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Enzymatic characterization and mutational studies of TruD – the fifth family of pseudouridine synthases
Archives of Biochemistry and Biophysics 489 (2009) 15–19
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Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
Enzymatic characterization and mutational studies of TruD – the fifth family of pseudouridine synthases Chio Mui Chan a, Raven H. Huang a,b,* a b
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
a r t i c l e
i n f o
Article history: Received 25 June 2009 and in revised form 28 July 2009 Available online 5 August 2009 Keywords: RNA modification Pseudouridine Pseudouridine synthase Enzyme mechanism Site-directed mutagenesis
a b s t r a c t Pseudouridine (W) is formed through isomerization of uridine (U) catalyzed by a class of enzymes called pseudouridine synthases (WS). TruD is the fifth family of WS. Studies of the first four families (TruA, TruB, RsuA, and RluA) of WS reveal a conserved Asp and Tyr are critical for catalysis. However, in TruD family, the tyrosine is not conserved. In this study, we measured the enzymatic parameters for TruD in Escherichia coli, and carried out enzymatic assays for a series of single, double, and triple TruD mutants. Our studies indicate that a Glu, strictly conserved in only TruD family is likely to be the general base in TruD. We also proposed a possible distinct mechanism of TruD-catalyzed W formation compared to the first four families. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Pseudouridine (W),1 also called the fifth nucleoside, is one of the most abundant modified nucleosides in RNAs [1]. To date, W has been found in tRNAs, rRNAs, and snoRNAs, and it plays important roles for the proper biological functions of these RNAs [2–4]. W is formed through isomerization of the naturally occurring uridine (U) in RNA, catalyzed by WS. Four families of WS, named after the Escherichia coli enzymes TruA, TruB, RsuA, and RluA, were first discovered [5–8]. Sequence alignments of these four WS families identified a strictly conserved aspartic acid, which was shown to be essential for catalysis [9–11]. Subsequent structural studies confirmed the structural conservation of this conserved aspartic acid [12–15] (Fig. 1A). Structural studies, however, also revealed structural conservation of two additional amino acids, K or R and Y in the K/RxY motif (Fig. 1A). Crystal structures of TruB in complex with a stem-loop RNA from our lab as well as others indicated that the conserved K/R in the K/RxY motif interacted with the phosphate group in the targeted nucleotide [13,16,17]. Our subsequent biochemical and structural studies showed that, in addition to the structural role played by the hydrophobic phenyl ring of the conserved Y in the K/RxY motif, the OH group in the side chain
* Corresponding author. Address: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. Fax: +1 217 244 5858. E-mail address: [email protected] (R.H. Huang). 1 Abbreviations used: W, pseudouridine; U, uridine; WS, pseudouridine synthases; 2D TLC, two-dimensional thin layer chromatography; 5FhW, 5-fluoro-6-hydroxylpseudouridine; RRM, RNA Recognition Motif. 0003-9861/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2009.07.023
of Y is required for the last step of U-to-W conversion reaction: abstracting the proton attached to C5 of the targeted U base to complete isomerization [18]. Based upon these prior studies, the roles of the conserved amino acids K/R and Y in the K/RxY motif appeared to be well understood until the identification of the fifth family of WS, TruD. TruD was discovered to be the enzyme responsible for formation of W at position 13 of E. coli tRNAGlu [19]. This enzyme is found in other bacteria, archaea, and eukarya. Because of its highly divergent amino acid sequence when comparing to the previously identified four families of WS, it is classified as the fifth family. Soon after the discovery of TruD, three research groups reported the crystal structure of the enzyme [20–22]. The structure reveals that, despite no amino acid sequence homology to the other four families of enzymes, the catalytic domain of TruD has a similar overall folding as the first four families of WS. Our detailed structural analysis near the active site of TruD revealed a significant difference in TruD from the other four families of WS. Instead of the K/RxY motif in the other four families of WS, TruD contains an NxF motif (Fig. 1B). Furthermore, this NxF motif is strictly conserved within the TruD family, supported by amino acid sequence alignments of TruD with 58 other TruD homologs from bacteria, archaea, and eukarya [19]. The existence of an NxF motif in TruD, instead of the K/RxY in the other four families of WS, presents a dilemma to understand how TruD carries out its enzymatic reaction. Based on our previous study on TruB, Y in the K/RxY motif is absolutely required for catalysis with a OH group [18]. However, F in the NxF motif of TruD is not able to abstract a proton due to its lack of a OH group, another
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C.M. Chan, R.H. Huang / Archives of Biochemistry and Biophysics 489 (2009) 15–19
Fig. 1. Structural comparison of TruD to the other four families of WS. (A) Superposition of representative structures from the first four families of WS shows conservation of residues in the active site. Only the side chains of the conserved residues in the active site are shown in stick, with TruA, TruB, RluA, and RsuA colored red, blue, cyan and yellow, respectively. The structure of 5FhW from the crystal structure of TruB–RNA complex (PDB ID: 1K8W) is shown in green to highlight the relative positions of the conserved residues in WS to the targeted nucleotide. The residues in thin stick (not labeled) are conserved hydrophobic residues (L, I, V, and F) for structural roles near the active site of enzymes. (B) Superposition of the structures of TruB–RNA complex (blue and green) with TruD (magenta). Also shown in the figure is a strictly conserved glutamate unique to TruD. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
amino acid(s) near the active site of TruD must be responsible for the proton abstraction. We report here that a strictly conserved glutamate near the active site in TruD is likely to act as a general base for the proton abstraction at C5 of the U base, playing the role previously filled by the conserved Y in the K/RxY motif in the other four families of WS. Thus, the mechanism of a TruD-catalyzed reaction appears to be different from the first four families of WS.
