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Protein Engineering, Vol. 12, No. 11, 999-1004, November 1999
© 1999 Oxford University Press

Role of Arg163 in the N-glycosidase activity of neo-trichosanthin

Hui-Guang Li, Shi-Zhen Xu, Shen Wu1, Li Yan, Jian-Hui Li, Richy N.-S. Wong2, Qing-Li Shi2 and Yi-Cheng Dong

Department of Protein Engineering, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101 and 2 Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China

Three mutant crystals of neo-trichosanthin (n-TCS), R163K, R163H and R163Q, were obtained by the hanging drop vapor diffusion method. Structure determination indicated that there are no significant differences between the mutants and n-TCS except in the active pocket. All of them were also soaked in sodium citrate buffer (pH 4.5) containing 20% KCl and 10 mg/ml AMP. Structure determination suggests that in the active pocket of the crystals of R163K and R163H, parallel to the aromatic ring of Tyr70, each mutant possesses an adenine. The relationship between structure and function is discussed. Biochemical analysis reveals that the mutants R163K and R163H have N-glycosidase activity, while R163Q does not. This suggests that R163 is a crucial residue for the enzyme activity of n-TCS, and its role is providing proton.

Keywords: mutants/neo-trichosanthin/N-glycosidase activity/protein crystallography/RIPs

1 To whom correspondence should be addressed;email: wushen{at}sun5.ibp.ac.cn


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