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.
Raffinose (O-alpha- D-galactopyranosyl-(1-->6)- O-alpha- D-glucopyranosyl-(1<-->2)- O-beta- 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-alpha- D-galactopyranosyl-(1-->1)- L- myo-inositol), p-nitrophenyl alpha- 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 alpha-galactosidases (EC 3.2.1.22). The physiological implications of these observations are discussed.
Peterbauer
Thomas
T
Institute of Ecology, University of Vienna, Althanstrasse 14, Austria.
Mach
Lukas
L
Mucha
Jan
J
Richter
Andreas
A
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004187:Disaccharides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005700:Galactosyltransferases / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006026:Glycoside Hydrolases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology* / Q000235:genetics; D011887:Raffinose / Q000096:biosynthesis*; D012639:Seeds / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism; D000519:alpha-Galactosidase / Q000037:antagonists & inhibitors / Q000378:metabolism
D018076:DNA, Complementary; D004187:Disaccharides; D004791:Enzyme Inhibitors; C013536:6 beta-galactinol; D013395:Sucrose; D005700:Galactosyltransferases; C113913:galactinol -raffinose galactosyltransferase; C468749:raffinose synthase; D006026:Glycoside Hydrolases; C022514:raffinase; D000519:alpha-Galactosidase; D011887:Raffinose