Planta

, Volume 215, Issue 5, pp 839–846

Functional expression of a cDNA encoding pea (Pisum sativum L.) raffinose synthase, partial purification of the enzyme from maturing seeds, and steady-state kinetic analysis of raffinose synthesis

  • Thomas Peterbauer
  • Lukas Mach
  • Jan Mucha
  • Andreas Richter
Original Article

DOI: 10.1007/s00425-002-0804-7

Cite this article as:
Peterbauer, T., Mach, L., Mucha, J. et al. Planta (2002) 215: 839. doi:10.1007/s00425-002-0804-7

Abstract.

Raffinose (O-α-D-galactopyranosyl-(1→6)-O-α-D-glucopyranosyl-(1↔2)-O-β-D-fructofuranoside) is a widespread oligosaccharide in plant seeds and other tissues. Raffinose synthase (EC 2.4.1.82) is the key enzyme that channels sucrose into the raffinose oligosaccharide pathway. We here report on the isolation of a cDNA encoding for raffinose synthase from maturing pea (Pisum sativum L.) seeds. The coding region of the cDNA was expressed in Spodoptera frugiperda Sf21 insect cells. The recombinant enzyme, a protein of glycoside hydrolase family 36, displayed similar kinetic properties to raffinose synthase partially purified from maturing seeds by anion-exchange and size-exclusion chromatography. Apart from the natural galactosyl donor galactinol (O-α-D-galactopyranosyl-(1→1)-L-myo-inositol), p-nitrophenyl α-D-galactopyranoside, an artificial substrate, was utilized as a galactosyl donor. An equilibrium constant of 4.1 was determined for the galactosyl transfer reaction from galactinol to sucrose. Steady-state kinetic analysis suggested that raffinose synthase is a transglycosidase operating by a ping-pong reaction mechanism and may also act as a glycoside hydrolase. The enzyme was strongly inhibited by 1-deoxygalactonojirimycin, a potent inhibitor for α-galactosidases (EC 3.2.1.22). The physiological implications of these observations are discussed.

cDNA cloning Enzyme kinetics Galactinol Pisum Raffinose synthase Seed 

Copyright information

© Springer-Verlag 2002

Authors and Affiliations

  • Thomas Peterbauer
    • 1
  • Lukas Mach
    • 2
  • Jan Mucha
    • 2
  • Andreas Richter
    • 1
  1. 1.Institute of Ecology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria
  2. 2.Centre for Applied Genetics, University of Agricultural Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria

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