viously [27]. The radioactivity of the radiolabeled UMP and WMP were quantified using a PhosphorImager system (Molecular Dynamics). Selected 2D TLC analyses are shown in Fig. 2. For the kinetic parameters of the wild-type enzyme, we have carried out the time-dependent reactions with the concentration of TruD constant but varying the substrate concentration. Km and kcat were derived using the Lineweaver–Burk plot based on 2D TLC data.
Materials and methods
Results and discussion
Materials
Kinetic characterization of TruD
The TruD plasmid was a gift from Dr. Ferre-D’Amare (Fred Hutchinson Cancer Research Center, Seattle). The E. coli strain BL21(DE3) transformed with pET15b-TruD (with N-terminal Histagged) was grown at 37 °C to an optical density of 0.6 and induced with 0.5 mM IPTG for 3 h at 37 °C. Cells were lysed in Buffer A (20 mM Tris–HCl, pH 7.6, 50 mM NaCl) and cell lysate was centrifuged. The resulting supernatant was filtered and applied to a HISSELECTTM nickel affinity gel (Sigma). The resin-bound protein was washed with 25 mM imidazole in Buffer A and then eluted with 200 mM imidazole in Buffer A. The protein was purified more than 98% pure. Plasmids containing TruD mutants were obtained by the QuickChange method. Purification of the TruD mutants was the same as that of the wild-type enzyme described above. E. coli tRNAGlu was prepared through in vitro transcription by T7 polymerase analogous to other tRNAs prepared in our lab [18,23]. RNase T2 was purified based on a published protocol [24].
We first determined kinetic constants for TruD, which have not been reported previously. Wild-type TruD has a Km and kcat of 380 nM and 0.058 min1, respectively (Table 1). The other WS whose kinetic data have been reported, are TruB and RluA. TruB has Km and kcat of 146 nM and 7.2 min1, respectively [28]. RluA has Km and kcat of 108 nM and 5.9 min1, respectively [28]. While the Km of TruD is roughly in the same range of TruB and RluA, the kcat is much smaller. Thus, compared to TruB and RluA, TruD is roughly a 100-fold slower enzyme, possibly due to the fact that U13 in tRNA is less accessible compared to other modification targets, such as the TWC loop modified by TruB. Alternatively, TruD may be a less efficient enzyme compared to the other four families of WS due to its possibly distinct reaction mechanism, as discussed below.
Analysis of pseudouridine formation by two-dimensional thin layer chromatography (2D TLC)
Based on their differences from other four families of WS (Fig.1) as well as their strict conservation within the TruD family, we have chosen five strictly conserved amino acids for mutational studies in TruD: E31, K79, Q87, N129, and F131. As indicated in Table 1 and Fig. 3, the enzymatic effect of a single mutation in TruD varies, ranging from no enzymatic activity to maintaining most of the activity of the wild-type enzyme. Our mutational studies indicate that E31 is likely the general base instead of the conserved Y in the K/RxY motif in the other four families of WS. First, a conservative E31Q mutation, which maintains the same length of the side chain but changes the functional group of the side chain from a carboxyl group to an amide, resulted in a total loss of enzymatic activity (Table 1 and Fig. 3A). On the other hand, the E31D mutant, which maintains the functional group of the side chain but reduces the length of the side chain, still retains 30% activity of the wild-type enzyme (Table 1 and
A method combining 32P-AMP-radiolabeled tRNAGlu and RNase T2 digestion, followed by 2D TLC, was employed for the enzymatic assay of TruD-catalyzed reaction [25–27]. A reaction mixture in 50 ll scale containing 20 mM Tris–HCl (pH 7.6), 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 2 mM DTT, 0.1 mg/ml BSA, 0.5 lM wildtype TruD or mutants (0.025 nmol), 0.3–0.9 lM [32P]AMP-radiolabeled tRNAGlu substrate, was incubated at 37 °C for 0, 10, 20, and 40 min. The tRNA was recovered through phenol extraction and ethanol precipitation and the purified tRNA was suspended in 2.5 ll of 50 mM ammonium acetate (pH 4.5) and digested with RNase T2 overnight at room temperature. After digestion, the 30 -nucleotide monophosphates were analyzed by 2D TLC using PEI Cellulose F TLC plastic sheets (EM Science) used a solvent system reported pre-
Effect of single conserved amino acid mutation on the enzymatic activity and a likely mechanism of TruD-catalyzed W formation
C.M. Chan, R.H. Huang / Archives of Biochemistry and Biophysics 489 (2009) 15–19
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Fig. 2. Representatives of 2D TLC analysis of the enzymatic reactions carried out by the wild-type (WT) and mutated TruD.
