title: Strictosidine synthase from Catharanthus roseus: purification and characterization of multiple forms.
abstract: Multiple (six) forms of strictosidine synthase from Catharanthus roseus cell suspension cultures were purified and characterized. A purification protocol is presented composed of hydrophobic-interaction, gel-permeation and ion-exchange chromatography and chromatofocusing. Four of six isoforms were purified to apparent homogeneity, whereas two others were nearly homogeneous. All strictosidine synthase isoforms were found to be glycoproteins. The isoforms were also found in leaves and roots of the plant, in seedlings and in hairy root cultures. The ratio of the different isoforms differed slightly between these sources. The kinetic parameters of the isoforms showed no significant differences. The maximal velocity (300-400 nkat/mg of protein) is the highest reported so far. It was demonstrated that the apparent Michaelis constant for tryptamine (approx. 9 microM) is much lower than values reported previously. The presence of weak product inhibition (Kp approx. 35 times Km) was established, whereas substrate inhibition was not detected.
authors.affiliation: Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Leiden University, Gorlaeus Laboratories, The Netherlands.
authors.forename: A
authors.initials: A
authors.lastname: de Waal
authors.forename: A H
authors.initials: AH
authors.lastname: Meijer
authors.forename: R
authors.initials: R
authors.lastname: Verpoorte
mesh_terms: D019759:Carbon-Nitrogen Lyases; D002845:Chromatography; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D018515:Plant Leaves / Q000201:enzymology; D018517:Plant Roots / Q000201:enzymology; D010944:Plants / Q000201:enzymology*; D017421:Sequence Analysis; D014166:Transferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D014166:Transferases; D019759:Carbon-Nitrogen Lyases; C022684:strictosidine synthetase