Identification of amino acids essential for enzymatic activity of plant invertases

The role of two conserved sequence motifs in plant invertases, the NDPN and WEC-P/V-D boxes, was studied by a structure-function analysis of extracellular invertase CIN1 from Chenopodium rubrum. Specific amino acid substitutions were introduced by site-directed mutagenesis, and the mutated genes were heterologously expressed in an invertase deficient Saccharomyces cerevisiae strain. It was shown that the aspartate from the first and the glutamate and the cysteine from the second box are essential for enzyme activity. The possible function of these amino acid residues is discussed with respect to the inhibitory effect of sulfhydryl group blocking reagents on invertase activity, the interaction between amino acids in the active centre, and their participation in the catalytic mechanism.