Fig. 3A). This indicates that the carboxyl group in the side chain of E31 is critical for enzymatic activity. On the other hand, the length of the side chain is less important. Since no crystal structure of TruD in complex with a tRNA substrate is available, we modeled 5-fluoro-6-hydroxylpseudouridine (5FhW) from the structure of TruB–RNA complex into the crystal structure of TruD (Fig. 1B). In this model, the side chain of E31 is 6 Å away from the F atom attached to the C5 of 5FhW (presumably the position of the proton in a real tRNA substrate). Although the distance is too long for the side chain of E31 to be involved in catalysis, it can be shortened with a different conformation of E31 side chain when TruD binds the tRNA substrate. For example, the side chain of E31 can be 4 Å away from the F atom if another rotamer was chosen using program O [29]. We conclude that E31 may function as the general base, abstracting the proton attached to C5 of the targeted nucleotide to complete the reaction of U-to-W conversion. Mutations of other conserved amino acids in the active site were less severe, while N129 mutation abolishes the enzymatic activity (Table 1). The conservative K79R mutation reduces the Table 1 Kinetic parameters of the wild-type TruD and the relative activities of the TruD mutants. Kinetic parameters Km kcat kcat/Km
0.38 (lM) 0.058 (min1) 1.5 105 (min1 M1)
Relative activity (%) Wild-type Single mutant E31Q E31D K79R K79L Q87E N129K F131Y Double mutant E31Q/F131Y K79L/F131Y K79L/N129K Triple mutant K79L/N129K/F131Y
100 0 27 10 5 29 0 68 0 0 0 0
Fig. 3. Time course of enzymatic reactions carried out by the wild-type and mutated TruD. The data is presented in two separate panels to show clarity. (A) The wild-type enzyme (j), the F131Y mutant (h), the E31D mutant ( ), the E31Q mutant (d), and the E31Q/F131Y mutant ( ). (B) The wild-type enzyme (j), the Q87E mutant (), the K79R mutant (4), the K79L mutant (d), the N129K mutant ( ), and the K79L/N129K mutant (}).
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enzymatic activity by 10-fold compared to the wild-type enzyme (Table 1 and Fig. 3B). Further mutation of K79L only resulted in two additional fold loss of activity (Table 1 and Fig. 3B), indicating that K79 is important, but not essential. Although the current structure of TruD does not show hydrogen bonding between E31 and Q87, an alternative conformation of E31 may bring these two in a hydrogen bonding distance. The conservative mutation of Q87E resulted in the loss of 70% enzymatic activity (Table 1 and Fig. 3B), indicating its important role in catalysis. We have also created single mutants within the NxF motif. Because a side chain of an asparagine was previously shown to interact with the phosphate backbone in structures of other protein–nucleic acid complexes, we expected N129 in the NxF motif of TruD to play a similar role. Surprisingly, the N129K mutation resulted in an inactive enzyme (Table 1 and Fig. 3B). On the other hand, F131Y mutation is the least severe one among all mutants, retaining 68% of the wildtype enzymatic activity (Table 1 and Fig. 3A). Assuming the overall requirement for the reaction catalyzed by TruD is the same as other four families of WS, a general base must be present in TruD in order to complete the reaction. Our mutational studies indicated that E31 is the best candidate as the general base for the abstraction of the proton attached to C5 to complete U-to-W conversion. Thus, a likely mechanism of the TruD-catalyzed reaction is shown in Fig. 4. Nucleophilic attack by the strictly conserved D80 on the C10 of the ribose detaches the uracil base from the ribose (Fig. 4, Step 1). Rotation of the detached base and reattachment of the rotated base results in formation of the C5–C10 bond between the base and the ribose (Fig. 4, Steps 2 and 3). The side chain of the strictly conserved E31 abstracts the proton attached to C5 of the base to complete the reaction (Fig. 4, Step 4). However, this is a tentative model based on the data presented here, and further experiments are required to verify it. We have also considered possibilities of other conserved amino acids near the active site to serve as the general base, but we have ruled out those possibilities based on our data and the following logics. Our structural analysis of TruD with 5FhW modeled indicated that only four amino acids contain functional groups in their side chains within 8 Å distance from F atom in 5FhW: E31, K79, T85, and N129. Therefore, besides E31, K79 is another potential candidate for the general base (to the best of our knowledge, it is
