SummaryGalactomannan biosynthesis in vitro is catalysed by membrane preparations from developing fenugreek seed endosperms. Two enzymes interact: a GDP-mannose dependent (1®4)-b-D-mannan synthase and a UDPgalactose dependent (1®6)-a-D-galactosyltransferase. The statistical distribution of galactosyl substituents along the mannan backbone, and the degree of galactose substitution of the primary product of galactomannan biosynthesis appear to be regulated by the speci®city of the galactosyltransferase. We now report the detergent solubilisation of the fenugreek galactosyltransferase with retention of activity, the identi®cation on gels of a putative 51 kDa galactosyltransferase protein, and the isolation, cloning and sequencing of the corresponding cDNA. The solubilised galactosyltransferase has an absolute requirement for added acceptor substrates. Beta-(1®4)-linked D-manno-oligosaccharides with chain lengths greater than or equal to 5 acted as acceptors, as did galactomannans of low to medium galactosesubstitution. The putative galactosyltransferase cDNA encodes a 51282 Da protein, with a single transmembrane alpha helix near the N terminus. We have also con®rmed the identity of the galactosyltransferase by inserting the cDNA in frame into the genome of the methylotrophic yeast Pichia pastoris under the control of an AOX promoter and the yeast alpha secretion factor and observing the secretion of galactomannan agalactosyltransferase activity. Particularly high activities were observed when a truncated sequence, lacking the membrane-spanning helix, was expressed.
A novel xyloglucan-specific endo-(1-->4)-beta-D-glucanase, involved in the post-germinative mobilization of xyloglucan storage reserves, has previously been isolated from nasturtium (Tropaeolum majus L.) seed. Its mode of action has been shown, in vitro, to be one of transglycosylation except at low substrate (glycosylacceptor) concentrations when hydrolysis predominates. Here it is shown that this nasturtium seed xyloglucan endo-transglycosylase is encoded by a single gene which is transcribed and processed to a 1.5 kb mRNA. The isolation and DNA sequence analysis of a cDNA copy of the nasturtium xyloglucan endo-transglycosylase transcript is described. The cDNA encodes a 33.5 kDa precursor polypeptide which is subsequently processed to a 31 kDa mature protein. The precursor incorporates an N-terminal signal sequence which probably contains information relevant to the targeting of the enzyme to the cell wall. The computer-predicted isoelectric point (5.14) and low (approximately 0%) alpha-helix content of the deduced mature protein are in excellent agreement with the experimental data obtained using the purified enzyme. The deduced protein sequence lacks homology with known plant endo-(1-->4)-beta-D-glucanases, consistent with the unique properties of the enzyme. Database searches have revealed that a Brassica protein (meri-5) of previously unknown function, but abundantly expressed in expanding tissue, shares structural identity with the nasturtium xyloglucan endo-transglycosylase. The expression of a xyloglucan endo-transglycosylase in expanding tissue would be consistent with the contention that enzymes of this type are involved in cell wall loosening.
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