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 UDPgalactose (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 (136)-␣, 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.Galactomannans are the principal polysaccharide component of the walls of endosperm cells of legume seeds (Reid, 1985). Their structures are relatively simple, consisting of a linear (134)-␤-linked d-mannan backbone with single-unit (136)-linked ␣-d-galactopyranosyl side chains. The Man-to-Gal (Man/Gal) ratio is variable between 1.1 (high Gal) and about 3.4 (low Gal), and is taxonomically significant (Reid and Meier, 1970;Bailey, 1971). The galactomannans are multifunctional molecules, serving as the molecular basis of a water imbibition and drought avoidance mechanism before and during germination, and then as a source of storage carbohydrate for the developing seedling (Reid and Bewley, 1979). When isolated, galactomannans are water soluble, with solution properties that have industrial applications. For example, the galactomannans from guar (Cyamopsis tetragonoloba) and locust bean (Ceratonia siliqua) are used in the food industry as viscosifiers, stabilizers, and gelling agents, alone and in combination with other polysaccharides (Dea and Morrison, 1975;. The Man/Gal ratio is an important determinant of commercial functionality, with high Man/Gal ratios (low Gal substitution) favoring valuable mixed-polymer interactions (Dea et al., 1977).Galactomannan biosynthesis is catalyzed in vitro by membrane preparations isolated from the developin...

Poplar Carbohydrate-Active Enzymes. Gene Identification and Expression Analyses

“…Poplar CSLA-like genes had ESTs that were broadly distributed in active tissues but absent in dormant meristems (Table III). The side chains of galactomannan are synthesized by an a-1,6-galactosyl transferase belonging to the GT34 family (Edwards et al, 2002;Reid et al, 2003). Two homologous genes were found in poplar, with only one EST detected in the TW tissues (Table III).…”

The Seeds of Lotus japonicus Lines Transformed with Sense, Antisense, and Sense/Antisense Galactomannan Galactosyltransferase Constructs Have Structurally Altered Galactomannans in Their Endosperm Cell Walls

“…The nascent mannan backbone is exposed to GMGT action as it emerges from the MS, whereas the transfer specificity of the GMGT (Edwards et al, 2002) determines the statistical distribution of Gal residues according to a second order Markov chain model Edwards et al, 2002). In fenugreek, guar, and senna, the observed degree of Gal substitution of the primary product (Edwards et al, 1992) of galactomannan biosynthesis in vivo is very close to the theoretical maximum permitted by the four Markov probabilities scaled to let the highest one of the four equal 1.00 .…”

“…Gal substitution at the Man residues occupying the second and/or the fourth position within the binding sequence prevents hydrolysis, with the result that only certain well-defined fragment oligosaccharides are released from galactomannans by the action of the enzyme (McCleary, 1979;McCleary and Matheson, 1983). The smallest of these allowed oligosaccharides are M2, M3, and the three Gal-substituted manno-oligosaccharides monogalactosylmannobiose, monogalactosylmannotriose, and digalactosylmannopentaose, the molecular structures of which are illustrated in Edwards et al, 2002. Figure 3 documents the labeled manno-and galactomanno-oligosaccharides released from the labeled in vitro products on digestion with the Aspergillus niger endo-␤-mannanase.…”

“…Furthermore, the maximum degrees of Gal substitution allowed by the deduced Markov probabilities for fenugreek, guar, and senna membranes are very close to those observed for the primary product (Edwards et al, 1992) of galactomannan biosynthesis in vivo . Thus, the biosynthesis of galactomannans requires a specific functional interaction between MS and GMGT, within which the transfer specificity of the GMGT is important in determining the statistical distribution of galactosyl residues along the mannan backbone and the Man/Gal ratio (Edwards et al, 2002).…”

“…Neither the ␣-helix nor the short N-terminal domain are required for catalytic action . The detergentsolubilized fenugreek GMGT has a principal acceptor substrate recognition sequence of six (134)-␤-linked Man residues, with transfer occurring at the third Man residue from the nonreducing end of the sequence (Edwards et al, 2002).…”