Critical Aspartic Acid Residues in Pseudouridine Synthases

Abstract

The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411–2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996)Nucleic Acids Res. 24, 3756–3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998)Biochemistry37, 344–351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function. The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411–2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996)Nucleic Acids Res. 24, 3756–3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998)Biochemistry37, 344–351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function. All organisms chemically modify their RNA after transcription, and the isomerization of uridine to its C-glycoside isomer pseudouridine (Ψ) 1The abbreviations used are: Ψ, pseudouridine; [3H]tRNA, E. coli tRNAPhetranscript containing [5-3H]uridine is the most prevalent modification, Fig. 1 (1Grosjean H. Benne R. Modification and Editing of RNA. ASM Press, Washington, D. C.1998Crossref Google Scholar). This isomerization is catalyzed by the pseudouridine synthases, enzymes that display specificity for U residues at particular positions in certain RNA molecules, a specificity that can range from handling a single specific site to mild promiscuity (2Nurse K. Wrzesinski J. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 102-112PubMed Google Scholar, 3Wrzesinski J. Nurse K. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 437-448PubMed Google Scholar, 4Wrzesinski J. Bakin A. Nurse K. Lane B.G. Ofengand J. Biochemistry. 1995; 34: 8904-8913Crossref PubMed Scopus (85) Google Scholar, 5Simos G. Tekotte H. Grosjean H. Segref A. Sharma K. Tollervey D. Hurt E.C. EMBO J. 1996; 15: 2270-2284Crossref PubMed Scopus (155) Google Scholar, 6Raychaudhuri S. Conrad J. Hall B.G. Ofengand J. RNA. 1998; 4: 1407-1417Crossref PubMed Scopus (112) Google Scholar, 7Huang L.X. Ku J. Pookanjanatavip M. Gu X.R. Wang D. Greene P.J. Santi D.V. Biochemistry. 1998; 37: 15951-15957Crossref PubMed Scopus (52) Google Scholar). Physiological ramifications resulting from the lack of Ψ at particular locations have recently become evident, mandating a fuller understanding of Ψ generation in particular and RNA modification generally. In Escherichia coli, severe growth inhibition results from disruption of rluD (formerly denoted sfhB oryfiI), which encodes the Ψ synthase responsible for isomerization of U residues at positions 1911, 1915, and 1917 of 23 S rRNA (6Raychaudhuri S. Conrad J. Hall B.G. Ofengand J. RNA. 1998; 4: 1407-1417Crossref PubMed Scopus (112) Google Scholar, 7Huang L.X. Ku J. Pookanjanatavip M. Gu X.R. Wang D. Greene P.J. Santi D.V. Biochemistry. 1998; 37: 15951-15957Crossref PubMed Scopus (52) Google Scholar). In eukaryotes, Steitz and co-workers (8Yu Y.T. Shu M.D. Steitz J.A. EMBO J. 1998; 17: 5783-5795Crossref PubMed Scopus (209) Google Scholar) have elegantly demonstrated that the presence of Ψ in the U2 small nuclear RNA is required for proper assembly of the spliceosome, work that relied on the inhibition of the responsible Ψ synthase(s) by U2 transcripts containing 5-fluorouridine. Such inhibition of Ψ synthases by RNA containing 5-fluorouracil is well precedented (9Kammen H.O. Marvel C.C. Hardy L. Penhoet E.E. J. Biol. Chem. 1988; 263: 2255-2263Abstract Full Text PDF PubMed Google Scholar, 10Samuelsson T. Nucleic Acids Res. 1991; 19: 6139-6144Crossref PubMed Scopus (49) Google Scholar, 11Patton J.R. Biochem. J. 1993; 290: 595-600Crossref PubMed Scopus (29) Google Scholar), and this inhibition may account for a secondary mode of action of the anticancer drug 5-fluorouracil, which primarily acts by inhibiting thymidylate synthase (12Santi D.V. McHenry C.S. Sommer H. Biochemistry. 