Nucleic Acids Research, 2003, Vol. 31, No. 18 5247-5255
© 2003 Oxford University Press
The structural integrity exerted by N-terminal pyroglutamate is crucial for the cytotoxicity of frog ribonuclease from Rana pipiens
1 Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan and 2 Department of Life Sciences, National Tsing-Hua University, Hsin-Chu 300, Taiwan
*To whom correspondence should be addressed. Tel: +886 2 27899167; Fax: +886 2 27829142; Email: ydliao{at}ibms.sinica.edu.tw
Onconase, a cytotoxic ribonuclease from Rana pipiens, possesses pyroglutamate (Pyr) at the N-terminus and has a substrate preference for uridine–guanine (UG). To identify residues responsible for onconase’s cytotoxicity, we cloned the rpr gene from genomic DNA and expressed it in Escherichia coli BL21(DE3). The recombinant onconase with Met at the N-terminus had reduced thermostability, catalytic activity and antigenicity. Therefore, we developed two methods to produce onconase without Met. One relied on the endogeneous E.coli methionine aminopeptidase and the other relied on the cleavage of a pelB signal peptide. The Pyr1 substitutional variants maintained similar secondary structures to wild-type onconase, but with less thermostability and specific catalytic activity for the innate substrate UG. However, the non-specific catalytic activity for total RNAs varied depending on the relaxation of base specificity. Pyr1 promoted the structural integrity by forming a hydrogen bond network through Lys9 in 
1 and Val96 in ß6, and participated in catalytic activity by hydrogen bonds to Lys9 and P1 catalytic phosphate. Residues Thr35 and Asp67 determined B1 base specificity, and Glu91 determined B2 base specificity. The cytotoxicity of onconase is largely determined by structural integrity and specific catalytic activity for UG through Pyr1, rather than non-specific activity for total RNAs.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J.-H. Ahn, M.-Y. Hwang, K.-H. Lee, C.-Y. Choi, and D.-M. Kim Use of signal sequences as an in situ removable sequence element to stimulate protein synthesis in cell-free extracts Nucleic Acids Res., February 28, 2007; 35(4): e21 - e21. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. N. Suhasini and R. Sirdeshmukh Transfer RNA Cleavages by Onconase Reveal Unusual Cleavage Sites J. Biol. Chem., May 5, 2006; 281(18): 12201 - 12209. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-D. Liao, J.-C. Jeng, C.-F. Wang, S.-C. Wang, and S.-T. Chang Removal of N-terminal methionine from recombinant proteins by engineered E. coli methionine aminopeptidase Protein Sci., July 1, 2004; 13(7): 1802 - 1810. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Y. Gorbatyuk, C.-K. Tsai, C.-F. Chang, and T.-h. Huang Effect of N-terminal and Met23 Mutations on the Structure and Dynamics of Onconase J. Biol. Chem., February 13, 2004; 279(7): 5772 - 5780. [Abstract] [Full Text] [PDF]
|
|