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Ribozyme Catalysis Revisited: Is Water Involved?

2007; Elsevier BV; Volume: 28; Issue: 6 Linguagem: Inglês

10.1016/j.molcel.2007.12.001

1097-4164

Nils G. Walter,

Bacterial Genetics and Biotechnology

Enzymatic catalysis by RNA was discovered 25 years ago, yet mechanistic insights are emerging only slowly. Thought to be metalloenzymes at first, some ribozymes proved more versatile than anticipated when shown to utilize their own functional groups for catalysis. Recent evidence suggests that some may also judiciously place structural water molecules to shuttle protons in acid-base catalyzed reactions. Enzymatic catalysis by RNA was discovered 25 years ago, yet mechanistic insights are emerging only slowly. Thought to be metalloenzymes at first, some ribozymes proved more versatile than anticipated when shown to utilize their own functional groups for catalysis. Recent evidence suggests that some may also judiciously place structural water molecules to shuttle protons in acid-base catalyzed reactions. The "whodunit" is the most critical and challenging question in any murder story, even if it plays out on the molecular level. In the case of catalysis by RNA, a phenomenon whose discovery revolutionized our understanding of molecular biology 25 years ago, a key question has been "who" endows such a chemically monotonous, four-letter biopolymer with the ability to carry out (site-)specific chemistry. A prime example of an unsolved molecular murder mystery is found in the seemingly simple RNA backbone transesterification catalyzed by the five naturally occurring small ribozymes, the hammerhead, hairpin, hepatitis delta virus (HDV), Varkud satellite (VS), and glmS ribozymes (Figure 1A) (Lilley, 2004Lilley D.M. The Varkud satellite ribozyme.RNA. 2004; 10: 151-158Crossref PubMed Scopus (68) Google Scholar, Winkler et al., 2004Winkler W.C. Nahvi A. Roth A. Collins J.A. Breaker R.R. Control of gene expression by a natural metabolite-responsive ribozyme.Nature. 2004; 428: 281-286Crossref PubMed Scopus (694) Google Scholar, Doudna and Lorsch, 2005Doudna J.A. Lorsch J.R. Ribozyme catalysis: not different, just worse.Nat. Struct. Mol. Biol. 2005; 12: 395-402Crossref PubMed Scopus (124) Google Scholar, Fedor and Williamson, 2005Fedor M.J. Williamson J.R. The catalytic diversity of RNAs.Nat. Rev. Mol. Cell Biol. 2005; 6: 399-412Crossref PubMed Scopus (267) Google Scholar). Solving this mystery is not only of academic interest in the quest to understand all biological catalysis but also has practical implications for the use of ribozymes in gene therapy (where they can crack down on undesired viral RNA) and biosensor applications (where they can detect the presence of specific biomarkers). Evidence has recently started to arise that the water solvent, i.e., the "butler," may be involved in the case. Despite the apparent simplicity of their chemical makeup, ribozymes can form complex tertiary structures, as perhaps best exemplified by the ribosome, leading to the intricate placement of potential participants in reaction chemistry (Nissen et al., 2000Nissen P. Hansen J. Ban N. Moore P.B. Steitz T.A. The structural basis of ribosome activity in peptide bond synthesis.Science. 2000; 289: 920-930Crossref PubMed Scopus (1753) Google Scholar, Beringer and Rodnina, 2007Beringer M. Rodnina M.V. The ribosomal peptidyl transferase.Mol. Cell. 2007; 26: 311-321Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). When investigations into small ribozyme catalysis were launched, metal cations were obvious suspects as they often catalyze related chemistry in protein-based RNases (Figure 1B). Recent protein examples caught in the act include the tRNase Z family and endonuclease CPSF-73, where a binuclear zinc ion site activates a water molecule to site-specifically hydrolyze the 3′ end of pre-tRNA and pre-mRNA, respectively (Figure 1C) (Vogel et al., 2005Vogel A. Schilling O. Spath B. Marchfelder A. The tRNase Z family of proteins: physiological functions, substrate specificity and structural properties.Biol. Chem. 2005; 386: 1253-1264Crossref PubMed Scopus (85) Google Scholar, Mandel et al., 2006Mandel C.R. Kaneko S. Zhang H. Gebauer D. Vethantham V. Manley J.L. Tong L. Polyadenylation factor CPSF-73 is the pre-mRNA 3′-end-processing endonuclease.Nature. 