title: Novel insights on the mechanism of action of alpha-amylase inhibitors from the plant defensin family.
abstract: Plant defensins are small cysteine-rich proteins commonly synthesized in plants, encoded by large multigene families. Most plant defensins that have been characterized to date show potent antifungal and/or bactericidal activities. This report describes VuD1, an unusual defensin that is able to inhibit insect-pest alpha-amylases. VuD1 was cloned from cowpea (Vigna unguiculata) seeds and expressed in a heterologous system. Inhibitory enzyme assays showed that VuD1 efficiently inhibits alpha-amylases from the weevils Acanthoscelides obtectus and Zabrotes subfasciatus, caused low inhibition toward mammalian enzymes and was unable to inhibit the alpha-amylases from Callosobruchus maculatus and Aspergillus fumigatus. To shed some light over the mechanism of action of VuD1, molecular modeling analyses were performed, revealing that the N-terminus of the molecule is responsible for binding with the active site of weevil enzymes. Moreover, models of VuD1 and mammalian enzymes were also generated to elucidate the specificity mechanisms. The data presented herein suggests that this defensin has potential application in the development of transgenic plants for insect pest control.
authors.affiliation: Centro de Analises Proteômicas e Bioquímicas, Pós-Graduação em Ciências Genomicas e Biotecnologia, Universidade Católica de Brasília, Brasília-DF, Brazil.
authors.forename: Patrícia B
authors.initials: PB
authors.lastname: Pelegrini
authors.forename: Fung T
authors.initials: FT
authors.lastname: Lay
authors.forename: André M
authors.initials: AM
authors.lastname: Murad
authors.forename: Marilyn A
authors.initials: MA
authors.lastname: Anderson
authors.forename: Octavio L
authors.initials: OL
authors.lastname: Franco
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000235:genetics; D023082:Defensins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007887:Fabaceae / Q000378:metabolism*; D006801:Humans; D007313:Insecta / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012639:Seeds / Q000378:metabolism; D016415:Sequence Alignment; D000516:alpha-Amylases / Q000037:antagonists & inhibitors*
chemical_list: D018744:DNA, Plant; D023082:Defensins; D004791:Enzyme Inhibitors; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D000516:alpha-Amylases