unlikely that an Asparagine or a Threonine acts as a general base in catalysis of an enzymatic reaction). However, both K79R and K79L mutants are still enzymatically active (Table 1 and Fig. 3). If K79 was the general base, these mutations would have been expected to abolish the enzymatic activity. Attempts to engineer an active TruD mutant sharing the same reaction mechanism as the other four families of WS The fact that TruD has a similar overall folding of the WS domain made us wonder if it is possible to create an active TruD mutant, which has the same conserved amino acids in the active site as in other four families of WS. Therefore, we have created TruD double and triple mutants to test this hypothesis. We have created three double mutants. The first double mutant, E31Q/F131Y, was created to eliminate the general base of E31 and to introduce an OH group in F131 to test whether introduction of an OH group in F131 might rescue the enzymatic activity lost by E31Q mutation. The second double mutant, K79L/F131Y, was created to test whether K79 might affect the possible rescuing effect of F131Y mutation. The third double mutant, K79L/N129K, was created to test whether the essential mutation of N129K might have resulted from the possibly steric clash of K79 with the mutated K129. All of these mutants were enzymatically inactive (Table 1). Next, we created a triple mutant, K79L/N129K/F131Y, resulting in an enzyme with identical amino acids surrounding the two catalytic amino acids as in the other four families of WS: the LD motif and the K/RxY motif. The result of this triple mutation was still an inactive enzyme. Although the catalytic domain of TruD has the same overall fold as the other four families of WS, the sequential arrangement of the secondary structures in TruD is different. The overall folding of the core WS domain in all five families of WS can be depicted as two RNA Recognition Motif (RRM) stitched by a central b strand. While the C-terminus of the WS core domain provides the central b strand in the previous four families of WS, the central b strand in TruD comes from the N-terminus of the core domain. Therefore, the structure of the WS core domain of TruD is a circular permutation of a conserved folding found in the other four families of WS, a term first employed by Ferre-D’Amare and coworker [20]. Our fail-
Fig. 4. Proposed mechanism of W formation by TruD. As in the case of the other four families of WS, the conserved aspartic acid is likely to be the nucleophile for the detachment of the base. The conserved glutamate in TruD is proposed here to be the likely general base for the abstraction of the proton attached to C5 of the base in the final step of the reaction to form the product W.
C.M. Chan, R.H. Huang / Archives of Biochemistry and Biophysics 489 (2009) 15–19
ure to convert TruD into an enzyme that uses the same mechanism employed by the other four families of WS may indicate involvement of additional conserved amino acids near or beyond the active site of TruD in catalysis. Acknowledgments We thank Dr. Ferre-D’Amare (Fred Hutchinson Cancer Research Center, Seattle) for providing TruD expression plasmid and S. Kelley for critical reading of the manuscript. References [1] J. Rozenski, P.F. Crain, J.A. McCloskey, Nucleic Acids Res. 27 (1999) 196–197. [2] C.S. Yarian, M.M. Basti, R.J. Cain, G. Ansari, R.H. Guenther, E. Sochacka, G. Czerwinska, A. Malkiewicz, P.F. Agris, Nucleic Acids Res. 27 (1999) 3543–3549. [3] T.H. King, B. Liu, R.R. McCully, M.J. Fournier, Mol. Cell 11 (2003) 425–435. [4] Y.T. Yu, M.D. Shu, J.A. Steitz, EMBO J. 17 (1998) 5783–5795. [5] H.O. Kammen, C.C. Marvel, L. Hardy, E.E. Penhoet, J. Biol. Chem. 263 (1988) 2255–2263. [6] K. Nurse, J. Wrzesinski, A. Bakin, B.G. Lane, J. Ofengand, RNA 1 (1995) 102–112. [7] J. Wrzesinski, A. Bakin, K. Nurse, B.G. Lane, J. Ofengand, Biochemistry 34 (1995) 8904–8913. [8] J. Wrzesinski, K. Nurse, A. Bakin, B.G. Lane, J. Ofengand, RNA 1 (1995) 437–448.
