Structure-function analysis of trypanosoma brucei RNA triphosphatase and evidence for a two-metal mechanism

doi:10.1074/jbc.M309188200

HOME

HELP

FEEDBACK

SUBSCRIPTIONS

Structure-function analysis of trypanosoma brucei RNA triphosphatase and evidence for a two-metal mechanism

Chunling Gong, Alexandra Martins, and Stewart Shuman

Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021

Corresponding Author: s-shuman{at}ski.mskcc.org

Trypanosoma brucei RNA triphosphatase TbCet1 is a 252-amino acid polypeptide that catalyzes the first step in mRNA cap formation. By performing an alanine scan of TbCet1, we identify six amino acids that are essential for triphosphatase activity (Glu52, Arg127, Glu168, Arg186, Glu216, and Glu218). These results consolidate the proposal that protozoan, fungal, and Chlorella virus RNA triphosphatases belong to a single family of metal-dependent NTP phosphohydrolases with a unique tunnel active site composed of 8 b strands. Limited proteolysis of TbCet1 suggests that the hydrophilic N-terminus is surface exposed, whereas the catalytic core domain is tightly folded, except for a protease-sensitive loop (76WKGRRARKT84) between two of the putative tunnel strands. The catalytic domain of TbCet1 is extraordinarily thermostable; it remains active after heating for 2 h at 75°C. Analysis by zonal velocity sedimentation indicates that TbCet1 is a monomeric enzyme, unlike fungal RNA triphosphatases, which are homodimers. We show that tripolyphosphate is a potent competitive inhibitor of TbCet1 (Ki 1.4 µM) that binds more avidly to the active site than the ATP substrate (Km 25 µM). We present evidence of synergistic activation of the TbCet1 triphosphatase by manganese and magnesium, consistent with a two metal mechanism of catalysis. Our findings provide new insight to the similarities (in active site tertiary structure and catalytic mechanism) and differences (in quaternary structure and thermal stability) between different branches of the tunnel enzyme family.

Add to CiteULike CiteULike Add to Complore Complore Add to Connotea Connotea Add to Del.icio.us Del.icio.us Add to Digg Digg Add to Reddit Reddit Add to Technorati Technorati What's this?

This article has been cited by other articles:

R. Jain and S. Shuman
Polyphosphatase Activity of CthTTM, a Bacterial Triphosphate Tunnel Metalloenzyme
J. Biol. Chem., November 7, 2008; 283(45): 31047 - 31057.
[Abstract] [Full Text] [PDF]

B. Mittra, J. R. Zamudio, J. M. Bujnicki, J. Stepinski, E. Darzynkiewicz, D. A. Campbell, and N. R. Sturm
The TbMTr1 Spliced Leader RNA Cap 1 2 '-O-Ribose Methyltransferase from Trypanosoma brucei Acts with Substrate Specificity
J. Biol. Chem., February 8, 2008; 283(6): 3161 - 3172.
[Abstract] [Full Text] [PDF]

M. F. Souliere, J.-P. Perreault, and M. Bisaillon
Magnesium-binding studies reveal fundamental differences between closely related RNA triphosphatases
Nucleic Acids Res., February 2, 2008; 36(2): 451 - 461.
[Abstract] [Full Text] [PDF]

N. Keppetipola, R. Jain, and S. Shuman
Novel Triphosphate Phosphohydrolase Activity of Clostridium thermocellum TTM, a Member of the Triphosphate Tunnel Metalloenzyme Superfamily
J. Biol. Chem., April 20, 2007; 282(16): 11941 - 11949.
[Abstract] [Full Text] [PDF]

C. Gong, P. Smith, and S. Shuman
Structure-function analysis of Plasmodium RNA triphosphatase and description of a triphosphate tunnel metalloenzyme superfamily that includes Cet1-like RNA triphosphatases and CYTH proteins
RNA, August 1, 2006; 12(8): 1468 - 1474.
[Abstract] [Full Text] [PDF]

G. K. ARHIN, H. LI, E. ULLU, and C. TSCHUDI
A protein related to the vaccinia virus cap-specific methyltransferase VP39 is involved in cap 4 modification in Trypanosoma brucei
RNA, January 1, 2006; 12(1): 53 - 62.
[Abstract] [Full Text] [PDF]

S. Hausmann, M. A. Altura, M. Witmer, S. M. Singer, H. G. Elmendorf, and S. Shuman
Yeast-like mRNA Capping Apparatus in Giardia lamblia
J. Biol. Chem., April 1, 2005; 280(13): 12077 - 12086.
[Abstract] [Full Text] [PDF]