Structure–function analysis of Plasmodium RNA triphosphatase and description of a triphosphate tunnel metalloenzyme superfamily that includes Cet1-like RNA triphosphatases and CYTH proteins

Abstract

RNA triphosphatase catalyzes the first step in mRNA capping. The RNA triphosphatases of fungi and protozoa are structurally and mechanistically unrelated to the analogous mammalian enzyme, a situation that recommends RNA triphosphatase as an anti-infective target. Fungal and protozoan RNA triphosphatases belong to a family of metal-dependent phosphohydrolases exemplified by yeast Cet1. The Cet1 active site is unusually complex and located within a topologically closed hydrophilic b-barrel (the triphosphate tunnel). Here we probe the active site of Plasmodium falciparum RNA triphosphatase by targeted mutagenesis and thereby identify eight residues essential for catalysis. The functional data engender an improved structural alignment in which the Plasmodium counterparts of the Cet1 tunnel strands and active-site functional groups are located with confidence. We gain insight into the evolution of the Cet1-like triphosphatase family by noting that the heretofore unique tertiary structure and active site of Cet1 are recapitulated in recently deposited structures of proteins from Pyrococcus (PBD 1YEM) and Vibrio (PDB 2ACA). The latter proteins exemplify a CYTH domain found in CyaB-like adenylate cyclases and mammalian thiamine triphosphatase. We conclude that the tunnel fold first described for Cet1 is the prototype of a larger enzyme superfamily that includes the CYTH branch. This superfamily, which we name ''triphosphate tunnel metalloenzyme,'' is distributed widely among bacterial, archaeal, and eukaryal taxa. It is now clear that Cet1-like RNA triphosphatases did not arise de novo in unicellular eukarya in tandem with the emergence of caps as the defining feature of eukaryotic mRNA. They likely evolved by incremental changes in an ancestral tunnel enzyme that conferred specificity for RNA 59-end processing.

Triphosphate Tunnel Metalloenzyme Function in Senescence Highlights a Biological Diversification of This Protein Superfamily

“…The triphosphate tunnel metalloenzyme (TTM) superfamily comprises two groups of enzymes, RNA triphosphatases and CYTH phosphatases (CyaB adenylate cyclase, thiamine triphosphatase) that possess common characteristics in their catalytic sites (Iyer and Aravind, 2002;Gong et al, 2006). Members of this superfamily are able to hydrolyze a variety of triphosphate substrates, giving them important roles in cAMP formation, mRNA capping, and secondary metabolism (Iyer and Aravind, 2002;Gallagher et al, 2006;Gong et al, 2006;Song et al, 2008).…”

“…Members of this superfamily are able to hydrolyze a variety of triphosphate substrates, giving them important roles in cAMP formation, mRNA capping, and secondary metabolism (Iyer and Aravind, 2002;Gallagher et al, 2006;Gong et al, 2006;Song et al, 2008). Most TTMs possess a unique tunnel structure composed of eight antiparallel beta strands forming a beta barrel and a characteristic EXEXK motif (where X is any amino acid), which is important for catalytic activity (Lima et al, 1999;Iyer and Aravind, 2002;Gallagher et al, 2006).…”

“…However, it appears they have acquired the ability to act on a diverse range of nucleotide and organophosphate substrates (Iyer and Aravind, 2002;Bettendorff and Wins, 2013). Known functions of TTMs include fungal and protozoan RNA triphosphatases (Cet1; Lima et al, 1999;Gong et al, 2006), bacterial adenylate cyclases (CyaB from Aeromonas hydrophila and YpAC-IV from Yersinia pestis; Sismeiro et al, 1998;Gallagher et al, 2006), and mammalian thiamine triphosphatases (Lakaye et al, 2004;Song et al, 2008). More recently, tripolyphosphatase activity was discovered for CthTTM from Clostridium thermocellum and NeuTTM from Nitrosomonas europaea, highlighting the functional diversity of this superfamily (Keppetipola et al, 2007;Delvaux et al, 2011).…”

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

“…RNA triphosphatases of fungi, protozoa, and several DNA viruses are the founding members of the triphosphate tunnel metalloenzyme (TTM) 2 superfamily of phosphohydrolases (1). 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).…”

“…It was thought initially that the Cet1-like RNA triphosphatases arose de novo in unicellular eukarya in tandem with the emergence of caps as the defining feature of eukaryal mRNA. This notion has been eclipsed by the finding that the heretofore unique tertiary structure and active site of yeast RNA triphosphatase are recapitulated in the crystal structures of archaeal and bacterial proteins of unknown biochemical function, including proteins from Pyrococcus (Protein Data Bank (PDB) accession codes 1YEM and 2DC4), Vibrio (2ACA), and Nitrosomonas (2FBL) (1). The Cet1-like archaeal/bacterial proteins are usually annotated as belonging to the so-called CYTH family (19), which is defined by its two biochemically characterized founding members, an Aeromonas hydrophila adenylate cyclase CyaB and a mammalian thiamine triphosphatase (20,21).…”