2006; 444: 953-956Crossref PubMed Scopus (322) Google Scholar); and RNase H, where two magnesium ions appear to fulfill a similar role (Yang et al., 2006Yang W. Lee J.Y. Nowotny M. Making and breaking nucleic acids: two-Mg2+-ion catalysis and substrate specificity.Mol. Cell. 2006; 22: 5-13Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). Similarly, in the large group I intron ribozymes, an external and an internal if distal 3′-hydroxyl (3′-OH) functional group are thought to be activated as nucleophiles for phosphoryl transfer in the first and second steps of self-splicing, respectively, by two magnesium ions, in analogy to the mechanism employed by DNA and RNA polymerases (Figure 1D) (Stahley and Strobel, 2006Stahley M.R. Strobel S.A. RNA splicing: group I intron crystal structures reveal the basis of splice site selection and metal ion catalysis.Curr. Opin. Struct. Biol. 2006; 16: 319-326Crossref PubMed Scopus (74) Google Scholar, Yang et al., 2006Yang W. Lee J.Y. Nowotny M. Making and breaking nucleic acids: two-Mg2+-ion catalysis and substrate specificity.Mol. Cell. 2006; 22: 5-13Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). By comparison, the small ribozymes catalyze a simpler phosphoryl transfer that reversibly interconverts the nearly isoenergetic linear 3′,5′- and cyclic 2′,3′-phosphodiesters in a site-specific and nonhydrolytic reaction (Figure 1A). The similarities in the hydrolytic and nonhydrolytic phosphoryl transfers suggested early on that small ribozymes are metalloenzymes, just like their larger counterparts (Pyle, 1993Pyle A.M. Ribozymes: a distinct class of metalloenzymes.Science. 1993; 261: 709-714Crossref PubMed Scopus (438) Google Scholar, Pyle, 1996Pyle A.M. Role of metal ions in ribozymes.Met. Ions Biol. Syst. 1996; 32: 479-520PubMed Google Scholar). Indeed, experimental (Lott et al., 1998Lott W.B. Pontius B.W. von Hippel P.H. A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate.Proc. Natl. Acad. Sci. USA. 1998; 95: 542-547Crossref PubMed Scopus (84) Google Scholar) and theoretical considerations (Leclerc and Karplus, 2006Leclerc F. Karplus M. Two-metal-ion mechanism for hammerhead-ribozyme catalysis.J. Phys. Chem. B. 2006; 110: 3395-3409Crossref PubMed Scopus (32) Google Scholar) continue to be invoked to support the notion that small ribozymes judiciously place two magnesium ions to catalyze site-specific chemistry. Four potential roles may be ascribed to metal ions in the reaction (Emilsson et al., 2003Emilsson G.M. Nakamura S. Roth A. Breaker R.R. Ribozyme speed limits.RNA. 2003; 9: 907-918Crossref PubMed Scopus (166) Google Scholar) (Figure 1A): (1) structural stabilization of the in-line nucleophilic attack configuration; (2) deprotonation of the upstream 2′-OH by a general base such as a metal hydroxide; (3) Lewis-acid type stabilization of the developing negative charge in the transition state; and (4) protonation of the leaving group oxyanion by a general acid such as a hydrated metal ion. Due to its highly polyanionic character, RNA attracts nearly stoichiometric counter ion charges under physiologic conditions, with a preference for divalent magnesium ions. What functions do these many divalents have? Nonspecifically as well as site-specifically bound Mg2+ ions are crucial in aiding RNA fold into the complex three-dimensional structures required for biological activity (Draper et al., 2005Draper D.E. Grilley D. Soto A.M. Ions and RNA folding.Annu. Rev. Biophys. Biomol. Struct. 2005; 34: 221-243Crossref PubMed Scopus (386) Google Scholar). Most of these divalents bind dynamically quite distant from the RNA active site and so cannot be implicated in catalysis. Circumstantial evidence for a direct catalytic role of a few specific divalents in small ribozyme catalysis was provided by X-ray crystallography, placing metal ions near the catalytic cores of the hammerhead (Scott et al., 1996Scott W.G. Murray J.B. Arnold J.R.P. Stoddard B.L. Klug A. Capturing the structure of a catalytic RNA intermediate: the hammerhead ribozyme.Science. 1996; 274: 2065-2069Crossref PubMed Scopus (414) Google Scholar) and HDV ribozymes (Ke et al., 2004Ke A. Zhou K. Ding F. Cate J.H. Doudna J.A. A conformational switch controls hepatitis delta virus ribozyme catalysis.Nature. 2004; 429: 201-205Crossref PubMed Scopus (233) Google Scholar), and by the discovery of small RNA catalysts dependent on specific transition metal ions such as Mn2+ (Dange et al., 1990Dange V. Van Atta R.B. Hecht S.M. A Mn2+-dependent ribozyme.Science. 1990; 248: 585-588Crossref PubMed Scopus (49) Google Scholar) or Pb2+ (Pan and Uhlenbeck, 1992Pan T. Uhlenbeck O.C. In vitro selection of RNAs that undergo autolytic cleavage with Pb2+.Biochemistry. 