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

Abstract

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.

Crystal Structure of Barley Limit Dextrinase-Limit Dextrinase Inhibitor (LD-LDI) Complex Reveals Insights into Mechanism and Diversity of Cereal Type Inhibitors

“…Arg 34 and Leu 35 from this loop interact with the active site in trypsin (Fig. 9a) (30). Notably, the sequence of the RBI trypsin-binding loop is not conserved in the corresponding LDI loop 1 (Ser 37 and Arg 38 correspond to Arg 34 and Leu 35 , respectively, in RBI) (Fig.…”

“…the complex formed by ␣-amylase from yellow meal worm (TMA) and RBI, the LDI homologue from ragi (30), and the complex between pig pancreatic ␣-amylase and the wheat inhibitor 0.28 AI; the structural coordinates of the latter complex are not deposited in the PDB (41). Very surprisingly, superimposition of the complexes of LDI (LD-LDI) and its homologue from ragi (TMA-RBI; PDB code 1TMQ) showed their binding modes to be entirely different (Fig.…”

“…Thus, the N-terminal sequence 1 SVGTS 5 assumes a 3 10 -helical conformation upon binding to TMA but is unstructured in the free RBI (42,43). This rearrangement appears essential for the inhibition mechanism that involves contacts between the N-terminal amino group of RBI with the three acidic residues at the active site in TMA (30). Essentially the same binding mode is reported for an LDI homologue from wheat with its porcine ␣-amylase target (0.28 AI) (41).…”

Assessing of α-Amylase Activity of Midgut in Wheat Bug Eurygaster maura

“…These enzymes catalyze the hydrolysis of α-D-(1, 4)-glucan linkage in starch components, glycogen and various other related carbohydrates [16,30] .…”

“…The effect of temperature on α-amylase activity was determined either by incubating the reaction mixture at 10,15,20,25,30,35,40,45, 50, 55, 60 and 70°C for 30 min. The effect of temperature on stability of amylase activity was tested by pre-incubation of the enzyme at 10, 20, 30, 40, 50, 60 and 70°C for 30 min, followed by measurement of activity as mentioned before.…”