Enzymic transfer of α-L-arabinopyranosyl residues to exogenous 1,4-linked β-D-galacto-oligosaccharides using solubilized mung bean (Vigna radiata) hypocotyl microsomes and UDP-β-L-arabinopyranose

Abstract

A single alpha-L-arabinopyranosyl (alpha-L-Arap) residue was shown, by a combination of chemical and spectroscopic methods, to be transferred to O-4 of the nonreducing terminal galactosyl (Gal) residue of 2-aminobenzamide (2AB)-labeled galacto-oligosaccharides when these oligosaccharides were reacted with UDP-ss-L-arabinopyranose (UDP-ss-L-Arap) in the presence of a Triton X-100-soluble extract of microsomal membranes isolated from mung bean (Vigna radiata, L. Wilezek) hypocotyls. Maximum-(1-->4)-arabinopyranosyltransferase activity was obtained at pH 6.0-6.5 and 20 degrees C in the presence of 25 mM Mn2+. The enzyme had an apparent K m of 45 microM for the 2AB-labeled galactoheptasaccharide and 330 microM for UDP-ss-L-Arap. A series of 2AB-labeled galacto-oligosaccharides with a degree of polymerization (DP) between 6 and 10 that contained a single alpha-L-Arap residue linked to the former nonreducing terminal Gal residue were generated when the 2AB-labeled galactohexasaccharide (Gal6-2AB) was reacted with UDP- ss-L-Ara p in the presence of UDP-beta-D-Galp and the solubilized microsomal fraction. The mono-arabinosylated galacto-oligosaccharides are not acceptor substrates for the galactosyltransferase activities known to be present in mung bean microsomes. These results show that mung bean hypocotyl microsomes contain an enzyme that catalyzes the transfer of Arap to the nonreducing Gal residue of galacto-oligosaccharides and suggest that the presence of a alpha-L-Arap residue on the former terminal Gal residue prevents galactosylation of galacto-oligosaccharides.

Identification of a Mung Bean Arabinofuranosyltransferase That Transfers Arabinofuranosyl Residues onto (1, 5)-Linked α-l-Arabino-Oligosaccharides

“…2D). We have also shown that the arabinan side chains have an average DP of 6 in the RG-I isolated from 3-d-old mung bean hypocotyls (Ishii et al, 2005c). The availability of a facile assay for ArafT activity provides an opportunity to investigate arabinan synthesis in great detail.…”

“…The aqueous layer was freeze dried. The residue was dissolved in 75% CH 3 CNwater (v/v) and separated by normal-phase HPLC (Ishii et al, 2005c). The purified product was analyzed by LC-ESI-mass spectrometry and 1 H-NMR spectrometry (Ishii et al, 2005c).…”

“…The residue was dissolved in 75% CH 3 CNwater (v/v) and separated by normal-phase HPLC (Ishii et al, 2005c). The purified product was analyzed by LC-ESI-mass spectrometry and 1 H-NMR spectrometry (Ishii et al, 2005c). The enzymatically formed products from Ara 7 -2AB were treated with a-L-arabinofuranosidase (Yang et al, 2006) in 100 mM sodium acetate buffer (pH 5.0) at 50°C overnight and analyzed by HPLC as described above.…”

“…The reaction mixture was incubated for 30 min at 25°C and then the reaction was terminated by heating for 5 min at 100°C. The mixture was centrifuged and the supernatant then analyzed by normal-phase HPLC using fluorescence (l x 5 330 mm, l em 5 420 mm) detection as described (Ishii et al, 2005c). Enzyme activity is expressed as pmol Ara transferred min 21 mg protein 21 , based on the concentration curve of 2AB-Ara 3 as the calibration standard.…”

The Origin and Functions of Arabinofuranosyl Residues in Plant Cell Walls

“…A single Arap residue was attached to galacto-oligosaccharide ( Fig. 3B) (17). From these results we speculated that Araf residues are formed from UDP-Araf, not UDP-Arap.…”