1992; 31: 3887-3895Crossref PubMed Scopus (185) Google Scholar), thought to affect cleavage. In the late 1990s, however, it was discovered that the hammerhead, hairpin, and VS ribozymes, in contrast to their larger counterparts, undergo catalysis even in the complete absence of divalent metal ions, as long as sufficient monovalent countercharge is provided (Hampel and Cowan, 1997Hampel A. Cowan J.A. A unique mechanism for RNA catalysis: the role of metal cofactors in hairpin ribozyme cleavage.Chem. Biol. 1997; 4: 513-517Abstract Full Text PDF PubMed Scopus (184) Google Scholar, Nesbitt et al., 1997Nesbitt S. Hegg L.A. Fedor M.J. An unusual pH-independent and metal-ion-independent mechanism for hairpin ribozyme catalysis.Chem. Biol. 1997; 4: 619-630Abstract Full Text PDF PubMed Scopus (192) Google Scholar, Young et al., 1997Young K.J. Gill F. Grasby J.A. Metal ions play a passive role in the hairpin ribozyme catalysed reaction.Nucleic Acids Res. 1997; 25: 3760-3766Crossref PubMed Scopus (156) Google Scholar, Murray et al., 1998Murray J.B. Seyhan A.A. Walter N.G. Burke J.M. Scott W.G. The hammerhead, hairpin and VS ribozymes are catalytically proficient in monovalent cations alone.Chem. Biol. 1998; 5: 587-595Abstract Full Text PDF PubMed Scopus (370) Google Scholar). These observations rule out an obligatory role of metal ions in catalysis by these ribozymes and suggest instead that metal ions can support catalysis solely in an indirect, electrostatic mode, presumably through structure stabilization. The same is likely true for the glmS ribozyme (Roth et al., 2006Roth A. Nahvi A. Lee M. Jona I. Breaker R.R. Characteristics of the glmS ribozyme suggest only structural roles for divalent metal ions.RNA. 2006; 12: 607-619Crossref PubMed Scopus (86) Google Scholar), but a partial exception to this rule is the HDV ribozyme, which shows strong preference for divalents over monovalents (Nakano et al., 2000Nakano S. Chadalavada D.M. Bevilacqua P.C. General acid-base catalysis in the mechanism of a hepatitis delta virus ribozyme.Science. 2000; 287: 1493-1497Crossref PubMed Scopus (365) Google Scholar). The HDV ribozyme is thought to employ a hydrated Mg2+ ion either as general acid (Perrotta et al., 1999Perrotta A.T. Shih I. Been M.D. Imidazole rescue of a cytosine mutation in a self-cleaving ribozyme.Science. 1999; 286: 123-126Crossref PubMed Scopus (253) Google Scholar, Ke et al., 2004Ke A. Zhou K. Ding F. Cate J.H. Doudna J.A. A conformational switch controls hepatitis delta virus ribozyme catalysis.Nature. 2004; 429: 201-205Crossref PubMed Scopus (233) Google Scholar, Krasovska et al., 2005Krasovska M.V. Sefcikova J. Spackova N. Sponer J. Walter N.G. Structural dynamics of precursor and product of the RNA enzyme from the hepatitis delta virus as revealed by molecular dynamics simulations.J. Mol. Biol. 2005; 351: 731-748Crossref PubMed Scopus (57) Google Scholar) or base catalyst in its main reaction channel (Nakano et al., 2000Nakano S. Chadalavada D.M. Bevilacqua P.C. General acid-base catalysis in the mechanism of a hepatitis delta virus ribozyme.Science. 2000; 287: 1493-1497Crossref PubMed Scopus (365) Google Scholar, Nakano et al., 2003Nakano S. Cerrone A.L. Bevilacqua P.C. Mechanistic characterization of the HDV genomic ribozyme: classifying the catalytic and structural metal ion sites within a multichannel reaction mechanism.Biochemistry. 2003; 42: 2982-2994Crossref PubMed Scopus (69) Google Scholar, Das and Piccirilli, 2005Das S.R. Piccirilli J.A. General acid catalysis by the hepatitis delta virus ribozyme.Nat. Chem. Biol. 2005; 1: 45-52Crossref PubMed Scopus (193) Google Scholar, Liu et al., 2007Liu H. Robinet J.J. Ananvoranich S. Gauld J.W. Density functional theory investigation on the mechanism of the hepatitis delta virus ribozyme.J. Phys. Chem. B. 2007; 111: 439-445Crossref PubMed Scopus (17) Google Scholar, Wei et al., 2007Wei K. Liu L. Cheng Y.H. Fu Y. Guo Q.X. Theoretical examination of two opposite mechanisms proposed for hepatitis delta virus ribozyme.J. Phys. Chem. B. 2007; 111: 1514-1516Crossref PubMed Scopus (15) Google Scholar), although a minor reaction channel of residual self-cleavage activity persists at molar concentrations of monovalents, in the absence of Mg2+ (Nakano et al., 2000Nakano S. Chadalavada D.M. Bevilacqua P.C. General acid-base catalysis in the mechanism of a hepatitis delta virus ribozyme.Science. 2000; 287: 1493-1497Crossref PubMed Scopus (365) Google Scholar, Nakano et al., 2001Nakano S. Proctor D.J. Bevilacqua P.C. Mechanistic characterization of the HDV genomic ribozyme: assessing the catalytic and structural contributions of divalent metal ions within a multichannel reaction mechanism.Biochemistry. 2001; 40: 12022-12038Crossref PubMed Scopus (122) Google Scholar, Wadkins et al., 2001Wadkins T.S. Shih I. Perrotta A.T. Been M.D. A pH-sensitive RNA tertiary interaction affects self-cleavage activity of the HDV ribozymes in the absence of added divalent metal ion.J. Mol. Biol. 2001; 305: 1045-1055Crossref PubMed Scopus (76) Google Scholar, Perrotta and Been, 2006Perrotta A.T. Been M.D. HDV ribozyme activity in monovalent cations.Biochemistry. 