Strictosidine synthase from Catharanthus roseus: purification and characterization of multiple forms

Area of Science:

• Plant biochemistry
• Enzyme kinetics
• Secondary metabolism

Background:

• Strictosidine synthase (SS) is a key enzyme in the biosynthesis of indole alkaloids in Catharanthus roseus.
• Indole alkaloids play vital roles in plant defense mechanisms.
• Understanding SS isoforms is crucial for metabolic engineering of these valuable compounds.

Purpose of the Study:

• To purify and characterize multiple strictosidine synthase isoforms from Catharanthus roseus cell cultures.
• To investigate the kinetic properties and distribution of these isoforms within the plant.
• To establish the highest reported catalytic efficiency for strictosidine synthase.

Main Methods:

• Purification using hydrophobic-interaction, gel-permeation, ion-exchange chromatography, and chromatofocusing.
• Characterization of enzyme isoforms, including homogeneity assessment and glycoprotein analysis.
• Kinetic analysis to determine parameters like maximal velocity and Michaelis constant.

Main Results:

• Six strictosidine synthase isoforms were identified, with four purified to homogeneity.
• All isoforms were confirmed to be glycoproteins and present in various plant tissues.
• Enzymes displayed high maximal velocity (300-400 nkat/mg protein) and low apparent Michaelis constant for tryptamine (~9 microM).

Conclusions:

• The study presents a comprehensive purification and characterization of C. roseus strictosidine synthase isoforms.
• The identified isoforms exhibit significantly improved kinetic parameters compared to previous reports.
• Findings provide valuable insights into the catalytic efficiency and distribution of strictosidine synthase in C. roseus.