Colorado potato beetle alpha-amylase: Purification, action pattern and subsite mapping for exploration of active centre

• Main Authors: Gyémánt, G. (Author), Hámori, C. (Author), Kandra, L. (Author), Remenyik, J. (Author)
• Format: Article
• Language: English
• Published: Elsevier B.V. 2021
• Subjects: affinity chromatography; alpha-Amylases; amino acid sequence; Amino Acid Sequence; amylase; animal; Animals; Article; beetle; binding affinity; binding site; Binding Sites; Bond cleavage frequency; catalysis; Catalytic Domain; Coleoptera; Colorado potato beetle; controlled study; enzyme active site; enzyme analysis; enzyme purification; enzyme specificity; enzymology; genetics; high performance liquid chromatography; human; Humans; hydrolysis; Hydrolysis; Insect α-amylase; isolation and purification; metabolism; nonhuman; pig; protein binding; Protein Binding; protein cleavage; protein purification; sequence alignment; sequence homology; Sequence Homology, Amino Acid; Subsite structure; Substrate Specificity; Swine; Tenebrio
• Online Access: View Fulltext in Publisher

 Colorado potato beetle is an invasive insect herbivore and one of the most challenging agricultural pests globally. This study is the first characterization of the active centre of Colorado potato beetle (Leptinotarsa decemlineata) α-amylase (LdAmy). Bond cleavage frequency values for LdAmy were determined by HPLC product analysis on a chromophore labelled maltooligomer substrate series. Binding energies between amino acid moieties of subsites and glucose residues of substrate were calculated. Active site contains six subsites in the binding region of LdAmy; four glycone- (−4, −3, −2, −1) and two aglycone-binding sites (+1, +2). Subsite map calculation resulted in apparent binding energies −11.8 and − 11.0 kJ/mol for subsites (+2) and (−3), respectively, which revealed very favorable interactions at these positions. Structures of binding sites of LdAmy and mammalian α-amylases show similarity, but there are variations in the binding energies at subsite (−2) and (−4). Differences were interpreted by comparison of amino acid sequences of human salivary α-amylase (HSA) and porcine pancreatic α-amylase (PPA) and two insect (Leptinotarsa decemlineata and Tenebrio molitor) enzymes. The observed substitution of positively charged His305 in HSA at subsite (−2) with an acidic Asp in LdAmy in the same position may explain the obtained energy reduction. © 2020 The Authors