A motif rich in charged residues determines product specificity in isomaltulose synthase.

Isomaltulose synthase (PalI) catalyzes hydrolysis of sucrose and formation of alpha-1,6 and alpha-1,1 bonds to produce isomaltulose (alpha-D-glucosylpyranosyl-1,6-D-fructofranose) and small amount of trehalulose (alpha-D-glucosylpyranosyl-1,1-D-fructofranose). A potential isomaltulose synthase-specific motif ((325)RLDRD(329)), that contains a 'DxD' motif conserved in many glycosyltransferases, was identified based on sequence comparison with reference to the secondary structural features of PalI and homologs. Site-directed mutagenesis analysis of the motif showed that the four charged amino acid residues (Arg(325), Arg(328), Asp(327) and Asp(329)) influence the enzyme kinetics and determine the product specificity. Mutation of these four residues increased trehalulose formation by 17-61% and decreased isomaltulose by 26-67%. We conclude that the 'RLDRD' motif controls the product specificity of PalI.