Molecular investigation of coleopteran specific α-amylase inhibitors from Amaranthaceae members

α-Amylase inhibitors (α-AIs) target α-amylases and interfere with the carbohydrate digestion of insects. Among different classes of α-AIs, a knottin-type inhibitor from Amaranthus hypochondriacus (AhAI) is found to be specific against coleopteran storage pests. In this report, we have characterized three previously unidentified knottin-type α-AIs from various Amaranthaceae plants namely, Amaranthus hypochondriacus (AhAI2), Alternanthera sessilis (AsAI) and Chenopodium quinoa (CqAI). They contain a signal peptide, pro-peptide, and mature peptide. New α-AIs mature peptides share 68 to 78% identity with the AhAI and have highly variable pro-peptide regions. Along with the cystine-knot fold, they show the conservation of reactive site residues. We have successfully expressed active recombinant α-AIs using an oxidative cytoplasmic environment. Inhibition studies against diverse amylases revealed that these inhibitors showed selective inhibition of coleopteran recombinant insect α-amylases viz., Tribolium castaneum, and Callosobruchus chinensis. Tribolium castaneum α-amylase inhibition potency was highest for AhAI2 (Ki ~ 15 μM) followed by AsAI (Ki ~ 43 μM) and CqAI (Ki ~ 61 μM). Interaction analysis of these inhibitors illustrated that the reactive site of inhibitors makes several non-covalent interactions with the substrate-binding pocket of coleopteran α-amylases. The selectivity of these inhibitors against coleopteran α-amylases highlights their potential in storage grain pest control.

Keywords: Amaranthaceae; Coleoptera; Knottin-type; α-Amylase; α-Amylase inhibitor.