Activity of wheat alpha-amylase inhibitors towards bruchid alpha-amylases and structural explanation of observed specificities

Plant alpha-amylase inhibitors show great potential as tools to engineer re sistance of crop plants against pests. Their possible use is, however, comp licated by observed variations in specificity of enzyme inhibition, even wi thin closely related families of inhibitors. Five alpha-amylase inhibitors of the structural 0.19 family were isolated from wheat kernels, and assayed against three insect alpha-amylases and porcine pancreatic alpha-amylase, revealing several intriguing differences in inhibition profiles, even betwe en proteins sharing sequence identity of up to 98%. Inhibition of the enzym e from a commercially important pest, the bean weevil Acanthoscelides obtec tus, is observed for the first time. Using the crystal structure of an inse ct alpha-amylase in complex with a structurally related inhibitor, models w ere constructed and refined of insect and human alpha-amylases bound to 0.1 9 inhibitor. Four key questions posed by the differences in biochemical beh aviour between the five inhibitors were successfully explained using these models. Residue size and charge, loop lengths, and the conformational effec ts of a Cys to Pro mutation, were among the factors responsible for observe d differences in specificity. The improved structural understanding of the bases for the 0.19 structural family inhibitor specificity reported here ma y prove useful in the future for the rational design of inhibitors possessi ng altered inhibition characteristics.