Plant a-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Five a-amylase inhibitors of the structural 0.19 family were isolated from wheat kernels, and assayed against three insect a-amylases and porcine pancreatic a-amylase, revealing several intriguing differences in inhibition profiles, even between proteins sharing sequence identity of up to 98%. Inhibition of the enzyme from a commercially important pest, the bean weevil Acanthoscelides obtectus, is observed for the first time. Using the crystal structure of an insect a-amylase in complex with a structurally related inhibitor, models were constructed and refined of insect and human a-amylases bound to 0.19 inhibitor. Four key questions posed by the differences in biochemical behaviour between the five inhibitors were successfully explained using these models. Residue size and charge, loop lengths, and the conformational effects of a Cys to Pro mutation, were among the factors responsible for observed differences in specificity. The improved structural understanding of the bases for the 0.19 structural family inhibitor specificity reported here may prove useful in the future for the rational design of inhibitors possessing altered inhibition characteristics.Keywords: a-amylase; amylase inhibitor specificity; structural modeling; Acanthoscelides; bruchids.a-Amylases (a-1,4-glucan-4-glucanohydrolases; EC 3.2.1.1) are hydrolytic enzymes that are widespread in nature, being found in animals, microorganisms and plants. They are involved in the degradation of a-1,4-linked sugar polymers, such as starch and glycogen, into oligosaccharides. a-Amylases and related enzymes are widely used in biotechnology for starch degradation and in synthetic chemistry for the production of oligosaccharides by transglycosylation [1]. a-Amylases are also drug-design targets for the development of compounds for the treatment of diabetes, obesity and hyperlipaemia [2,3].Plant a-amylase inhibitors (a-AIs), particularly abundant in cereals [4±8] and leguminosae [9±13], have been extensively studied, in part because they play a role in plant resistance to insect and microbial pests [7,14] and also because they are major allergens involved in baker's asthma disease [15]. Some wheat a-AIs inhibit insect a-amylases strongly but do not inhibit mammalian a-amylases [7,16] suggesting that they could be used as tools of engineered resistance of crop plants against pests [17]. Their potential has already been illustrated by the resistance to pea weevil (Bruchus pisorum), the cowpea weevil (Callosobruchus maculatus) and the azuki bean weevil (Callosobruchus chinensis) exhibited by pea seeds expressing a-AI1 from common bean, Phaseolus vulgaris [18±20].The common bean contains two allelic variants of a-amylase inhibitors called a-AI1 and a-AI2, differing in their specificity towards a-amylases....