2006; 45: 11357-11365Crossref PubMed Scopus (45) Google Scholar, Nakano and Bevilacqua, 2007Nakano S. Bevilacqua P.C. Mechanistic characterization of the HDV genomic ribozyme: a mutant of the C41 motif provides insight into the positioning and thermodynamic linkage of metal ions and protons.Biochemistry. 2007; 46: 3001-3012Crossref PubMed Scopus (38) Google Scholar). With divalents no longer considered indispensable, researchers sought and found new suspects that may affect small ribozyme catalysis: RNA functional groups, in analogy to the histidine side chains 12 and 119 of RNase A, thought to serve as the base and acid catalysts, respectively, in the same reaction (Figure 2A) (Doudna and Lorsch, 2005Doudna J.A. Lorsch J.R. Ribozyme catalysis: not different, just worse.Nat. Struct. Mol. Biol. 2005; 12: 395-402Crossref PubMed Scopus (124) Google Scholar, Fedor and Williamson, 2005Fedor M.J. Williamson J.R. The catalytic diversity of RNAs.Nat. Rev. Mol. Cell Biol. 2005; 6: 399-412Crossref PubMed Scopus (267) Google Scholar). The apparent pKas of ribozyme reactions typically differ from those of ionizable functional groups in free nucleobases by several pH units, but it is thought that the latter pKas can become shifted by the negatively charged RNA environment (Bevilacqua and Yajima, 2006Bevilacqua P.C. Yajima R. Nucleobase catalysis in ribozyme mechanism.Curr. Opin. Chem. Biol. 2006; 10: 455-464Crossref PubMed Scopus (98) Google Scholar). Structural and mechanistic evidence for RNA functional group involvement was first discovered for the HDV ribozyme (Ferre-D'Amare et al., 1998Ferre-D'Amare A.R. Zhou K. Doudna J.A. Crystal structure of a hepatitis delta virus ribozyme.Nature. 1998; 395: 567-574Crossref PubMed Scopus (668) Google Scholar), where cytosine (C)75 was proposed to complement the hydrated Mg2+ ion by serving either as the general base (Perrotta et al., 1999Perrotta A.T. Shih I. Been M.D. Imidazole rescue of a cytosine mutation in a self-cleaving ribozyme.Science. 1999; 286: 123-126Crossref PubMed Scopus (253) Google Scholar) or acid (Nakano et al., 2000Nakano S. Chadalavada D.M. Bevilacqua P.C. General acid-base catalysis in the mechanism of a hepatitis delta virus ribozyme.Science. 2000; 287: 1493-1497Crossref PubMed Scopus (365) Google Scholar). Once a precedent was set, analogous suggestions were made for the hairpin ribozyme (Pinard et al., 2001Pinard R. Hampel K.J. Heckman J.E. Lambert D. Chan P.A. Major F. Burke J.M. Functional involvement of G8 in the hairpin ribozyme cleavage mechanism.EMBO J. 2001; 20: 6434-6442Crossref PubMed Scopus (105) Google Scholar) and the ribosome (Muth et al., 2000Muth G.W. Ortoleva-Donnelly L. Strobel S.A. A single adenosine with a neutral pKa in the ribosomal peptidyl transferase center.Science. 2000; 289: 947-950Crossref PubMed Scopus (238) Google Scholar, Nissen et al., 2000Nissen P. Hansen J. Ban N. Moore P.B. Steitz T.A. The structural basis of ribosome activity in peptide bond synthesis.Science. 2000; 289: 920-930Crossref PubMed Scopus (1753) Google Scholar), although the latter hypothesis could not be substantiated (Muth et al., 2001Muth G.W. Chen L. Kosek A.B. Strobel S.A. pH-dependent conformational flexibility within the ribosomal peptidyl transferase center.RNA. 2001; 7: 1403-1415PubMed Google Scholar, Rodnina et al., 2007Rodnina M.V. Beringer M. Wintermeyer W. How ribosomes make peptide bonds.Trends Biochem. Sci. 2007; 32: 20-26Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). For hairpin ribozyme catalysis, the protonation state of G8 was found to be important (Pinard et al., 2001Pinard R. Hampel K.J. Heckman J.E. Lambert D. Chan P.A. Major F. Burke J.M. Functional involvement of G8 in the hairpin ribozyme cleavage mechanism.EMBO J. 2001; 20: 6434-6442Crossref PubMed Scopus (105) Google Scholar). In a crystal structure of a ribozyme-inhibitor complex, G8 is found close to the 2′-OH of A-1, the functional group that needs to be deprotonated during the reaction (Rupert and Ferre-D'Amare, 2001Rupert P.B. Ferre-D'Amare A.R. Crystal structure of a hairpin ribozyme-inhibitor complex with implications for catalysis.Nature. 2001; 410: 780-786Crossref PubMed Scopus (391) Google Scholar). Of several models consistent with this observation, the notion that G8 stabilizes the transition state by electrostatic coordination and/or hydrogen bonding is supported by exogenous nucleobase rescue experiments (Kuzmin et al., 2004Kuzmin Y.I. Da Costa C.P. Fedor M.J. Role of an active site guanine in hairpin ribozyme catalysis probed by exogenous nucleobase rescue.J. Mol. Biol. 2004; 340: 233-251Crossref PubMed Scopus (81) Google Scholar), X-ray crystallography of a transition state analog (Rupert et al., 2002Rupert P.B. Massey A.P. Sigurdsson S.T. Ferre-D'Amare A.R. Transition state stabilization by a catalytic RNA.Science. 