1974; 13: 471-481Crossref PubMed Scopus (582) Google Scholar). Consistent with 5-fluorouracil cytotoxicity resulting from Ψ synthase inhibition is a long string of observations concerning cell lines treated with both 5-fluorouracil and thymidine (eliminating the need for thymidylate synthase). This treatment affects many RNA-mediated events, including disruption of rRNA maturation (13Wilkinson D.S. Pitot H.C. J. Biol. Chem. 1973; 248: 63-68Abstract Full Text PDF PubMed Google Scholar, 14Ghoshal K. Jacob S.T. Cancer Res. 1994; 54: 632-636PubMed Google Scholar), disruption of pre-mRNA splicing (15Doong S.L. Dolnick B.J. J. Biol. Chem. 1988; 263: 4467-4473Abstract Full Text PDF PubMed Google Scholar, 16Sierakowska H. Shukla R.R. Dominski Z. Kole R. J. Biol. Chem. 1989; 264: 19185-19191Abstract Full Text PDF PubMed Google Scholar, 17Lenz H.J. Manno D.J. Danenberg K.D. Danenberg P.V. J. Biol. Chem. 1994; 269: 31962-31968Abstract Full Text PDF PubMed Google Scholar), and, perhaps, reducing translational accuracy (18Dolnick B.J. Pink J.J. J. Biol. Chem. 1985; 260: 3006-3014Abstract Full Text PDF PubMed Google Scholar). The link is thus established between Ψ synthases and critical RNA-mediated cellular processes, the disruption of which can lead to dire consequences. One such consequence is likely the X-linked human disease dyskeratosis congenita. Young men suffering from this disease have blotchy skin, poor dental health, sparse hair (including a lack of eyebrows), and evanescent nails; these men also tend to develop gastrointestinal tumors and suffer bone marrow failure (19Luzzatto L. Karadimitris A. Nat. Genet. 1998; 19: 6-7Crossref PubMed Scopus (56) Google Scholar). The gene responsible for dyskeratosis congenita has recently been identified and encodes a protein dubbed dyskerin, which contains a nuclear localization sequence and has two stretches of amino acids highly similar to the E. coli Ψ synthase TruB (20Heiss N.S. Knight S.W. Vulliamy T.J. Klauck S.M. Wiemann S. Mason P.J. Poustka A. Dokal I. Nat. Genet. 1998; 19: 32-38Crossref PubMed Scopus (751) Google Scholar). Although the detected similarity to TruB is in itself rather weak evidence for concluding that dyskerin is a Ψ synthase, the sequence alignments of the known Ψ synthases support that assessment (see below), and probing the functional significance of these alignments was the purpose of the experiments described in this communication. A brief presentation of the alignments is, therefore, imperative before recounting and discussing the experiments. After Penhoet and co-workers (9Kammen H.O. Marvel C.C. Hardy L. Penhoet E.E. J. Biol. Chem. 1988; 263: 2255-2263Abstract Full Text PDF PubMed Google Scholar) and Ofengand and co-workers (2Nurse K. Wrzesinski J. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 102-112PubMed Google Scholar, 3Wrzesinski J. Nurse K. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 437-448PubMed Google Scholar, 4Wrzesinski J. Bakin A. Nurse K. Lane B.G. Ofengand J. Biochemistry. 1995; 34: 8904-8913Crossref PubMed Scopus (85) Google Scholar) cloned the first four Ψ synthase genes, both Koonin (21Koonin E.V. Nucleic Acids Res. 1996; 24: 2411-2415Crossref PubMed Scopus (203) Google Scholar) and Santi and co-workers (22Gustafsson C. Reid R. Greene P.J. Santi D.V. Nucleic Acids Res. 1996; 24: 3756-3762Crossref PubMed Scopus (120) Google Scholar) undertook alignments and data base searches with these Ψ synthases. Both studies found statistically insignificant identity between the four proteins over their entire length, but both studies also determined that three short stretches of amino acids (5–13 residues) displayed significant similarity. Interestingly, each of the four Ψ synthases (RluA, RsuA, TruB, and TruA, which was formerly named either HisT or Ψ synthase I) had clear homologs in the data base, which led Koonin (21Koonin E.V. Nucleic Acids Res. 1996; 24: 2411-2415Crossref PubMed Scopus (203) Google Scholar) to conclude that each of the first four cloned Ψ synthases represented a different family of these enzymes. Indeed, similarity to the known Ψ synthases has been used to target unidentified open reading frames as Ψ synthases, and this strategy has led to the cloning of eight more Ψ synthases fromE. coli, yeast, mouse, and human (as a partial clone) (6Raychaudhuri S. Conrad J. Hall B.G. Ofengand J. RNA. 1998; 4: 1407-1417Crossref PubMed Scopus (112) Google Scholar, 7Huang L.X. Ku J. Pookanjanatavip M. Gu X.R. Wang D. Greene P.J. Santi D.V. Biochemistry. 