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[9] E.V. Koonin, Nucleic Acids Res. 24 (1996) 2411–2415. [10] L. Huang, M. Pookanjanatavip, X. Gu, D.V. Santi, Biochemistry 37 (1998) 344– 351. [11] X. Gu, Y. Liu, D.V. Santi, Proc. Natl. Acad. Sci. USA 96 (1999) 14270–14275. [12] P.G. Foster, L. Huang, D.V. Santi, R.M. Stroud, Nat. Struct. Biol. 7 (2000) 23–27. [13] C. Hoang, A.R. Ferre-D’Amare, Cell 107 (2001) 929–939. [14] J. Sivaraman, V. Sauve, R. Larocque, E.A. Stura, J.D. Schrag, M. Cygler, A. Matte, Nat. Struct. Biol. 9 (2002) 353–358. [15] J. Sivaraman, P. Iannuzzi, M. Cygler, A. Matte, J. Mol. Biol. 335 (2004) 87–101. [16] H. Pan, S. Agarwalla, D.T. Moustakas, J. Finer-Moore, R.M. Stroud, Proc. Natl. Acad. Sci. USA 100 (2003) 12648–12653. [17] K. Phannachet, R.H. Huang, Nucleic Acids Res. 32 (2004) 1422–1429. [18] K. Phannachet, Y. Elias, R.H. Huang, Biochemistry 44 (2005) 15488–15494. [19] Y. Kaya, J. Ofengand, RNA 9 (2003) 711–721. [20] C. Hoang, A.R. Ferre-D’Amare, RNA 10 (2004) 1026–1033. [21] Y. Kaya, M. Del Campo, J. Ofengand, A. Malhotra, J. Biol. Chem. 279 (2004) 18107–18110. [22] U.B. Ericsson, P. Nordlund, B.M. Hallberg, FEBS Lett. 565 (2004) 59–64. [23] C. Zhou, R.H. Huang, Proc. Natl. Acad. Sci. USA 105 (2008) 16142–16147. [24] M. Hiramaru, T. Uchida, F. Egami, Anal. Biochem. 17 (1966) 135–142. [25] Y. Kuchino, N. Hanyu, S. Nishimura, Methods Enzymol. 155 (1987) 379–396. [26] G. Keith, Biochimie 77 (1995) 142–144. [27] H. Grosjean, G. Keith, L. Droogmans, Methods Mol. Biol. 265 (2004) 357–391. [28] V. Ramamurthy, S.L. Swann, C.J. Spedaliere, E.G. Mueller, Biochemistry 38 (1999) 13106–13111. [29] T.A. Jones, J.-Y. Zou, S.W. Cowan, M. Kjeldgaard, Acta Crystallogr. A 47 (1991) 110–119.
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Enzymatic characterization and mutational studies of TruD – the fifth family of pseudouridine synthases Chio Mui Chan a, Raven H. Huang a,b,* a b
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
a r t i c l e
i n f o
Article history: Received 25 June 2009 and in revised form 28 July 2009 Available online 5 August 2009 Keywords: RNA modification Pseudouridine Pseudouridine synthase Enzyme mechanism Site-directed mutagenesis
a b s t r a c t Pseudouridine (W) is formed through isomerization of uridine (U) catalyzed by a class of enzymes called pseudouridine synthases (WS). TruD is the fifth family of WS. Studies of the first four families (TruA, TruB, RsuA, and RluA) of WS reveal a conserved Asp and Tyr are critical for catalysis. However, in TruD family, the tyrosine is not conserved. In this study, we measured the enzymatic parameters for TruD in Escherichia coli, and carried out enzymatic assays for a series of single, double, and triple TruD mutants. Our studies indicate that a Glu, strictly conserved in only TruD family is likely to be the general base in TruD. We also proposed a possible distinct mechanism of TruD-catalyzed W formation compared to the first four families. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Pseudouridine (W),1 also called the fifth nucleoside, is one of the most abundant modified nucleosides in RNAs [1]. To date, W has been found in tRNAs, rRNAs, and snoRNAs, and it plays important roles for the proper biological functions of these RNAs [2–4]. W is formed through isomerization of the naturally occurring uridine (U) in RNA, catalyzed by WS. Four families of WS, named after the Escherichia coli enzymes TruA, TruB, RsuA, and RluA, were first discovered [5–8]. Sequence alignments of these four WS families identified a strictly conserved aspartic acid, which was shown to be essential for catalysis [9–11]. Subsequent structural studies confirmed the structural conservation of this conserved aspartic acid [12–15] (Fig. 1A). Structural studies, however, also revealed structural conservation of two additional amino acids, K or R and Y in the K/RxY motif (Fig. 1A). Crystal structures of TruB in complex with a stem-loop RNA from our lab as well as others indicated that the conserved K/R in the K/RxY motif interacted with the phosphate group in the targeted nucleotide [13,16,17]. Our subsequent biochemical and structural studies showed that, in addition to the structural role played by the hydrophobic phenyl ring of the conserved Y in the K/RxY motif, the OH group in the side chain
* Corresponding author. Address: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. Fax: +1 217 244 5858. E-mail address: [email protected] (R.H. Huang). 1 Abbreviations used: W, pseudouridine; U, uridine; WS, pseudouridine synthases; 2D TLC, two-dimensional thin layer chromatography; 5FhW, 5-fluoro-6-hydroxylpseudouridine; RRM, RNA Recognition Motif. 0003-9861/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2009.07.023
