Biosynthesis of pectic galactan by membrane-bound galactosyltransferase from soybean ( Glycine max Merr.) seedlings

Abstract

We investigated the properties of a galactosyltransferase (GalT) that is involved in the synthesis of beta-(1-->4)-galactan side chains of pectins. A membrane preparation of etiolated 6-day-old soybean ( Glycine max Merr.) hypocotyls transferred [(14)C]Gal from UDP-[(14)C]Gal into intact and partially hydrolyzed lupin beta-(1-->4)-galactans of various chain lengths as exogenous acceptors, while activity to endogenous acceptors was negligible. Maximal activity occurred at pH 6.5 and 20-25 degrees C in the presence of 25 mM Mn(2+) and 0.75% Triton X-100. The transfer reaction onto the unmodified commercial pectic galactan ( M(r)>150000) from lupin we used was very low but increased when the M(r) of the galactan was reduced by partial acid hydrolysis. Among the partially hydrolyzed galactans, high- M(r) (average M(r) 60000) beta-(1-->4)-galactan was a more efficient acceptor [specific activity 2000-3000 pmol min(-1) (mg protein)(-1)] than low- M(r) (average M(r) 10000 and 5000) polymers. Digestion of the radiolabeled product from high- M(r) galactan with endo-beta-(1-->4)-galactanase released mainly radioactive beta-(1-->4)-galactobiose and Gal, indicating that the transfer of [(14)C]Gal occurred through beta-(1-->4)-linkages. HPLC analysis showed that the enzyme also catalyzes incorporation of Gal into pyridylaminated (PA) beta-(1-->4)-galactooligomers with degree of polymerization at least 5. Gal(7)-PA chains were elongated by attachment of one, two, or three Gal residues leading to the formation of Gal(8-10)-PA.

Exploring the Ultrastructural Localization and Biosynthesis of β(1,4)-Galactan in Pinus radiata Compression Wood

“…This implies either that developing compression wood has a higher level of GalT than soybean or that galactan formation is not hindered in pine as compared with soybean. The biochemical properties of pine GalT were measured and found to be similar to the pectic GalT from soybean (Konishi et al, 2004). The optimal pH and temperature are 7 and 20°C for pine and 6.5 and 20°C to 25°C for soybean.…”

“…Whether those differences are the result of different enzyme properties, the lack of interfering enzymes in pine microsomal preparations, or some other factor(s) is currently unknown. An interesting observation of possible relevance in explaining this difference is that galactan chains in soybean have a DP of 43 to 47 (Konishi et al, 2004), while pine galactan is thought have a DP of approximately 380 (Nanayakkara, 2007).…”

“…Excess 2AP was removed from the reaction by extraction with 1:1 phenol:chloroform (3 3 5 mL), followed by desalting on a Sephadex G-10 column in a buffer of 10 mM ammonium bicarbonate. Separation of the 2AP-tagged oligosaccharides was accomplished by reverse-phase HPLC on a C 18 -PAQ column (10 mm 3 250 mm; Cosmosil; Konishi et al, 2004). The column was eluted with 50 mM ammonium acetate (pH 4.5) containing 0.025% 1-butanol at a flow rate of 2 mL min 21 and a temperature of 40°C.…”

“…An aliquot of the sample was used to verify that the reaction was in the pH range of the galactanase. The sample was digested with 100 milliunits of endo-b(1,4)-galactanase at 37°C for 3 h, and the reaction was stopped by heating at 95°C for 5 min (Konishi et al, 2004). The digestion was analyzed by HPLC with the TSKgel Amide-80 column.…”

“…Despite substantial efforts to characterize the biosynthesis of this polymer, b(1,4)-galactan biosynthetic enzymes and their corresponding genes are currently unknown (Peugnet et al, 2001;Geshi et al, 2002Geshi et al, , 2004Abdel-Massih et al, 2003;Kato et al, 2003;Ishii et al, 2004;Konishi et al, 2004Konishi et al, , 2007Gorshkova and Morvan, 2006). However, based on other cell wall polysaccharide biosynthetic enzymes, it is likely that the enzymes involved in the biosynthesis of b(1,4)-galactan are either Golgi localized or pass through the Golgi in transit to the apoplastic space (Reyes and Orellana, 2008).…”