2002; 298: 1421-1424Crossref PubMed Scopus (250) Google Scholar), and molecular dynamics (MD) simulations of the active ribozyme-substrate complex, where G8 is observed to hydrogen bond with a nonbridging oxygen of the scissile phosphate (Figure 2B) (Rhodes et al., 2006Rhodes M.M. Reblova K. Sponer J. Walter N.G. Trapped water molecules are essential to structural dynamics and function of a ribozyme.Proc. Natl. Acad. Sci. USA. 2006; 103: 13380-13385Crossref PubMed Scopus (81) Google Scholar). Furthermore, A38 is located adjacent to the 5′-oxyanion leaving group of the scissile phosphate (Figure 2B) (Rupert and Ferre-D'Amare, 2001Rupert P.B. Ferre-D'Amare A.R. Crystal structure of a hairpin ribozyme-inhibitor complex with implications for catalysis.Nature. 2001; 410: 780-786Crossref PubMed Scopus (391) Google Scholar, Rhodes et al., 2006Rhodes M.M. Reblova K. Sponer J. Walter N.G. Trapped water molecules are essential to structural dynamics and function of a ribozyme.Proc. Natl. Acad. Sci. USA. 2006; 103: 13380-13385Crossref PubMed Scopus (81) Google Scholar, Salter et al., 2006Salter J. Krucinska J. Alam S. Grum-Tokars V. Wedekind J.E. Water in the active site of an all-RNA hairpin ribozyme and effects of Gua8 base variants on the geometry of phosphoryl transfer.Biochemistry. 2006; 45: 686-700Crossref PubMed Scopus (71) Google Scholar), suggesting that it may act as the general acid (or possibly base for the 2′-OH). All of these assignments remain controversial, however, as nucleotide analog interference mapping and exogenous nucleobase rescue experiments instead support a model in which A38 stabilizes the transition state electrostatically (Ryder et al., 2001Ryder S.P. Oyelere A.K. Padilla J.L. Klostermeier D. Millar D.P. Strobel S.A. Investigation of adenosine base ionization in the hairpin ribozyme by nucleotide analog interference mapping.RNA. 2001; 7: 1454-1463PubMed Google Scholar, Kuzmin et al., 2005Kuzmin Y.I. Da Costa C.P. Cottrell J.W. Fedor M.J. Role of an active site adenine in hairpin ribozyme catalysis.J. Mol. Biol. 2005; 349: 989-1010Crossref PubMed Scopus (90) Google Scholar) and G8 serves in general acid-base catalysis (Wilson et al., 2006Wilson T.J. Ouellet J. Zhao Z.Y. Harusawa S. Araki L. Kurihara T. Lilley D.M. Nucleobase catalysis in the hairpin ribozyme.RNA. 2006; 12: 980-987Crossref PubMed Scopus (58) Google Scholar). Are there other suspects that may affect catalytic function in small ribozymes? An omnipresent participant in all of biology is water, the universal solvent of life. Water dissolves more substances than any other fluid due to its unique physicochemical properties, among them the capacity to form very strong hydrogen bonds combined with fast rotational diffusion, leading to rapid reorientation of hydrogen bonds. In addition, water as a polyprotic acid is equally likely to be in protonated (H3O+) and unprotonated (OH−) states at neutral (near-physiologic) pH, conducts protons by fast tunneling through chains of aligned water molecules (during the so-called Grotthus conduction mechanism), and is present at high concentrations (around 55 M) in all biological systems. Structurally ordered water molecules can be found associated with RNA in high-resolution crystal structures of tRNA (Westhof, 1988Westhof E. Water: an integral part of nucleic acid structure.Annu. Rev. Biophys. Biophys. Chem. 1988; 17: 125-144Crossref PubMed Scopus (254) Google Scholar) as well as MD simulations of RNA (Auffinger and Westhof, 2000Auffinger P. Westhof E. RNA solvation: a molecular dynamics simulation perspective.Biopolymers. 2000; 56: 266-274Crossref PubMed Scopus (50) Google Scholar). Water is thought to contribute to the hydrophobic collapse of a folding RNA (Sorin et al., 2005Sorin E.J. Rhee Y.M. Pande V.S. Does water play a structural role in the folding of small nucleic acids?.Biophys. J. 2005; 88: 2516-2524Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) and to the stabilization of a 2′-O-methylated over an unmodified RNA helix (Auffinger and Westhof, 2001Auffinger P. Westhof E. Hydrophobic groups stabilize the hydration shell of 2′-O-methylated RNA duplexes.Angew. Chem. Int. Ed. Engl. 2001; 40: 4648-4650Crossref PubMed Scopus (52) Google Scholar). It is also invoked in catalysis of the HDV ribozyme (Nakano et al., 2000Nakano S. Chadalavada D.M. Bevilacqua P.C. General acid-base catalysis in the mechanism of a hepatitis delta virus ribozyme.Science. 