of Y is required for the last step of U-to-W conversion reaction: abstracting the proton attached to C5 of the targeted U base to complete isomerization [18]. Based upon these prior studies, the roles of the conserved amino acids K/R and Y in the K/RxY motif appeared to be well understood until the identification of the fifth family of WS, TruD. TruD was discovered to be the enzyme responsible for formation of W at position 13 of E. coli tRNAGlu [19]. This enzyme is found in other bacteria, archaea, and eukarya. Because of its highly divergent amino acid sequence when comparing to the previously identified four families of WS, it is classified as the fifth family. Soon after the discovery of TruD, three research groups reported the crystal structure of the enzyme [20–22]. The structure reveals that, despite no amino acid sequence homology to the other four families of enzymes, the catalytic domain of TruD has a similar overall folding as the first four families of WS. Our detailed structural analysis near the active site of TruD revealed a significant difference in TruD from the other four families of WS. Instead of the K/RxY motif in the other four families of WS, TruD contains an NxF motif (Fig. 1B). Furthermore, this NxF motif is strictly conserved within the TruD family, supported by amino acid sequence alignments of TruD with 58 other TruD homologs from bacteria, archaea, and eukarya [19]. The existence of an NxF motif in TruD, instead of the K/RxY in the other four families of WS, presents a dilemma to understand how TruD carries out its enzymatic reaction. Based on our previous study on TruB, Y in the K/RxY motif is absolutely required for catalysis with a OH group [18]. However, F in the NxF motif of TruD is not able to abstract a proton due to its lack of a OH group, another
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C.M. Chan, R.H. Huang / Archives of Biochemistry and Biophysics 489 (2009) 15–19
Fig. 1. Structural comparison of TruD to the other four families of WS. (A) Superposition of representative structures from the first four families of WS shows conservation of residues in the active site. Only the side chains of the conserved residues in the active site are shown in stick, with TruA, TruB, RluA, and RsuA colored red, blue, cyan and yellow, respectively. The structure of 5FhW from the crystal structure of TruB–RNA complex (PDB ID: 1K8W) is shown in green to highlight the relative positions of the conserved residues in WS to the targeted nucleotide. The residues in thin stick (not labeled) are conserved hydrophobic residues (L, I, V, and F) for structural roles near the active site of enzymes. (B) Superposition of the structures of TruB–RNA complex (blue and green) with TruD (magenta). Also shown in the figure is a strictly conserved glutamate unique to TruD. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
amino acid(s) near the active site of TruD must be responsible for the proton abstraction. We report here that a strictly conserved glutamate near the active site in TruD is likely to act as a general base for the proton abstraction at C5 of the U base, playing the role previously filled by the conserved Y in the K/RxY motif in the other four families of WS. Thus, the mechanism of a TruD-catalyzed reaction appears to be different from the first four families of WS.
viously [27]. The radioactivity of the radiolabeled UMP and WMP were quantified using a PhosphorImager system (Molecular Dynamics). Selected 2D TLC analyses are shown in Fig. 2. For the kinetic parameters of the wild-type enzyme, we have carried out the time-dependent reactions with the concentration of TruD constant but varying the substrate concentration. Km and kcat were derived using the Lineweaver–Burk plot based on 2D TLC data.
Materials and methods
Results and discussion
Materials
Kinetic characterization of TruD
The TruD plasmid was a gift from Dr. Ferre-D’Amare (Fred Hutchinson Cancer Research Center, Seattle). The E. coli strain BL21(DE3) transformed with pET15b-TruD (with N-terminal Histagged) was grown at 37 °C to an optical density of 0.6 and induced with 0.5 mM IPTG for 3 h at 37 °C. Cells were lysed in Buffer A (20 mM Tris–HCl, pH 7.6, 50 mM NaCl) and cell lysate was centrifuged. The resulting supernatant was filtered and applied to a HISSELECTTM nickel affinity gel (Sigma). The resin-bound protein was washed with 25 mM imidazole in Buffer A and then eluted with 200 mM imidazole in Buffer A. The protein was purified more than 98% pure. Plasmids containing TruD mutants were obtained by the QuickChange method. Purification of the TruD mutants was the same as that of the wild-type enzyme described above. E. coli tRNAGlu was prepared through in vitro transcription by T7 polymerase analogous to other tRNAs prepared in our lab [18,23]. RNase T2 was purified based on a published protocol [24].
We first determined kinetic constants for TruD, which have not been reported previously. Wild-type TruD has a Km and kcat of 380 nM and 0.058 min1, respectively (Table 1). The other WS whose kinetic data have been reported, are TruB and RluA. TruB has Km and kcat of 146 nM and 7.2 min1, respectively [28]. RluA has Km and kcat of 108 nM and 5.9 min1, respectively [28]. While the Km of TruD is roughly in the same range of TruB and RluA, the kcat is much smaller. Thus, compared to TruB and RluA, TruD is roughly a 100-fold slower enzyme, possibly due to the fact that U13 in tRNA is less accessible compared to other modification targets, such as the TWC loop modified by TruB. Alternatively, TruD may be a less efficient enzyme compared to the other four families of WS due to its possibly distinct reaction mechanism, as discussed below.
