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The digestive tract of lepidopteran insects is extremely alkaline. In the present work, molecular adaptation of amylolytic enzymes to this environment was investigated in the flour moth Ephestia kuehniella, an important stored-product pest. Three digestive alpha-amylases [Ephestia kuehniella alpha-amylase isoenzymes 1-3 (EkAmy1-3)] with an alkaline pH optimum were purified from larvae and biochemically characterized. These isoenzymes differ significantly in their sensitivity to alpha-amylase inhibitors of plant origin that are directed against herbivores as antifeedants. Such functional variability renders the amylolytic system less vulnerable to suppression by plant defensive molecules. Moreover, we found that expression of alpha-amylases is upregulated in larvae feeding on a diet enriched with an alpha-amylase inhibitor. The alpha-amylases are secreted into the larval midgut by an exocytotic mechanism, as revealed by immunogold microscopy. The cDNA sequence of EkAmy3 was determined, and a homology model of EkAmy3 was built in order to analyze the structural features responsible for adaptation to alkaline pH. First, the overall fold was found to be stabilized by remodeling of ion pairs. Second, molecular simulations supported by activity measurements showed that EkAmy3 does not bind a Cl(-), owing to an Arg-to-Gln mutation in a conserved binding site. The Cl(-)-binding residues are in contact with the catalytic residues, and this change might help to fine-tune the catalytic pK(a) values to an alkaline pH optimum. We conclude that lepidopteran alpha-amylases are evolutionarily adapted in terms of structure and expression dynamics for effective functioning in the digestive system.

The primary structure and proteolytic processing of the alpha-amylase isoinhibitor alpha AI-1 from common bean (Phaseolus vulgaris cv. Magna) was determined by protein chemistry techniques. The inhibitory specificity of alphaAI-1 was screened with a panel of the digestive alpha-amylases from 30 species of insects, mites, gastropod, annelid worm, nematode and fungal phytopathogens with a focus on agricultural pests and important model species. This in vitro analysis showed a selective inhibition of alpha-amylases from three orders of insect (Coleoptera, Hymenoptera and Diptera) and an inhibition of alpha-amylases of the annelid worm. The inhibitory potential of alphaAI-1 against several alpha-amylases was found to be modulated by pH. To understand how alphaAI-1 discriminates among closely related alpha-amylases, the sequences of the alpha-amylases sensitive, respectively, insensitive to alphaAI-1 were compared, and the critical determinants were localized on the spatial alpha-amylase model. Based on the in vitro analysis of the inhibitory specificity of alphaAI-1, the in vivo activity of the ingested alphaAI-1 was demonstrated by suppression of the development of the insect larvae that expressed the sensitive digestive alpha-amylases. The first comprehensive mapping of alphaAI-1 specificity significantly broadens the spectrum of targets that can be regulated by alpha-amylase inhibitors of plant origin, and points to potential application of these protein insecticides in plant biotechnologies.