Transfer specificity of detergent-solubilized fenugreek galactomannan galactosyltransferase.
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.
Department of Biological Sciences, University of Stirling, Stirling FK9 4LA, Scotland.
Mary E
ME
Edwards
Elaine
E
Marshall
Michael J
MJ
Gidley
J S Grant
JS
Reid
D000818:Animals; D001234:Aspergillus niger / Q000201:enzymology; D002250:Carbon Radioisotopes; D003902:Detergents / Q000494:pharmacology*; D007887:Fabaceae / Q000201:enzymology; D005690:Galactose / Q000378:metabolism* / Q000031:analogs & derivatives; D005700:Galactosyltransferases / Q000378:metabolism*; D006026:Glycoside Hydrolases; D008351:Mannans / Q000096:biosynthesis*; D008361:Mannosidases / Q000494:pharmacology; D008364:Mannosyltransferases / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D012908:Snails / Q000201:enzymology; D013379:Substrate Specificity; D029910:Trigonella / Q000187:drug effects / Q000201:enzymology*; D014531:Uridine Diphosphate Galactose / Q000378:metabolism; D000519:alpha-Galactosidase / Q000494:pharmacology; D001616:beta-Galactosidase / Q000494:pharmacology; D044902:beta-Mannosidase
D002250:Carbon Radioisotopes; D003902:Detergents; D008351:Mannans; D009844:Oligosaccharides; C012990:galactomannan; D014531:Uridine Diphosphate Galactose; D005700:Galactosyltransferases; D008364:Mannosyltransferases; C021542:mannan synthetase; D006026:Glycoside Hydrolases; D008361:Mannosidases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D000519:alpha-Galactosidase; D001616:beta-Galactosidase; D044902:beta-Mannosidase; D005690:Galactose