Molecular cloning of bruchid (Zabrotes subfasciatus) alpha-amylase cDNA and interactions of the expressed enzyme with bean amylase inhibitors

Abstract

alpha-Amylases are important digestive enzymes in weevils that infest starchy seeds, and plants have evolved proteinaceous alpha-amylase inhibitors (alpha AI) for protection. To gain a better understanding of the interaction between weevil alpha-amylases and alpha AIs, we cloned the alpha-amylase cDNA of Zabrotes subfasciatus larvae. Larvae of this bruchid infest seeds of cultivated varieties of the common bean, Phaseolus vulgaris, although the seeds contain high levels of an alpha AI. The alpha-amylase cDNA, called ZsAmy, encodes a mature protein of 466 amino acids with a signal peptide of 17 amino acids. This protein has 50-60% amino acid identity with the other five known insect alpha-amylases. Three amino acid residues known to be important for catalysis and three histidine residues involved in substrate binding are conserved in the derived amino acid sequence of ZsAmy. Expression of ZsAmy with a baculovirus vector in cultured insect cells resulted in the production of active alpha-amylase, alpha AI-1, the form of the inhibitor found in cultivated beans, does not inhibit larval or expressed bruchid alpha-amylase, but alpha AI-2, a form of the inhibitor found in certain wild bean accessions, does inhibit the larval, as well as the expressed bruchid alpha-amylase. These and other observations lead to the conclusion that ZsAmy encodes the major larval amylase of this bruchid species.