2000; 287: 1493-1497Crossref PubMed Scopus (365) Google Scholar) as well as group I intron ribozymes (Figure 1D) (Stahley and Strobel, 2006Stahley M.R. Strobel S.A. RNA splicing: group I intron crystal structures reveal the basis of splice site selection and metal ion catalysis.Curr. Opin. Struct. Biol. 2006; 16: 319-326Crossref PubMed Scopus (74) Google Scholar) through proton transfers involving the inner solvation sphere of the catalytic magnesium ions. Yet until recently water was rarely observed in crystal structures of ribozymes, mostly due to the difficulty of assigning the residual electron densities observed at moderate resolutions to water molecules rather than small ions associated with the RNA. This "invisibility" of water kept it largely out of the sights of ribozyme researchers. This contrasts with the attention that structurally ordered (low-B factor) water molecules in protein enzymes and proton pumps have attracted, where they were suggested (Meyer, 1992Meyer E. Internal water molecules and H-bonding in biological macromolecules: a review of structural features with functional implications.Protein Sci. 1992; 1: 1543-1562Crossref PubMed Scopus (181) Google Scholar, Frank et al., 2004Frank R.A. Titman C.M. Pratap J.V. Luisi B.F. Perham R.N. A molecular switch and proton wire synchronize the active sites in thiamine enzymes.Science. 2004; 306: 872-876Crossref PubMed Scopus (148) Google Scholar, Wang et al., 2007Wang L. Yu X. Hu P. Broyde S. Zhang Y. A water-mediated and substrate-assisted catalytic mechanism for sulfolobus solfataricus DNA polymerase IV.J. Am. Chem. Soc. 2007; 129: 4731-4737Crossref PubMed Scopus (110) Google Scholar) and observed (Garczarek and Gerwert, 2006Garczarek F. Gerwert K. Functional waters in intraprotein proton transfer monitored by FTIR difference spectroscopy.Nature. 2006; 439: 109-112Crossref PubMed Scopus (455) Google Scholar) to participate in proton shuttling through "relays" and "wires." RNase A, for example, harbors several chains of conserved structural water molecules radiating from its catalytic core (Figure 2A) (Zegers et al., 1994Zegers I. Maes D. Dao-Thi M.H. Poortmans F. Palmer R. Wyns L. The structures of RNase A complexed with 3′-CMP and d(CpA): active site conformation and conserved water molecules.Protein Sci. 1994; 3: 2322-2339Crossref PubMed Scopus (177) Google Scholar) that have been proposed to serve as proton shuttles to bulk water at the enzyme's surface (Meyer, 1992Meyer E. Internal water molecules and H-bonding in biological macromolecules: a review of structural features with functional implications.Protein Sci. 1992; 1: 1543-1562Crossref PubMed Scopus (181) Google Scholar). Water wires are thought particularly suitable to conduct protons by the Grotthus mechanism, as the initial proton transfer can occur at neutral pH in under 150 fs (Mohammed et al., 2005Mohammed O.F. Pines D. Dreyer J. Pines E. Nibbering E.T. Sequential proton transfer through water bridges in acid-base reactions.Science. 2005; 310: 83-86Crossref PubMed Scopus (442) Google Scholar) and is followed by proton transfer to other water molecules within 30 fs (Geissler et al., 2001Geissler P.L. Dellago C. Chandler D. Hutter J. Parrinello M. Autoionization in liquid water.Science. 2001; 291: 2121-2124Crossref PubMed Scopus (608) Google Scholar, Garczarek and Gerwert, 2006Garczarek F. Gerwert K. Functional waters in intraprotein proton transfer monitored by FTIR difference spectroscopy.Nature. 2006; 439: 109-112Crossref PubMed Scopus (455) Google Scholar), keeping each water molecule in a wire uncharged during most of the process. The hairpin ribozyme exemplifies the mounting evidence for an extended functionality of water solvent in RNA catalysis (Park and Lee, 2006Park H. Lee S. Role of solvent dynamics in stabilizing the transition state of RNA hydrolysis by hairpin ribozyme.RNA. 2006; 2: 858-862Google Scholar, Rhodes et al., 2006Rhodes M.M. Reblova K. Sponer J. Walter N.G. Trapped water molecules are essential to structural dynamics and function of a ribozyme.Proc. Natl. Acad. Sci. USA. 2006; 103: 13380-13385Crossref PubMed Scopus (81) Google Scholar, Salter et al., 2006Salter J. Krucinska J. Alam S. Grum-Tokars V. Wedekind J.E. Water in the active site of an all-RNA hairpin ribozyme and effects of Gua8 base variants on the geometry of phosphoryl transfer.Biochemistry. 2006; 45: 686-700Crossref PubMed Scopus (71) Google Scholar, Torelli et al., 2007Torelli A.T. Krucinska J. Wedekind J.E. A comparison of vanadate to a 2′-5′ linkage at the active site of a small ribozyme suggests a role for water in transition-state stabilization.RNA. 2007; 13: 1052-1070Crossref PubMed Scopus (46) Google Scholar). During MD simulation several water molecules were found to become trapped in the solvent-protected catalytic core for many consecutive nanoseconds as an inherent part of the RNA structure, only occasionally exchanging with bulk solvent (Figure 2B) (by comparison, typical water residency times on the outside of RNA last only fractions of nanoseconds [Auffinger and Westhof, 2001Auffinger P. Westhof E. Hydrophobic groups stabilize the hydration shell of 2′-O-methylated RNA duplexes.Angew. Chem. Int. Ed. Engl. 2001; 40: 4648-4650Crossref PubMed Scopus (52) Google Scholar, Rhodes et al., 2006Rhodes M.M. Reblova K. Sponer J. Walter N.G. Trapped water molecules are essential to structural dynamics and function of a ribozyme.Proc. Natl. Acad. Sci. USA. 2006; 103: 13380-13385Crossref PubMed Scopus (81) Google Scholar]). These water molecules are also observed in higher-resolution crystal structures (Salter et al., 2006Salter J. Krucinska J. Alam S. Grum-Tokars V. Wedekind J.E. Water in the active site of an all-RNA hairpin ribozyme and effects of Gua8 base variants on the geometry of phosphoryl transfer.Biochemistry. 