Analysis of pseudouridine formation by two-dimensional thin layer chromatography (2D TLC)
Based on their differences from other four families of WS (Fig.1) as well as their strict conservation within the TruD family, we have chosen five strictly conserved amino acids for mutational studies in TruD: E31, K79, Q87, N129, and F131. As indicated in Table 1 and Fig. 3, the enzymatic effect of a single mutation in TruD varies, ranging from no enzymatic activity to maintaining most of the activity of the wild-type enzyme. Our mutational studies indicate that E31 is likely the general base instead of the conserved Y in the K/RxY motif in the other four families of WS. First, a conservative E31Q mutation, which maintains the same length of the side chain but changes the functional group of the side chain from a carboxyl group to an amide, resulted in a total loss of enzymatic activity (Table 1 and Fig. 3A). On the other hand, the E31D mutant, which maintains the functional group of the side chain but reduces the length of the side chain, still retains 30% activity of the wild-type enzyme (Table 1 and
A method combining 32P-AMP-radiolabeled tRNAGlu and RNase T2 digestion, followed by 2D TLC, was employed for the enzymatic assay of TruD-catalyzed reaction [25–27]. A reaction mixture in 50 ll scale containing 20 mM Tris–HCl (pH 7.6), 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 2 mM DTT, 0.1 mg/ml BSA, 0.5 lM wildtype TruD or mutants (0.025 nmol), 0.3–0.9 lM [32P]AMP-radiolabeled tRNAGlu substrate, was incubated at 37 °C for 0, 10, 20, and 40 min. The tRNA was recovered through phenol extraction and ethanol precipitation and the purified tRNA was suspended in 2.5 ll of 50 mM ammonium acetate (pH 4.5) and digested with RNase T2 overnight at room temperature. After digestion, the 30 -nucleotide monophosphates were analyzed by 2D TLC using PEI Cellulose F TLC plastic sheets (EM Science) used a solvent system reported pre-
Effect of single conserved amino acid mutation on the enzymatic activity and a likely mechanism of TruD-catalyzed W formation
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Fig. 2. Representatives of 2D TLC analysis of the enzymatic reactions carried out by the wild-type (WT) and mutated TruD.
Fig. 3A). This indicates that the carboxyl group in the side chain of E31 is critical for enzymatic activity. On the other hand, the length of the side chain is less important. Since no crystal structure of TruD in complex with a tRNA substrate is available, we modeled 5-fluoro-6-hydroxylpseudouridine (5FhW) from the structure of TruB–RNA complex into the crystal structure of TruD (Fig. 1B). In this model, the side chain of E31 is 6 Å away from the F atom attached to the C5 of 5FhW (presumably the position of the proton in a real tRNA substrate). Although the distance is too long for the side chain of E31 to be involved in catalysis, it can be shortened with a different conformation of E31 side chain when TruD binds the tRNA substrate. For example, the side chain of E31 can be 4 Å away from the F atom if another rotamer was chosen using program O [29]. We conclude that E31 may function as the general base, abstracting the proton attached to C5 of the targeted nucleotide to complete the reaction of U-to-W conversion. Mutations of other conserved amino acids in the active site were less severe, while N129 mutation abolishes the enzymatic activity (Table 1). The conservative K79R mutation reduces the Table 1 Kinetic parameters of the wild-type TruD and the relative activities of the TruD mutants. Kinetic parameters Km kcat kcat/Km
0.38 (lM) 0.058 (min1) 1.5 105 (min1 M1)
Relative activity (%) Wild-type Single mutant E31Q E31D K79R K79L Q87E N129K F131Y Double mutant E31Q/F131Y K79L/F131Y K79L/N129K Triple mutant K79L/N129K/F131Y
100 0 27 10 5 29 0 68 0 0 0 0
Fig. 3. Time course of enzymatic reactions carried out by the wild-type and mutated TruD. The data is presented in two separate panels to show clarity. (A) The wild-type enzyme (j), the F131Y mutant (h), the E31D mutant ( ), the E31Q mutant (d), and the E31Q/F131Y mutant ( ). (B) The wild-type enzyme (j), the Q87E mutant (), the K79R mutant (4), the K79L mutant (d), the N129K mutant ( ), and the K79L/N129K mutant (}).