1998; 37: 15951-15957Crossref PubMed Scopus (52) Google Scholar,23Chen J. Patton J.R. RNA. 1999; 5: 409-419Crossref PubMed Scopus (45) Google Scholar, 24Conrad J. Sun D.H. Englund N. Ofengand J. J. Biol. Chem. 1998; 273: 18562-18566Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 25Lecointe F. Simos G. Sauer A. Hurt E.C. Motorin Y. Grosjean H. J. Biol. Chem. 1998; 273: 1316-1323Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 26Becker H.F. Motorin Y. Planta R.J. Grosjean H. Nucleic Acids Res. 1997; 25: 4493-4499Crossref PubMed Google Scholar). The degree of relatedness among the four families, however, was rather ambiguous. In fact, as a result of the relatively low levels of similarity, each report of these alignments omitted at least one of the genes from the figure showing the alignments: Koonin (21Koonin E.V. Nucleic Acids Res. 1996; 24: 2411-2415Crossref PubMed Scopus (203) Google Scholar) excluded TruA, and Santi and co-workers (22Gustafsson C. Reid R. Greene P.J. Santi D.V. Nucleic Acids Res. 1996; 24: 3756-3762Crossref PubMed Scopus (120) Google Scholar) excluded both TruA and TruB. Reexamination of the sequence data by Santi and co-workers (27Huang L.X. Pookanjanatavip M. Gu X.G. Santi D.V. Biochemistry. 1998; 37: 344-351Crossref PubMed Scopus (132) Google Scholar) led to the insight that among all of the Ψ synthasesand their identified homologs, a single aspartic is found aligned in all This aspartic acid residue in TruA was to and and all of the mutant TruA proteins catalytically inactive to bind tRNA with wild-type (27Huang L.X. Pookanjanatavip M. Gu X.G. Santi D.V. Biochemistry. 1998; 37: 344-351Crossref PubMed Scopus (132) Google Scholar). The critical catalytic of the aligned aspartic acid residue the functional significance of the aligned sequence Fig. the which Koonin (21Koonin E.V. Nucleic Acids Res. 1996; 24: 2411-2415Crossref PubMed Scopus (203) Google Scholar) named motif containing the aligned aspartic acid including the first four cloned Ψ synthases and dyskerin, which similarity with TruB in this (20Heiss N.S. Knight S.W. Vulliamy T.J. Klauck S.M. Wiemann S. Mason P.J. Poustka A. Dokal I. Nat. Genet. 1998; 19: 32-38Crossref PubMed Scopus (751) Google Scholar). Interestingly, this motif was also identified in both and on this Koonin that this of amino acids was in uridine (21Koonin E.V. Nucleic Acids Res. 1996; 24: 2411-2415Crossref PubMed Scopus (203) Google Scholar), which to with the ability of the TruA to bind tRNA (27Huang L.X. Pookanjanatavip M. Gu X.G. Santi D.V. Biochemistry. 1998; 37: 344-351Crossref PubMed Scopus (132) Google Scholar). This between and results and the weak similarity between the four families of Ψ synthases further of the and the aspartic acid residue To this we undertook the of the aligned aspartic acid residues in two Ψ synthases of different families, and TruB. was from was from and E. coli from The enzyme was from and from from the from was from was from and from E. coli from and all from or its A was used for the of the was in the of using a The which contains the gene coli a was a from Biochemistry. PubMed Scopus Google Scholar). containing the (2Nurse K. Wrzesinski J. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 102-112PubMed Google Scholar) and J. Nurse K. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 437-448PubMed Google Scholar) in by J. the of these do not they be to as and The for of RNA gene was a from T. and the RNA was as described G. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). All of these proteins have a to their to was using the to the for the the between at different additional is required for the to with a single which the as the is to the The for the was as for 1 of for 1 for and for and at The containing a to a by of the entire TruB was for the was The used for each are with the into and the its on the denoted in TruB and TruB and and and and The of the was by the entire two of three had the and no The mutant Ψ synthases and the of the are as TruB TruB and of wild-type TruB and with or used to and the in a was at the was 1 After the at for at and at of the the cell was and in containing and All at was and the was for to cell After to the was for to cell was to the and the was for to bind the enzyme to the After for the was in the of used for cell and into a The was with containing and enzyme was then with a of containing and TruB was containing and and at was containing This was either used one or was to with and then of and at The and of the mutant Ψ synthases to that of the wild-type enzyme that the was which small of that a treatment with was not The enzymes to be by with and of protein was from of was by in catalyzed by RNA as The was in containing and RNA After at protein was into and the was and with RNA was from the by of and then After at for at least the was by and the was The was in containing and the tRNA was using the to the of [5-3H]uridine was by for (27Huang L.X. Pookanjanatavip M. Gu X.G. Santi D.V. Biochemistry. 