A novel strategy for inhibition of α-amylases: Yellow meal worm α-amylase in complex with the Ragi bifunctional inhibitor at 2.5 Å resolution

Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland.
Structure (Impact Factor: 6.79). 08/1998; 6(7):911-21. DOI: 10.1016/S0969-2126(98)00092-6
Source: PubMed

ABSTRACT alpha-Amylases catalyze the hydrolysis of alpha-D-(1,4)-glucan linkages in starch and related compounds. There is a wide range of industrial and medical applications for these enzymes and their inhibitors. The Ragi bifunctional alpha-amylase/trypsin inhibitor (RBI) is the prototype of the cereal inhibitor superfamily and is the only member of this family that inhibits both trypsin and alpha-amylases. The mode of inhibition of alpha-amylases by these cereal inhibitors has so far been unknown.
The crystal structure of yellow meal worm alpha-amylase (TMA) in complex with RBI was determined at 2.5 A resolution. RBI almost completely fills the substrate-binding site of TMA. Specifically, the free N terminus and the first residue (Ser1) of RBI interact with all three acidic residues of the active site of TMA (Asp185, Glu222 and Asp287). The complex is further stabilized by extensive interactions between the enzyme and inhibitor. Although there is no significant structural reorientation in TMA upon inhibitor binding, the N-terminal segment of RBI, which is highly flexible in the free inhibitor, adopts a 3(10)-helical conformation in the complex. RBI's trypsin-binding loop is located opposite the alpha-amylase-binding site, allowing simultaneous binding of alpha-amylase and trypsin.
The binding of RBI to TMA constitutes a new inhibition mechanism for alpha-amylases and should be general for all alpha-amylase inhibitors of the cereal inhibitor superfamily. Because RBI inhibits two important digestive enzymes of animals, it constitutes an efficient plant defense protein and may be used to protect crop plants from predatory insects.

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    • "enzymes that are found in microorganisms, plants and animals. This enzyme catalyses the hydrolysis of a-D-(1,4)-glucan linkage in starch and related carbohydrates (Strobl et al. 1998). a-Amylase activity has been described from different species of several insect orders including Coleoptera, Hymenoptera, Diptera, Lepidoptera and Hemiptera (Baker and Woo 1985; Terra et al. 1988; Mendiola-Olaya et al. 2000; Oliveira-Neto et al. 2003; Kazzazi et al. 2005). "
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    ABSTRACT: The Red Palm Weevil, Rhynchophorus ferrugineus (Oliver) (Coleoptera: Curculionidae), is a serious pest of a wide range of plant species including coconut,sago, date and oil alms. The a-amylases are the hydrolytic enzymes that are involved in carbohydrate metabolism i n i nsects. So far nothing is done to demonstrate a-amylase activity of R. ferrugineus . Thus, the aim of the current study was to identify and characterise the a-amylase activity to gain a better understanding of digestive physiology of the insect. Thus, the a-amylase in the gut of red palm weevil was isolated and characterised using starch as a substrate. The study showed that the a-amylase is present in the gut of the insect for carbohydrate digestion. The a-amylase has an optimum pH and temperature of 5 and 408 C. The activity of a-amylase was increased by NaCl and KCl and inhibited by other compounds such as MgCl 2, CaCl 2, urea, ethylenediaminete-traacetic acid and sodium odecylsulfate. Native-PAGE electrophoresis of a-amylase showed two isoenzymes, one major and one minor band showing a-amylase importance in the carbohydrate etabolism of the insect. Understanding of the digestive physiology and a-amylase activity of Red Palm Weevil is important when new management strategies for this economically important pest are devised.
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    • "α-Amylase is an important group of digestive enzymes (Franco et al. 2000; Bandani et al. 2009). These enzymes catalyse the hydrolysis of α-D-(1, 4)-glucan linkage in starch components, glycogen and other carbohydrates and are found in animals, plants and micro-organisms (Strobl et al. 1998). Different patterns of activity and its isoforms depends on the type of diet (Mendiola-Olaya et al. 2000; Kotkar et al. 2009). "
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    ABSTRACT: Papilio machaon (Linnaeus) (Lepidoptera: Papilionidae) feeds on different plant species of the family Apiaceae. It also rarely feeds on the Rutaceae family such as rue (Ruta chalepensis) and fennel. In the current study, α-amylase activity of the larval midgut was investigated and characterised. Also, the effect of the wheat seed extracts on the larval midgut was explored. Results showed that the amylase activity was present in the midgut. Gel assays showed that more than one amylase band (three) was present in the larval midgut showing their importance in the insect feeding. Characterisation of the amylase showed that this enzyme was active in a wide range of the pHs. However, optimal pH for the enzyme activity was alkaline pH (pH 8.0). The effect of wheat seed extracts on the amylase activity showed that the insect amylase is highly sensitive to wheat protein extract. These results showed that wheat seed extract have a good potential to be explored more in order to purify its products and these products could be used in IPM programme in order to combat insect pest.
    Archives of Phytopathology and Plant Protection 06/2014; 47(10). DOI:10.1080/03235408.2013.840097
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    • "A comprehensive study (Strobl et al. 1998) on larval digestive !-amylase of Tenebrio molitor L. (Coleoptera: Tenebrionidae) showed that the enzyme has three domains. The central domain (domain A) is an (b/a)8-barrel that comprises the core of the molecule and includes catalytic amino acid residues, and domains B and C are almost opposite to each other, on each side of domain A (Strobl et al. 1998). Abundance and activity of insect !-amylases in the gut are dependent on food sources, so feeding on wool and plant tissues causes the lowest and highest amylolytic activity, respectively (Chapman 1998; Zibaee et al. 2008). "
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    ABSTRACT: α-Amylases are widespread enzymes that catalyze endohydrolysis of long α-1,4-glucan chains such as starch and glycogen. The highest amylolytic activity was found in 5th instar nymphs and midgut of the predatory bug, Andrallus spinidens F. (Hemiptera: Pentatomidae). The α-amylase was purified following a three-step procedure. The purified α-amylase had a specific activity of 13.46 U/mg protein, recovery of 4.21, purification fold of 13.87, and molecular weight of 21.3 kDa. The enzyme had optimal pH and temperature of 7 and 45°C, respectively. Na+, Mn+, Mg2+, and Zn2+ significantly decreased activity of the purified α-amylase, but some concentrations of K+, Ca2+, and Cu2+ had the opposite effect. EDTA, EGTA, and DTC significantly decreased enzymatic activity, showing the presence of metal ions in the catalytic site of the enzyme. Kinetic parameters of the purified α-amylase showed a Km of 3.71% in starch and 4.96% for glycogen, suggesting that the enzyme had a higher affinity for starch.
    Journal of Insect Science 05/2014; 14(65). DOI:10.1673/031.014.65 · 0.92 Impact Factor
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