Molecular characterisation of a membrane‐bound galactosyltransferase of plant cell wall matrix polysaccharide biosynthesis

Plant Journal - Tập 19 Số 6 - Trang 691-697 - 1999
Mary E. Edwards1, Cathryn A. Dickson1, Sumant Chengappa2,1, C. Sidebottom2, Michael J. Gidley2, J. S. Grant Reid1
1University of Stirling, Department of Biological Sciences, Stirling FK9 4LA, UK, and
2Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford, MK44 1LQ, UK

Tóm tắt

SummaryGalactomannan biosynthesis in vitro is catalysed by membrane preparations from developing fenugreek seed endosperms. Two enzymes interact: a GDP‐mannose dependent (1→4)‐β‐d‐mannan synthase and a UDP‐galactose dependent (1→6)‐α‐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 specificity of the galactosyltransferase. We now report the detergent solubilisation of the fenugreek galactosyltransferase with retention of activity, the identification 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 galactose‐substitution. The putative galactosyltransferase cDNA encodes a 51282 Da protein, with a single transmembrane alpha helix near the N terminus. We have also confirmed 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 α‐galactosyltransferase activity. Particularly high activities were observed when a truncated sequence, lacking the membrane‐spanning helix, was expressed.

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Plant Journal

Tập 19 Số 6

691-697

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