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enzymatic activity by 10-fold compared to the wild-type enzyme (Table 1 and Fig. 3B). Further mutation of K79L only resulted in two additional fold loss of activity (Table 1 and Fig. 3B), indicating that K79 is important, but not essential. Although the current structure of TruD does not show hydrogen bonding between E31 and Q87, an alternative conformation of E31 may bring these two in a hydrogen bonding distance. The conservative mutation of Q87E resulted in the loss of 70% enzymatic activity (Table 1 and Fig. 3B), indicating its important role in catalysis. We have also created single mutants within the NxF motif. Because a side chain of an asparagine was previously shown to interact with the phosphate backbone in structures of other protein–nucleic acid complexes, we expected N129 in the NxF motif of TruD to play a similar role. Surprisingly, the N129K mutation resulted in an inactive enzyme (Table 1 and Fig. 3B). On the other hand, F131Y mutation is the least severe one among all mutants, retaining 68% of the wildtype enzymatic activity (Table 1 and Fig. 3A). Assuming the overall requirement for the reaction catalyzed by TruD is the same as other four families of WS, a general base must be present in TruD in order to complete the reaction. Our mutational studies indicated that E31 is the best candidate as the general base for the abstraction of the proton attached to C5 to complete U-to-W conversion. Thus, a likely mechanism of the TruD-catalyzed reaction is shown in Fig. 4. Nucleophilic attack by the strictly conserved D80 on the C10 of the ribose detaches the uracil base from the ribose (Fig. 4, Step 1). Rotation of the detached base and reattachment of the rotated base results in formation of the C5–C10 bond between the base and the ribose (Fig. 4, Steps 2 and 3). The side chain of the strictly conserved E31 abstracts the proton attached to C5 of the base to complete the reaction (Fig. 4, Step 4). However, this is a tentative model based on the data presented here, and further experiments are required to verify it. We have also considered possibilities of other conserved amino acids near the active site to serve as the general base, but we have ruled out those possibilities based on our data and the following logics. Our structural analysis of TruD with 5FhW modeled indicated that only four amino acids contain functional groups in their side chains within 8 Å distance from F atom in 5FhW: E31, K79, T85, and N129. Therefore, besides E31, K79 is another potential candidate for the general base (to the best of our knowledge, it is
unlikely that an Asparagine or a Threonine acts as a general base in catalysis of an enzymatic reaction). However, both K79R and K79L mutants are still enzymatically active (Table 1 and Fig. 3). If K79 was the general base, these mutations would have been expected to abolish the enzymatic activity. Attempts to engineer an active TruD mutant sharing the same reaction mechanism as the other four families of WS The fact that TruD has a similar overall folding of the WS domain made us wonder if it is possible to create an active TruD mutant, which has the same conserved amino acids in the active site as in other four families of WS. Therefore, we have created TruD double and triple mutants to test this hypothesis. We have created three double mutants. The first double mutant, E31Q/F131Y, was created to eliminate the general base of E31 and to introduce an OH group in F131 to test whether introduction of an OH group in F131 might rescue the enzymatic activity lost by E31Q mutation. The second double mutant, K79L/F131Y, was created to test whether K79 might affect the possible rescuing effect of F131Y mutation. The third double mutant, K79L/N129K, was created to test whether the essential mutation of N129K might have resulted from the possibly steric clash of K79 with the mutated K129. All of these mutants were enzymatically inactive (Table 1). Next, we created a triple mutant, K79L/N129K/F131Y, resulting in an enzyme with identical amino acids surrounding the two catalytic amino acids as in the other four families of WS: the LD motif and the K/RxY motif. The result of this triple mutation was still an inactive enzyme. Although the catalytic domain of TruD has the same overall fold as the other four families of WS, the sequential arrangement of the secondary structures in TruD is different. The overall folding of the core WS domain in all five families of WS can be depicted as two RNA Recognition Motif (RRM) stitched by a central b strand. While the C-terminus of the WS core domain provides the central b strand in the previous four families of WS, the central b strand in TruD comes from the N-terminus of the core domain. Therefore, the structure of the WS core domain of TruD is a circular permutation of a conserved folding found in the other four families of WS, a term first employed by Ferre-D’Amare and coworker [20]. Our fail-
Fig. 4. Proposed mechanism of W formation by TruD. As in the case of the other four families of WS, the conserved aspartic acid is likely to be the nucleophile for the detachment of the base. The conserved glutamate in TruD is proposed here to be the likely general base for the abstraction of the proton attached to C5 of the base in the final step of the reaction to form the product W.
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ure to convert TruD into an enzyme that uses the same mechanism employed by the other four families of WS may indicate involvement of additional conserved amino acids near or beyond the active site of TruD in catalysis. Acknowledgments We thank Dr. Ferre-D’Amare (Fred Hutchinson Cancer Research Center, Seattle) for providing TruD expression plasmid and S. Kelley for critical reading of the manuscript. References [1] J. Rozenski, P.F. Crain, J.A. McCloskey, Nucleic Acids Res. 27 (1999) 196–197. [2] C.S. Yarian, M.M. Basti, R.J. Cain, G. Ansari, R.H. Guenther, E. Sochacka, G. Czerwinska, A. Malkiewicz, P.F. Agris, Nucleic Acids Res. 27 (1999) 3543–3549. [3] T.H. King, B. Liu, R.R. McCully, M.J. Fournier, Mol. Cell 11 (2003) 425–435. [4] Y.T. Yu, M.D. Shu, J.A. Steitz, EMBO J. 17 (1998) 5783–5795. [5] H.O. Kammen, C.C. Marvel, L. Hardy, E.E. Penhoet, J. Biol. Chem. 263 (1988) 2255–2263. [6] K. Nurse, J. Wrzesinski, A. Bakin, B.G. Lane, J. Ofengand, RNA 1 (1995) 102–112. [7] J. Wrzesinski, A. Bakin, K. Nurse, B.G. Lane, J. Ofengand, Biochemistry 34 (1995) 8904–8913. [8] J. Wrzesinski, K. Nurse, A. Bakin, B.G. Lane, J. Ofengand, RNA 1 (1995) 437–448.
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