It has been suggested that proteins from plants and microorganisms that inhibit α-amylases exert their natural roles in the control of endogenous α-amylase activity or are involved in plant defence mechanisms against pathogens and pests (Garcia-Olmedo et al.
; Whitaker et al.
; Franco et al.
; Svensson et al.
). Inhibitors of certain types have been reported to be anti-nutritional factors for humans (Garcia-Olmedo et al.
; Ho et al.
; Yoshikawa et al.
) and hence have received much attention for potential applications in therapies against obesity and certain diseases (Garcia-Olmedo et al.
; Layer et al.
). However, no definitive functions for these proteins has yet been identified in plants. According to sequence and three-dimensional structure similarities, these inhibitors can be classified into seven types: microbial, knottin-like, γ-thionin-like, CM proteins, Kunitz-type, thaumatin-like and legume lectin-like inhibitors (for a review, see Svensson et al.
). An inhibitor of one of these types, called α-amylase/subtilisin inhibitor, was discovered in barley (Yoshikawa et al.
). It possesses the ability to inhibit subtilisin (Bacillus subtilis
serine protease) and has sequence similarity to the Kunitz soybean trypsin inhibitor family (Onesti et al.
); it was also identified in a complex with an endogenous α-amylase (Weselake et al.
; Mundy et al.
). Rice, like most other cereals, contains several types of enzyme inhibitors (Tashiro et al.
; Abe et al.
; Feng et al.
; Yu et al.
), including a 20 kDa molecular-weight rice bifunctional α-amylase/subtilisin inhibitor (RASI) that can inhibit both α-amylase from the larvae of the red flour beetle (Tribolium castaneum
) and subtilisin from B. subtilis
(Ohtsubo & Richardson, 1992
). Yamagata and coworkers performed a detailed characterization of RASI and suggested that the expression of RASI occurred during the late milky stage in developing seeds (Yamagata et al.
). RASI was shown to be located/accumulated in the outermost part of the rice grain and the subcellular site of aleurone cells in the form of aleurone particles. This is the opposite of the distribution of BASI (barley α-amylase/subtilisin inhibitor from barley seed), which accumulates in the starchy endosperm (Leah & Mundy, 1989
BASI is the best characterized inhibitor of the α-amylase/subtilisin family (Mundy et al.
; Weselake et al.
). It contains a single chain of 181 amino acids with two disulfide bridges (Svendsen et al.
) and shows 58% sequence identity to RASI (Ohtsubo & Richardson, 1992
). BASI is highly specific for an endogenous high-pI α-amylase isoenzyme 2 (AMY2), but shows no effect on isoenzyme 1 (AMY1) that shares 80% sequence identity to AMY2 (Mundy et al.
). The K
for BASI acting on barley AMY2 was determined to be ~0.1 nM
(Abe et al.
; Bonsager et al.
), whereas WASI (wheat α-amylase/subtilisin inhibitor, 92% sequence identity to BASI; Mundy et al.
) was less potent. RASI showed even less activity against endogenous plant α-amylase (Yamagata et al.
). A rapid and tight simple two-step mechanism was proposed for the binding of BASI to AMY2 (Sidenius et al.
), which differs from the mode of action of α-AI1, AAI and RBI (lectin-type, knottin-type and cereal-type α-amylase inhibitors, respectively) as the inhibition process of these proteins involves the insertion of inhibition loops into the α-amylase active site (Franco et al.
), while BASI does not interact directly with any catalytic acidic residues of the enzyme (Valle et al.
). Further properties of BASI have been reviewed by Nielsen et al.
Details of the function of RASI and the related BASI and WASI in plant seeds remain unclear. RASI shows a sequence, enzymatic and physicochemical properties and physiological expression patterns that differ from homologous proteins from other cereals. Here, we describe the successful purification, crystallization and crystallographic characterization of 176-amino-acid RASI, which is the first reported α-amylase/subtilisin inhibitor from rice seed. The objective of our present work is to provide an alternative structural basis for and ultimately a functional illustration of α-amylase–inhibitor interactions in rice plants.