ENZYME SPECIFICITY IN GALACTOMANNAN BIOSYNTHESIS

Membrane-bound enzymes from developing legume-seed endosperms catalyse galactomannan biosynthesis in vitro from GDP-mannose and UDP-galactos e. A mannosyltransferase [mannan synthase] catalyses the extension of the linear (1-->4)-beta-linked D-mannan backbone towards the non-reduc ing end. A specific alpha-galactosyltransferase brings about the galac tosyl-substitution of the backbone by catalysing the transfer of a (1- ->6)-alpha-D-galactosyl residue to an acceptor mannosyl residue at or close to the non-reducing terminus of the growing backbone. Labelled g alactomannans with a range of mannose/galactose (Man/Gal) ratios were formed in vitro from GDP-[C-14]mannose and UDP-[C-14]galactose using m embrane-bound enzyme preparations from fenugreek (Trigonella foenum-gr aecum L.), guar (Cyamopsis tetragonoloba (L.) Taub.) and senna (Senna occidentalis (L.) Link.), species which in vivo, form galactomannans w ith Man/Gal ratios of 1.1, 1.6 and 3.3 respectively. The labelled gala ctomannans were fragmented using a structure-sensitive endo-(1-->4)-be ta-D-mannanase and the quantitative fragmentation data were processed using a computer algorithm which simulated the above model for galacto mannan biosynthesis on the basis of a second-order Markov chain proces s, and also the subsequent action of the endo-mannanase. For each gala ctomannan data-set processed, the algorithm generated a set of four co nditional probabilities required by the Markov model. The need for a s econd-order Markov chain description indicated that the galactomannan subsite recognition sequence of the galactosyltransferase must encompa ss at least three backbone mannose residues, i.e. the site of substitu tion and the two preceding ones towards the reducing end of the growin g galactomannan chain. Data-sets from the three plant species generate d three distinctly different sets of probabilities, and hence galactos e-substitution rules. For each species, the maximum degree of galactos e-substitution consistent with these rules was closely similar to that observed for the primary product of galactomannan biosynthesis in viv o. The data provide insight into the mechanism of action and the spati al organisation of membrane-bound polysaccharide synthases.