Structure of the bifunctional inhibitor of trypsin and α-amylase from ragi seeds at 2.2 Å resolution

Abstract

The crystal structure of a bifunctional inhibitor of -amylase and trypsin (RATI) from ragi seeds (Indian ®nger millet, Eleusine coracana Gaertneri) has been determined by X-ray diffraction at 2.2 A Ê resolution. The inhibitor consists of 122 amino acids, with ®ve disul®de bridges, and belongs to the plant -amylase/trypsin inhibitor family. The crystals were grown by the microdialysis method using ammonium sulfate as a precipitating agent. The structure was determined by the molecular-replacement method using as models the structures of Corn Hageman factor inhibitor (CHFI) and of RATI at 2.9 A Ê resolution determined previously. It has been re®ned to an R factor of 21.9%. The structure shows an r.m.s. deviation for C atoms of 2.0 A Ê compared with its own NMR structure, whereas the corresponding value compared with CHFI is found to be 1.4 A Ê . The r.m.s. difference for C atoms when compared with the same protein in the structure of the complex with -amylase is 0.7 A Ê . The conformations of trypsin-binding loop and the -amylase-binding N-terminal region were also found to be similar in the crystal structures of native RATI and its complex with -amylase. These regions differed considerably in the NMR structure.

WCI, a novel wheat chymotrypsin inhibitor: purification, primary structure, inhibitory properties and heterologous expression

“…4, ragi -amylase/trypsin inhibitor (P01087) and maize trypsin/factor IIA inhibitor (P01088) have known tertiary structures (Strobl et al 1995;Behnke et al 1998;Gourinath et al 2000). The structures of these two inhibitors, as well as those of the wheat alphaamylase/inhibitor 0.19 (Oda et al 1997) and the binary complex T. molitor a-amylase/ragi bifunctional inhibitor (Strobl et al 1998), have been utilized to propose a general model for the simultaneous interaction of the ragi inhibitor (P01087) with the two cognate enzymes, namely -amylase and trypsin (Strobl et al 1998).…”

“…4, have known tertiary structures (Strobl et al 1995;Behnke et al 1998;Gourinath et al 2000). In order to carry out a structure prediction study on WCI, we used the online I-TAS-SER service; the program built a 3D model of WCI by selecting (automatically) the 3D structure of the maize inhibitor as template.…”

Differential in vitro and in vivo effect of barley cysteine and serine protease inhibitors on phytopathogenic microorganisms

“…Their members can be classified as trypsin inhibitors, a-amylase inhibitors, and dual trypsin/a-amylase inhibitors [22]. Three different roles have been attributed to the family 16 inhibitors: i) regulators of seed germination; ii) storage proteins, and iii) defence proteins.…”

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

“…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).…”