2006; 45: 686-700Crossref PubMed Scopus (71) Google Scholar, Torelli et al., 2007Torelli A.T. Krucinska J. Wedekind J.E. A comparison of vanadate to a 2′-5′ linkage at the active site of a small ribozyme suggests a role for water in transition-state stabilization.RNA. 2007; 13: 1052-1070Crossref PubMed Scopus (46) Google Scholar) and appear to participate in extensive hydrogen bonding networks similar to those observed in protein enzymes (Meyer, 1992Meyer E. Internal water molecules and H-bonding in biological macromolecules: a review of structural features with functional implications.Protein Sci. 1992; 1: 1543-1562Crossref PubMed Scopus (181) Google Scholar). These networks form the basis for correlated motions that amplify local chemical modifications into functionally relevant structural rearrangements throughout the catalytic core (Rhodes et al., 2006Rhodes M.M. Reblova K. Sponer J. Walter N.G. Trapped water molecules are essential to structural dynamics and function of a ribozyme.Proc. Natl. Acad. Sci. USA. 2006; 103: 13380-13385Crossref PubMed Scopus (81) Google Scholar). In addition, the trapped water molecules line up in a hydrogen-bonded chain, reminiscent of the "proton wire" found in protein enzymes (Frank et al., 2004Frank R.A. Titman C.M. Pratap J.V. Luisi B.F. Perham R.N. A molecular switch and proton wire synchronize the active sites in thiamine enzymes.Science. 2004; 306: 872-876Crossref PubMed Scopus (148) Google Scholar). Coincidentally, a central water molecule in this wire accepts a hydrogen bond from the 2′-OH of A-1, engaging the proton that needs to be removed during catalysis (Figure 2B). The same water is also found to frequently donate a hydrogen bond to N1 of A38 (Rhodes et al., 2006Rhodes M.M. Reblova K. Sponer J. Walter N.G. Trapped water molecules are essential to structural dynamics and function of a ribozyme.Proc. Natl. Acad. Sci. USA. 2006; 103: 13380-13385Crossref PubMed Scopus (81) Google Scholar), opening the possibility of a mechanism in which a proton is first shuttled from the 2′-OH of A-1 through the water molecule to N1 of A38 and then onto the 5′-oxyanion leaving group in a concerted mechanism (Figure 2B). In this fashion, a water-assisted A38 could serve both as general base and acid in the reaction. Additionally, or alternatively, the trapped water molecules may stabilize the enhanced negative phosphate charge in the transition state as suggested by MD simulation and X-ray crystallography (Park and Lee, 2006Park H. Lee S. Role of solvent dynamics in stabilizing the transition state of RNA hydrolysis by hairpin ribozyme.RNA. 2006; 2: 858-862Google Scholar, Torelli et al., 2007Torelli A.T. Krucinska J. Wedekind J.E. A comparison of vanadate to a 2′-5′ linkage at the active site of a small ribozyme suggests a role for water in transition-state stabilization.RNA. 2007; 13: 1052-1070Crossref PubMed Scopus (46) Google Scholar). Higher-resolution electron density maps from X-ray crystallography have recently begun to detect water molecules in the catalytic cores of other ribozymes, including the hammerhead (Figure 2C) (Martick and Scott, 2006Martick M. Scott W.G. Tertiary contacts distant from the active site prime a ribozyme for catalysis.Cell. 2006; 126: 309-320Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar) and glmS ribozymes (Figure 2D) (Klein and Ferre-D'Amare, 2006Klein D.J. Ferre-D'Amare A.R. Structural basis of glmS ribozyme activation by glucosamine-6-phosphate.Science. 