Abstract
Rice seeds are rich sources of different proteinaceous inhibitors with characteristic bifunctional enzyme-inhibiting activities such as α–amylase/subtilisin and trypsin/chymotrypsin inhibitors. These inhibitors were generally expressed in developing seeds and other organs, and were thought to play an important role in plant’s defense system. It was generally believed that these inhibitors not only playing a role in plant development but also conferring a broad spectrum of resistance against pests and pathogens. Nevertheless, high-resolution structures of these inhibitors and in complex with target enzymes are available in other species but not in rice. Therefore, in this study, we purified RASI (rice alpha-amylase/subtilisin inhibitor, 18.9 kDa) from rice bran into homogenous and crystallized using the handing drop vapor diffusion method. According to X-ray diffraction of RASI crystals at resolution 1.80 Å, the unit cell belongs to space group P21212 with parameters a=79.99Å, b=62.95Å and c=66.70Å. Preliminary analysis of the protein indicate that there are two RASI molecules in an asymmetric unit with a solvent content of 44.05 %. One monomer contains 10 beta-sheet structures which are five anti-beta-sheet pairs. Detail of the function of RASI in plant seeds seems to remain unclear. Because of its inhibition to Subtilisin and alpha-amylase of certain bacteria, RASI was considered playing a defense role. In this research, we find that RASI might have some interaction to thioredoxin. But there were still no reference which improved the relationship directly. Despite it close structural relationship to other cereals showed distinct differences in sequences, in enzymatic and physico-chemical properties as well as the physiological expression to other species. Here we describe the successful purification and crystallographic characterization of RASI, and the goal of this project is to provide an alternative structural bases and ultimately functional illustration of enzyme-inhibitor interactions in rice plant which is one of the three most valuable cereal crops grown worldwide. In comparing between RASI and the homologous proteins, we can get more information about the protein functions.