Transfer specificity of detergent-solubilized fenugreek galactomannan galactosyltransferase

Abstract

The current experimental model for galactomannan biosynthesis in membrane-bound enzyme systems from developing legume-seed endosperms involves functional interaction between a GDP-mannose (Man) mannan synthase and a UDP-galactose (Gal) galactosyltransferase. The transfer specificity of the galactosyltransferase to the elongating mannan chain is critical in regulating the distribution and the degree of Gal substitution of the mannan backbone of the primary biosynthetic product. Detergent solubilization of the galactosyltransferase of fenugreek (Trigonella foenum-graecum) with retention of activity permitted the partial purification of the enzyme and the cloning and sequencing of the corresponding cDNA with proof of functional identity. We now document the positional specificity of transfer of ((14)C)Gal from UDP-((14)C)Gal to manno-oligosaccharide acceptors, chain lengths 5 to 8, catalyzed by the detergent-solubilized galactosyltransferase. Enzymatic fragmentation analyses of the labeled products showed that a single Gal residue was transferred per acceptor molecule, that the linkage was (1-->6)-alpha, and that there was transfer to alternative Man residues within the acceptor molecules. Analysis of the relative frequencies of transfer to alternative Man residues within acceptor oligosaccharides of different chain length allowed the deduction of the substrate subsite recognition requirement of the galactosyltransferase. The enzyme has a principal recognition sequence of six Man residues, with transfer of Gal to the third Man residue from the nonreducing end of the sequence. These observations are incorporated into a refined model for enzyme interaction in galactomannan biosynthesis.

Figure 1

α-Galactosidase-catalyzed release of labeled Gal from the purified product of incubating mannoheptaose (M7) with UDP-(¹⁴C)Gal in the presence of digitonin-solubilized fenugreek GMGT. The α-galactosidase was diluted to give a slow reaction. Samples of the α-galactosidase reaction mixture were removed at various times and subjected to TLC. The figure shows a digital autoradiogram of the TLC plate. M7prod, Purified transfer product.

Figure 2

β-Mannosidase digestion of a purified sample of labeled monogalactosylmannohexaose (M6G) formed by incubating mannohexaose with UDP-(¹⁴C)Gal in presence of Triton X-100-solubilized fenugreek GMGT. Samples of the β-mannosidase reaction mixture were taken at various times and subjected to TLC. The figure is a digital autoradiogram of the TLC plate. R, Reference standards of (¹⁴C)Man-labeled galactomannan oligosaccharides from endo-β-mannanase digestion of galactomannan biosynthesized in vitro (Reid et al., 1995).

Figure 3

Observed pattern of Gal substitution of M5, M6, M7, and M8 by detergent-solubilized fenugreek GMGT in relation to a hypothetical GMGT acceptor substrate subsite recognition sequence comprising six Man residues (numbered 1–6 from the nonreducing end of the sequence), with transfer occurring to the Man residue at site 3 of the recognition sequence. An asterisk indicates an observed position of minor substitution. A double asterisk indicates an observed position of major substitution.

Figure 4

A model for the interaction of MS and GMGT during galactomannan biosynthesis in fenugreek.