1998; 37: 344-351Crossref PubMed Scopus (132) Google Scholar). This was with tRNA to of specific activity for the Ψ synthase The for Ψ synthase activity was a modification of the release by Nurse (2Nurse K. Wrzesinski J. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 102-112PubMed Google Scholar), which the of from U in RNA is to A was containing and After for at was by of a small of a of a Ψ synthase to to and by into containing for at in a and the a of The was by in by The from these the and with the of the was with and to A of containing [5-3H]uridine was to a of Ψ synthase in containing After at of each was a which with the The was with 1 After the was into a with and then in a protein three data and the was used to the data and to the is the enzyme is the of tRNA to is the of tRNA to and is the for the The of this is the of tRNA is of the enzyme that the enzyme the enzyme was as described that the was A mutant Ψ synthase was and after of the was and as described To the of the wild-type Ψ synthase was after additional of the was as described The the and mutant TruB and and mutant of all four mutant enzymes to by over a of in the or of the mutant enzymes the wild-type enzymes. V. C. Grosjean H. Biochemistry. 1998; 37: PubMed Scopus Google Scholar) used to the ability of the wild-type and mutant Ψ synthases to bind substrate. A small of was with of and protein was to which to to A is in Fig. of by enzyme at of the which well with the release of only of the this of substrate was with wild-type TruB. Although the presence of RNA might this the of the for to the by of the in the for a on the of for Ψ synthase in no of on the not in the mutant Ψ synthases all bind tRNA substrate with The of wild-type TruB I) likely that the is with in the with substrate in The are the to from the enzyme at this with the [3H]tRNA, a significant of the mutant Ψ synthases be by of for determined by V. C. Grosjean H. Biochemistry. 1998; 37: PubMed Scopus Google for in a The Ψ synthase activity of each mutant was using the well established release (2Nurse K. Wrzesinski J. Bakin A. Lane B.G. Ofengand J. RNA. 1995; 1: 102-112PubMed Google Scholar). in Fig. the not catalyze the release of from the levels of the To the of catalytic the of mutant Ψ synthase in the was that of the wild-type enzyme in the the not from the of the in one in which the was with mutant enzyme for wild-type and mutant TruB and of of from was used to the After a or at the was by of in data have been at enzyme and tRNA and at different tRNA specific in a of from was used to the After a or at the was by of in data have been at enzyme and tRNA and at different tRNA specific The release to and uracil of a mutant enzyme with tRNA also be We or not the mutant enzymes release uracil at a rate to the rate of by the wild-type Ψ synthases. of of with mutant Ψ synthase was and wild-type Ψ synthase was to the which was In all the with wild-type enzyme in release of the of from as a with wild-type enzyme and substrate The mutant Ψ synthases described and to the of the sequence alignments (21Koonin E.V. Nucleic Acids Res. 1996; 24: 2411-2415Crossref PubMed Scopus (203) Google Scholar, C. Reid R. Greene P.J. Santi D.V. Nucleic Acids Res. 1996; 24: 3756-3762Crossref PubMed Scopus (120) Google Scholar) of the first identified Ψ synthases. The of to the data base for between proteins that do not display global similarity. A work on the can of but imperative to the functional or significance of aligned amino after many such studies can and develop that of as or To the of the aligned aspartic acid residue in the highly families of Ψ synthases in TruB and in each to alanine and was as a to all to the of The of was by a for the aspartic acid a that may functional of the of by the of the a critical for the aligned aspartic acid residues in TruB and from the four in TruA and (27Huang L.X. Pookanjanatavip M. Gu X.G. Santi D.V. Biochemistry. 