Biosynthesis of legume-seed galactomannans in vitro

Abstract

Particulate enzyme preparations were isolated from developing fenugreek (Trigonella foenum-graecum L.) and guar (Cyamopsis tetragonoloba [L.] Taub.) seed endosperms during the period of galactomannan deposition in vivo. These preparations catalysed the formation of polysacharide products from guanosine 5'-diphosphate (GDP)-mannose, from uridine 5'-diphosphate (UDP)-galactose and from mixtures of the two nucleotides. The products were analysed by solubility, by complete acid hydrolysis, and by selective enzymatic cleavage using pure enzymes of known specificity. With GDP-[U-(14)C]-D-mannose as substrate and a divalent metal cation (Mg(+2), Mn(+2), or Ca(+2)) a highly efficient transfer of labelled D-mannosyl residues was obtained to give a product identified as linear (1→4)-β-linked D-mannan. No transfer of galactosyl residues was obtained when GDP-[U-(14)C]-D-galactose was the only substrate, although very low and variable amounts of an unidentified product which released labelled glucose on acid hydrolysis were formed. In the presence of UDP-galactose, GDP-mannose and Mn(+2) ions, products were formed which have been characterised as galactomanans - a linear (1→4)-β-D-mannan backbone carrying D-galactopyranosyl substituents linked (1→6)-α to mannose. The degree of galactose substitution of the D-mannan backbone was manipulated in vitro by varying GDP-mannose concentrations at constant (saturating) UDP-galactose levels. The transfer of D-galactosyl residues from UDP-galactose to galactomannan was absolutely dependent upon the simultaneous transfer of D-mannosyl residues from GDP-mannose. D-Mannan sequences pre-formed in situ using the mannosyltransferase in the absence of UDP-galactose could not become galactose-substituted in a subsequent incubation either with UDP-galactose alone or with UDP-galactose plus GDP-mannose A model for the interaction of GDP-mannose mannosyltransferase and UDP-galactose galactosyltransferase in galactomannan biosynthesis is proposed.