Yi-Hung Lin (
林易弘
) 1 , Hsin-Tai Li (
李欣黛
) 2 , Wen-Yan Peng (
彭文彥
) 2 ,
Yen-Chieh Huang (
黃彥杰
)
1
, Ying-Cheng Hsieh (
謝殷程
)
2
, Hong-Hsiang Guan (
管泓翔
)
2
,
Ming-Yih Liu (
劉明毅
) 1 , Tschining Chang (
張俊英
) 3 ,
Andrew H.-J. Wang (
王惠鈞
) 3 , and Chun-Jung Chen (
陳俊榮
) 1,4
1 Biology Group, National Synchrotron Radiation Research Center, Hsinchu, Taiwan
2 Institute of Bioinformatics and Structural Biology, National TsingHua University, Hsinchu, Taiwan
3 Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
4 Institute of Physics, National TsingHua University, Hsinchu, Taiwan
Rice seeds are rich sources of different proteinaceous inhibitors with characteristic bifunctional enzyme-inhibiting activities such as

-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 two bi-functional enzyme inhibitors, RASI (rice

-amylase/subtilisin inhibitor, 18.9 KDa) and RBTI (rice bran trypsin inhibitor, 15 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 1.81 Å, the unit cell belongs to space group
P2
1
2
1
2 with parameters a=79.99 Å, b=62.95 Å and c=66.70 Å, whereas for RBTI crystals at a resolution of 2.07 Å, the unit cell belongs to space group P2
1
2
1
2
1
with parameters a=74.37 Å, b=96.69 Å and c=100.36 Å. Preliminary analysis of both proteins indicate that there are two RASI molecules in an asymmetric unit with a solvent content of 44.05 %, and four RBTI molecules in an asymmetric unit with a solvent content of 58.29 %. Details of the function of RASI and RBTI in plant seeds seems to remain unclear. Despite their close structural relationship to other cereals, both proteins however, 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 RBTI, 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.
References:
[1] Lin, Y.-H., Li, H.-T., Huang, Y.-C., Hsieh, Y.-C., Guan, H.-H., Liu, M.-Y., Chang, T., Wang,
A. H.-J. & Chen, C.-J. (2006). Acta Cryst.
F62, 522-524.
[2] Lin, Y.-H., Peng, W.-Y., Huang, Y.-C., Guan, H.-H., Hsieh, Y.-C., Liu, M.-Y., Chang, T. &
Chen, C.-J. (2006). Acta Cryst.
F62, 743-745.
[3] Yamasaki, T., Deguchi, M., Fujimoto, T., Masumura, T. U., Kanamaru, K., & Yamagata, H.
(2006). Biosci. Biotechnol. Biochem.
, 70, 1200-1209.
[4] Song, H. K., Kim, Y. S., Yang, J. K., Moon, J., Lee, J. Y. & Suh, S. W. (1999). J. Mol. Biol.
293, 1133-1144.