependent monoxygenase is necessary.

Molecular cloning and functional bacterial expression of a plant glucosidase specifically involved in alkaloid biosynthesis.

2000

Monoterpenoid indole alkaloids are a vast and structurally complex group of plant secondary compounds. In contrast to other groups of plant products which produce many glycosides, indole alkaloids rarely occur as glucosides. Plants of Rauvolfia serpentina accumulate ajmaline as a major alkaloid, whereas cell suspension cultures of Rauvolfia mainly accumulate the glucoalkaloid raucaffricine at levels of 1.6 g/l. Cell cultures do contain a specific glucosidase. known as raucaffricine-O-beta-D-glucosidase (RG), which catalyzes the in vitro formation of vomilenine, a direct intermediate in ajmaline biosynthesis. Here, we describe the molecular cloning and functional expression of this enzyme in…

Crystallization and preliminary X-ray crystallographic analysis of strictosidine synthase from Rauvolfia: the first member of a novel enzyme family.

2004

Strictosidine synthase is a central enzyme involved in the biosynthesis of almost all plant monoterpenoid indole alkaloids. Strictosidine synthase from Rauvolfia serpentina was heterologously expressed in Escherichia coli. Crystals of the purified recombinant enzyme have been obtained by the hanging-drop technique at 303 K with potassium sodium tartrate tetrahydrate as precipitant. The crystals belong to the space group R3 with cell dimensions of a=b=150.3 A and c=122.4 A. Under cryoconditions (120 K), the crystals diffract to about 2.95 A.