Protein Engineering vol. 16 no. 6 pp. 391-396, 2003
© 2003 Oxford University Press
Substrate binding and catalysis in trichosanthin occur in different sites as revealed by the complex structures of several E85 mutants
1Laboratory of Structural Biology and the MOE Laboratory of Protein Science, School of Life Science and Engineering, Tsinghua University, Beijing 100084, 2Department of Biochemistry, Chinese University of Hong Kong, Hong Kong and 3National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing, 100101, China
4 To whom correspondence should be addressed. e-mail: raozh{at}xtal.tsinghua.edu.cn; pcshaw{at}cuhk.edu.hk
Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) which possesses rRNA N-glycosidase activity. In recent years, its immunomodulatory, anti-tumor and anti-HIV properties have been revealed. Here we report the crystal structures of several E85 mutant TCS complexes with adenosine-5'-monophosphate (AMP) and adenine. In E85Q TCS/AMP and E85A TCS/AMP, near the active site of the molecule and parallel to the aromatic ring of Tyr70, an AMP molecule is bound to the mutant without being hydrolyzed. In the E85R TCS/adenine complex, the hydrolyzed product adenine is located in the active pocket where it occupies a position similar to that in the TCS/NADPH complex. Significantly, AMP is bound in a position different to that of adenine. In comparison with these structures, we suggest that there are at least two subsites in the active site of TCS, one for initial substrate recognition as revealed by the AMP site and another for catalysis as represented by the NADPH site. Based on these complex structures, the function of residue 85 and the mechanism of catalysis are proposed.
Received December 10, 2002; accepted May 20, 2003.