1998; 37: 344-351Crossref PubMed Scopus (132) Google Scholar) the global dissimilarity among TruA, TruB, and The and of TruB and the and of therefore, and for Ψ synthase The that all four are inactive the significance of the one aspartic acid residue that is aligned among all of the known Ψ synthases and their the of aligned amino acids containing the critical aspartic acid residue also a in Ψ synthase for experiments to this of amino acids a a more in or further experiments are required to this of amino acids is of the Ψ synthases in the three aligned or not which a to the three for on four different protein be in this The of TruA L.X. Santi D.V. J. 1997; Scholar) and of a catalytically of the Ψ synthase D. M. Conrad J. T. Sun D.H. Wang L. Ofengand J. R. D. 1999; PubMed Scopus Google Scholar) have been and the of of of the Ψ synthases families or not they their statistically insignificant global similarity. The of the critical aspartic acid residues also the of experiments. The data Fig. that the mutant enzymes bind tRNA which a the not of the wild-type and mutant enzymes in the of the for secondary suggesting that either the perhaps, the degree of is by may be a of or or from in between the and the of the of the or the results that the aligned aspartic acid residue is, critical for Ψ synthase function. This work not the of the aligned aspartic acid which as a as a as a or as a The of aspartic acid with was in to or not the group for the The lack of activity by TruB and however, not to this for the of the of relative to that of aspartic acid may a for the mutant enzymes to a The of Koonin (21Koonin E.V. Nucleic Acids Res. 1996; 24: 2411-2415Crossref PubMed Scopus (203) Google Scholar) that the motif containing the aspartic acid residue a uridine sequence of the aspartic acid residue essential for the uracil the mutant enzymes might catalyze at a rate by but then their on a (27Huang L.X. Pookanjanatavip M. Gu X.G. Santi D.V. Biochemistry. 1998; 37: 344-351Crossref PubMed Scopus (132) Google Scholar) and release into The uracil be the the and no be detected (as was of the aligned aspartic a Ψ synthase into a To test this was with each of the described Ψ synthase The of by the wild-type enzyme was the for the of substrate present and was or not the substrate was with mutant enzymes can be therefore, that the mutant enzymes do not release into at a rate to in the wild-type enzymes. The that the aligned aspartic acid residue is required for the of substrate tRNA for of the uridine in the In this only with tRNA is detected by the The results however, clearly the that of the aligned aspartic acid residue the Ψ synthases into The results of this also the of Ψ synthase to dyskerin, the protein the of which to the human disease dyskeratosis for the similarity to TruB in the containing the critical aspartic acid residue is (19Luzzatto L. Karadimitris A. Nat. Genet. 1998; 19: 6-7Crossref PubMed Scopus (56) Google Scholar, N.S. Knight S.W. Vulliamy T.J. Klauck S.M. Wiemann S. Mason P.J. Poustka A. Dokal I. Nat. Genet. 1998; 19: 32-38Crossref PubMed Scopus (751) Google Scholar). (20Heiss N.S. Knight S.W. Vulliamy T.J. Klauck S.M. Wiemann S. Mason P.J. Poustka A. Dokal I. Nat. Genet. 1998; 19: 32-38Crossref PubMed Scopus (751) Google Scholar) that dyskerin is the human of the protein have as a Ψ synthase that small RNA on the of its localization in a protein and similarity to TruB in the of the critical aspartic acid residue A. G. P. M. R. RNA. 1998; 4: PubMed Scopus Google Scholar). (20Heiss N.S. Knight S.W. Vulliamy T.J. Klauck S.M. Wiemann S. Mason P.J. Poustka A. Dokal I. Nat. Genet. 1998; 19: 32-38Crossref PubMed Scopus (751) Google Scholar) and A. G. P. M. R. RNA. 1998; 4: PubMed Scopus Google Scholar) also similarity with TruB in of amino acids identified by Santi and co-workers (22Gustafsson C. Reid R. Greene P.J. Santi D.V. Nucleic Acids Res. 1996; 24: 3756-3762Crossref PubMed Scopus (120) Google Scholar) and by Koonin (21Koonin E.V. Nucleic Acids Res. 1996; 24: 2411-2415Crossref PubMed Scopus (203) Google Scholar), named motif The experiments support the of dyskerin and as Ψ synthases by that the motif containing the aligned aspartic acid residue has functional significance in two Ψ synthase families that represented by TruA, families that display no statistically significant global sequence similarity to each other. In fact, the presence of the amino acid motif may as a prerequisite for Ψ synthase activity to genes of function.