2006; 313: 1752-1756Crossref PubMed Scopus (303) Google Scholar) as well as the ribosome (Schmeing et al., 2005Schmeing T.M. Huang K.S. Kitchen D.E. Strobel S.A. Steitz T.A. Structural insights into the roles of water and the 2′ hydroxyl of the P site tRNA in the peptidyl transferase reaction.Mol. Cell. 2005; 20: 437-448Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). In the hammerhead ribozyme, for example, a protonated water molecule has been proposed to serve as a proton source in a proton relay involving the 2′-OH of G8 as general acid catalyst (Figure 2C) (Martick and Scott, 2006Martick M. Scott W.G. Tertiary contacts distant from the active site prime a ribozyme for catalysis.Cell. 2006; 126: 309-320Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). In the case of the glmS ribozyme, a proposal has been made that the external glucosamine-6-phosphate ligand acts as general acid catalyst as well as, through a proton wire involving two structural water molecules, remote general base catalyst (Figure 2D). Notably, the underlying crystal structure contains a 2′-deoxy modification at the cleavage site (similar to the crystal structure of RNase A shown in Figure 2A) and lacks the amino functionality of the ligand (Klein and Ferre-D'Amare, 2006Klein D.J. Ferre-D'Amare A.R. Structural basis of glmS ribozyme activation by glucosamine-6-phosphate.Science. 2006; 313: 1752-1756Crossref PubMed Scopus (303) Google Scholar); it is known that such minor chemical modifications can lead to RNA conformational changes (Rueda et al., 2004Rueda D. Bokinsky G. Rhodes M.M. Rust M.J. Zhuang X. Walter N.G. Single-molecule enzymology of RNA: essential functional groups impact catalysis from a distance.Proc. Natl. Acad. Sci. USA. 2004; 101: 10066-10071Crossref PubMed Scopus (127) Google Scholar, Rhodes et al., 2006Rhodes M.M. Reblova K. Sponer J. Walter N.G. Trapped water molecules are essential to structural dynamics and function of a ribozyme.Proc. Natl. Acad. Sci. USA. 2006; 103: 13380-13385Crossref PubMed Scopus (81) Google Scholar). A more recent crystal structure carrying a cleavage site 2′-O-methyl modification and the native glucosamine-6-phosphate ligand does not display these water molecules and thus predicts direct general base catalysis by G40 (Figure 2D; G33 in the authors' numbering scheme) (Cochrane et al., 2007Cochrane J.C. Lipchock S.V. Strobel S.A. Structural investigation of the GlmS ribozyme bound to its catalytic cofactor.Chem. Biol. 2007; 14: 97-105Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Is there proof for whether or not structural water molecules are active participants in ribozyme catalysis? Of course any proton shifted to or from a hydrated metal ion or an RNA functional group will reside in bulk water sooner or later; that is, water molecules from the ubiquitous solvent will for sure indirectly be involved in any of the plausible mechanisms discussed above. But the open "whodunit" mystery is whether or not strategically placed, long-residency water molecules play a more direct role by, for example, shuttling protons during catalysis. The jury is still out, but water is an intriguing suspect to join the lineup with metal ions and RNA functional groups as potential catalytic effectors. In light of the molecular heterogeneities observed in RNA by single-molecule techniques (Zhuang, 2005Zhuang X. Single-molecule RNA science.Annu. Rev. Biophys. Biomol. Struct. 2005; 34: 399-414Crossref PubMed Scopus (144) Google Scholar) it is even plausible that different ribozyme molecules in an ensemble follow alternative reaction pathways. Clearly, additional experimental approaches such as neutron scattering (Zaccai, 1986Zaccai G. Measurement of density and location of solvent associated with biomolecules by small-angle neutron scattering.Methods Enzymol. 1986; 127: 619-629Crossref PubMed Scopus (7) Google Scholar), NMR (Newby and Greenbaum, 2002Newby M.I. Greenbaum N.L. Investigation of Overhauser effects between pseudouridine and water protons in RNA helices.Proc. Natl. Acad. Sci. USA. 2002; 99: 12697-12702Crossref PubMed Scopus (84) Google Scholar), and Fourier transform infrared (FTIR) spectroscopy (Garczarek and Gerwert, 2006Garczarek F. Gerwert K. Functional waters in intraprotein proton transfer monitored by FTIR difference spectroscopy.Nature. 2006; 439: 109-112Crossref PubMed Scopus (455) Google Scholar) are needed to further pinpoint the locations and roles of water molecules in RNA catalysis. Studies on RNA will undoubtedly be guided by the extensive prior work on protein enzymes. It is tempting to speculate that evolution has found ways to exploit highly abundant solvent molecules with unique physicochemical properties to enhance the catalytic capabilities of both protein and RNA. Given our limited understanding of even the bulk properties of water (Leetmaa et al., 2006Leetmaa M. Ljungberg M. Ogasawara H. Odelius M. Naslund L.A. Nilsson A. Pettersson L.G. Are recent water models obtained by fitting diffraction data consistent with infrared/Raman and x-ray absorption spectra?.J. Chem. Phys. 2006; 125: 244510Crossref PubMed Scopus (60) Google Scholar), it may well be that isolated structural water molecules bound to the surface of a biopolymer become quite unique chemical reagents (Buch et al., 2007Buch V. Milet A. Vacha R. Jungwirth P. Devlin J.P. Water surface is acidic.Proc. Natl. Acad. Sci. USA. 2007; 104: 7342-7347Crossref PubMed Scopus (305) Google Scholar).