Tertiary and Quaternary Structures of 0.19 α-Amylase Inhibitor from Wheat Kernel Determined by X-ray Analysis at 2.06 Å Resolution^,

Abstract

The crystal structure of 0.19 alpha-amylase inhibitor (0.19 AI) from wheat kernel was determined by the multiple-isomorphous replacement method coupled with density modification and noncrystallographic symmetry averaging and then refined by simulated annealing using diffraction data to 2.06 A resolution (R = 18.7%, free R = 22.3%). The asymmetric unit has four molecules of 0.19 AI, each comprised of 124 amino acid residues. Electron density for residues 1-4 and 69-77 is absent in all subunits, probably because of the intrinsic flexibility of these segments. Each subunit has four major alpha-helices and one one-turn helix which are arranged in the up-and-down manner, maintaining the favorable packing modes of the alpha-helices. 0.19 AI, however, has two short antiparallel beta-strands. All 10 cysteine residues in 0.19 AI form disulfide bonds (C6-C52, C20-C41, C28-C83, C42-C99, and C54-C115), consistent with the assignments made biochemically for 0.28 AI from wheat kernel and by NMR analysis of the bifunctional alpha-amylase/trypsin inhibitor from ragi seeds (RBI). The disulfide bond patterns in these AIs are similar to those in the hydrophobic protein from soybean (HPS), which lack only the bond corresponding to C28-C83 in 0.19 AI. Extensive interactions occurred between particular pairs of 0.19 AI subunits, mainly involving hydrophobic residues. Comparisons of the structures of 0.19 AI, RBI, and HPS showed that the arrangements of the major alpha-helices are similar but the conformations of the remaining residues differ markedly. The present X-ray analysis for 0.19 AI and the NMR analysis for RBI suggest that all the AIs in this family have a common fold. The alpha-amylase binding site is discussed on the basis of the tertiary and quaternary structures of 0.19 AI together with biochemical and spectroscopic data for AIs.

Analyses of trace metals, peptide ligands of trace metals and mercury speciation in home prepared bread

“…All identified inhibitors (except for the xylanase inhibitor) and barwin, antifungal and gliadin proteins are characterised by the high content of cysteine. Their composition and structure are detailed elsewhere (Bonsager et al, 2003;Hejgaard et al, 1991;Kasarda and D'ovidio, 1999;Maeda et al, 1985;Maeda and Finnie, 2005;Oda et al, 1997;Salcedo et al, 2007;Strobl et al, 1995;Svensson et al, 1992). The xylanase inhibitor found is rich in dicarboxylic amino acids, aspartic and glutamic acids (McLauchlan et al, 1999).…”

Wheat Amylase Trypsin Inhibitors as Nutritional Activators of Innate Immunity

“…by the gastric/enteric proteases pepsin and trypsin, maintaining their biological activity of TLR4 activation throughout their intestinal passage after oral ingestion. This protease resistance is due to their compact secondary structure which is characterized by 5 intra-chain disulfide bonds [18]. The modern wheat genome encodes 17 different ATI species of ∼120- to 150-amino acid length that vary in amino acid sequence but have similar secondary structures [19].…”

Effects of single and long-term administration of wheat albumin on blood glucose control: randomized controlled clinical trials

“…The specific a-amylase inhibiting proteins were identified as 0.19-, 0.28-, 0.36-, and 0.53-inhibitors according to the results of gel electrophoresis (Sodini et al, 1970). The primary and tertiary structures of these inhibitors were then clarified (Oda et al, 1997;Maeda et al, 1983;Kashlan & Richardson, 1981). Koike et al (1995) found that WA inhibited the increase of postprandial hyperglycemia, and 0.19-inhibitor (0.19-albumin) was revealed to exert the main a-amylase inhibiting activity in WA (Choudhury et al, 1996).…”