Structure and Oxidative Folding of AAI, the Major Alfa-Amylase Inhibitor From Amaranth Seeds

Abstract

AAI, the major alpha-amylase inhibitor (AAI) present in the seeds of the Mexican crop plant Amaranthus hypocondriacus is a 32-residue-long polypeptide with three disulfide bridges. Its structure is most closely related to the plant amylase inhibitor subfamily of knottins characterized by a topological knot formed by one disulfide bridge threading through a loop formed by the peptide chain as well as a short three-stranded beta sandwich core. AAI is specific against insect amylases and does not act on corresponding human or mammalian enzymes. It was found that the oxidative folding of AAI seems to follow a hirudine-like pathway with many non-native intermediates, but notably it proceeds through a major folding intermediate (MFI) that contains a vicinal disulfide bridge. Based on a review of the pertinent literature, the known vicinal disulfides in native proteins as well as well as the network of disulfide interchanges, we propose that MFI is a kinetic trap corresponding to a compact molten globule-like state which constrains the peptide chain to a smaller number of conformations that in turn can be rapidly funneled toward the native state. AAI: THE MAJOR ALPHA AMYLASE INHIBITOR OF Amaranthus hypochondriacusAAI (amaranth amylase inhibitor) is an alpha-amylase inhibitor isolated from the Mexican crop plant Amaranthus hypochondriacus. Amaranth grains are known to be cultivated for about 8,000 years and were part of the diet of the Aztecs. Today, Amaranth is grown in Mexico, Peru, and Bolivia, but the world's largest producer is China with an estimated annual production of 87 million metric tons. Amaranths are classified as pseudo cereals as they are not in the same botanical family as true cereals to which their grains bear similarity.Aqueous extracts of Amaranth grains were found to inhibit insect alpha-amylases extracted from the larvae of the red flour beetle (Dibolium castaneum) and of the grain borer (Prostephanus truncatus). The alpha-amylase was purified with classical techniques (Chagolla-Lopez et al., 1994). Briefly, crude extracts of amaranth flour were fractionated by ammonium sulfate precipitation (35-65%), fractionated on G75 Sepharose columns and the lyophilized active fractions were subjected to ion exchange chromatography The majority of inhibitory activity was found in one major peak which was further purified with reverse phase HPLC (RP-HPLC). The inhibitory activity was resistant to heat. As amino acid analysis revealed a high percentage of cysteine with no free sulfhydryl groups, the samples were reduced

Ligand‐induced transition in conformations of vicinal cysteine disulfides in proteins

“…28 vicinal disulfide intermediate in the oxidative folding of disulfide-rich peptide alpha-amylase inhibitor (AAI). 41 Analysis of the role of conformation of vicinal cysteine disulfide on the formation of compact molten globule-like state may also help for better understanding of its effects in funneling the oxidative folding of protein towards the native state.…”

Structure, Function and Protein Engineering of Cereal-Type Inhibitors Acting on Amylolytic Enzymes

“…This kind of α-amylase inhibitors were recognized long ago. They have been collectively covered in several reviews as well as in recent publications on specific inhibitors presenting rather different levels of structural and mechanistic insights (Kneen and Sandstedt, 1946;Garcia-Olmedo et al, 1987;Blanco et al, 1991;Carbonero and García-Olmedo, 1999;Svensson et al, 2004;Juge and Svensson, 2006;de Oliveira Carvalho and Gomes, 2009;Rehm et al, 2009;dos Santos et al, 2010;Kumar et al, 2010;Wang et al, 2014;Gadge et al, 2015;Sun et al, 2015;da Silva et al, 2018;Panwar et al, 2018;Tysoe and Withers, 2018;Tsvetkov and Yarullina, 2019;Juhász et al, 2020;Rane et al, 2020;Aguieiras et al, 2021;Geisslitz et al, 2021). The biological role of the plant hydrolase inhibitors is primarily in defense against insect pests and pathogenic fungi, whereas they are rarely involved in regulation of the activity of endogenous plant enzymes.…”