Structure-Function Analysis of Trypanosoma brucei RNA Triphosphatase and Evidence for a Two-metal Mechanism

Abstract

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 identified six amino acids that are essential for triphosphatase activity (Glu-52, Arg-127, Glu-168, Arg-186, Glu-216, and Glu-218). These results consolidate the proposal that protozoan, fungal, and Chlorella virus RNA triphosphatases belong to a single family of metaldependent NTP phosphohydrolases with a unique tunnel active site composed of eight ␤ strands. Limited proteolysis of TbCet1 suggests that the hydrophilic N terminus is surface-exposed, whereas the catalytic core domain is tightly folded with the exception of a protease-sensitive loop ( 76 WKGRRARKT 84 ) 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 (K i 1.4 M) that binds more avidly to the active site than the ATP substrate (K m 25 M). 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) among the different branches of the tunnel enzyme family.The m 7 GpppN cap structure (cap 0) 1 is a defining feature of eukaryotic mRNA that is required for mRNA stability and efficient translation. The cap is formed by three enzymes. The 5Ј-triphosphate end of the nascent pre-mRNA is hydrolyzed to a diphosphate by RNA triphosphatase. The diphosphate end is capped with GMP by RNA guanylyltransferase, and the GpppN cap is methylated by RNA (guanine-N7) methyltransferase (1, 2). The mRNAs of kinetoplastid protozoa such as Trypanosoma and Leishmania contain a unique hypermodified "cap 4" structure, which is derived from the m 7 GpppN cap by co-transcriptional methylation of seven sites within the first four nucleosides of the spliced leader RNA (3-6). Kinetoplastid mRNAs acquire their 5Ј caps via trans-splicing of an RNA leader sequence containing the cap 4 structure. The hypermethylated cap is required for trans-splicing (7-9).RNA guanylyltransferase and RNA triphosphatase components of the kinetoplastid capping apparatus have been identified (10, 11), but the proteins that catalyze the several methylation steps have not. Whereas the Trypanosoma brucei guanylyltransferase is mechanistically and structurally related to the guanylyltransferases of all other eukaryal species (1, 10), the T. brucei RNA triphosphatase (TbCet1) bears no resemblance whatsoever, structurally or mechanistically, to the analogous enzyme of the human host (11, 12). RNA triphosphatase is thereby recommended as a potential drug t...

A protein related to the vaccinia virus cap-specific methyltransferase VP39 is involved in cap 4 modification in Trypanosoma brucei

“…One of the most remarkable features in the SL RNA is the presence of an elaborate cap 4 structure, consisting of a terminal m 7 G cap followed by four nucleotides that carry a total of seven methyl groups (Bangs et al 1992). Since RNA guanylyltransferase and RNA triphosphatase components of the trypanosomatid capping apparatus have been identified (Silva et al 1998;Gong et al 2003), it is reasonable to postulate that m 7 G capping of the SL RNA is similar to the capping reaction for mRNAs in yeast and mammalian cells (Shuman 2001). In contrast to the well understood process of m 7 G cap formation, what remains largely unexplored in any eukaryotic system, with the exception of viruses, is the biogenesis of more complex mRNA cap structures.…”

Novel Triphosphate Phosphohydrolase Activity of Clostridium thermocellum TTM, a Member of the Triphosphate Tunnel Metalloenzyme Superfamily

“…This strong preference for an inorganic triphosphate is unprecedented among TTM proteins studied previously. For example, T. brucei Cet1 can hydrolyze PPP i but only 0.5% as well as it hydrolyzes ATP (9). Chlorella virus RNA triphosphatase cleaves PPP i 3% as well as it hydrolyzes ATP (15).…”

“…Members of this family that have been characterized biochemically include the triphosphatase components of the cellular mRNA capping systems of Saccharomyces cerevisiae (2,3), Schizosaccharomyces pombe (4), Candida albicans (5), Plasmodium falciparum (1,6,7), Trypanosoma brucei (8,9), Encephalitozoon cuniculi (10), and Giardia lamblia (11) plus the triphosphatases of the Chlorella virus, poxvirus, and baculovirus mRNA capping systems (12)(13)(14)(15)(16)(17). The signature biochemical property of this branch of the TTM superfamily is the ability to hydrolyze NTPs to nucleoside diphosphates and P i in the presence of manganese (2).…”

Yeast-like mRNA Capping Apparatus in Giardia lamblia

“…The PCR product was digested with NdeI and BamHI and then inserted into a customized T7 RNA polymerase-based pET-His 10 -Smt3 expression vector, wherein the GlCet1 was fused to an N-terminal His 10 -Smt3 domain (18,19). An E58A missense mutation was introduced into the GlCET1 gene by the PCR-based two-stage overlap extension method.…”