Insect pests and pathogens (fungi, bacteria and viruses) are responsible for severe crop losses. Insects feed directly on the plant tissues, while the pathogens lead to damage or death of the plant. Plants have evolved a certain degree of resistance through the production of defence compounds, which may be aproteic, e.g. antibiotics, alkaloids, terpenes, cyanogenic glucosides or proteic, e.g. chitinases, b-1,3-glucanases, lectins, arcelins, vicilins, systemins and enzyme inhibitors. The enzyme inhibitors impede digestion through their action on insect gut digestive a-amylases and proteinases, which play a key role in the digestion of plant starch and proteins. The natural defences of crop plants may be improved through the use of transgenic technology. Current research in the area focuses particularly on weevils as these are highly dependent on starch for their energy supply. Six dierent a-amylase inhibitor classes, lectin-like, knottin-like, cereal-type, Kunitz-like, c-purothionin-like and thaumatin-like could be used in pest control. These classes of inhibitors show remarkable structural variety leading to dierent modes of inhibition and dierent speci®city pro®les against diverse a-amylases. Speci®city of inhibition is an important issue as the introduced inhibitor must not adversely aect the plant's own a-amylases, nor the nutritional value of the crop. Of particular interest are some bifunctional inhibitors with additional favourable properties, such as proteinase inhibitory activity or chitinase activity. The area has bene®ted from the recent determination of many structures of a-amylases, inhibitors and complexes. These structures highlight the remarkable variety in structural modes of a-amylase inhibition. The continuing discovery of new classes of a-amylase inhibitor ensures that exciting discoveries remain to be made. In this review, we summarize existing knowledge of insect a-amylases, plant a-amylase inhibitors and their interaction. Positive results recently obtained for transgenic plants and future prospects in the area are reviewed.Keywords: a-amylase inhibitors; knottin-like; lectin-like; thaumatin-like; Kunitz; cereal-type; bean weevils; bifunctional inhibitors.Insect pests and pathogens such as fungi, bacteria and viruses are together, responsible for severe crop losses. Worldwide, losses in agricultural production due to pest attack are around 37%, with small-scale farmers hardest hit [1]. Starchy leguminous seeds are an important staple food and a source of dietary protein in many countries. These seeds are rich in protein, carbohydrate and lipid and therefore suffer extensive predation by bruchids (weevils) and other pests. The larvae of the weevil burrow into the seedpods and seeds and the insects usually continue to multiply during seed storage. The damage causes extensive losses, especially if the seeds are stored for long periods.In general, plants contain a certain degree of resistance against insect predation, which is re¯ected in the limited number of insects capable of feeding on a gi...

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....

Higher plants produce several families of proteins with toxic properties, which act as defense compounds against pests and pathogens. The thionin family represents one family and comprises low molecular mass cysteine-rich proteins, usually basic and distributed in different plant tissues. Here, we report the purification and characterization of a new thionin from cowpea (Vigna unguiculata) with proteinase inhibitory activity. Cowpea thionin inhibits trypsin, but not chymotrypsin, binding with a stoichiometry of 1:1 as shown with the use of mass spectrometry. Previous annotations of thionins as proteinase inhibitors were based on their erroneous identification as homologues of Bowman-Birk family inhibitors. Molecular modeling experiments were used to propose a mode of docking of cowpea thionin with trypsin. Consideration of the dynamic properties of the cowpea thionin was essential to arrive at a model with favorable interface characteristics comparable with structures of trypsin-inhibitor complexes determined by X-ray crystallography. In the final model, Lys11 occupies the S1 specificity pocket of trypsin as part of a canonical style interaction.

No abstract