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• W2006703609 abstract "Background:Although temperature-sensitive (ts) mutations of TrmD exist, deficient in converting G37- to m1G37-tRNA, the basis of their phenotype is unknown.Results: The ts-S88L mutation, while conferring thermal lability, caused a stronger defect on catalysis.Conclusion: The catalytic defect of the ts-S88L mutation reduced the quantity and quality of tRNA methylation.Significance: ts mutations leading to catalytic defects are useful for studying enzyme mechanism.Conditional temperature-sensitive (ts) mutations are important reagents to study essential genes. Although it is commonly assumed that the ts phenotype of a specific mutation arises from thermal denaturation of the mutant enzyme, the possibility also exists that the mutation decreases the enzyme activity to a certain level at the permissive temperature and aggravates the negative effect further upon temperature upshifts. Resolving these possibilities is important for exploiting the ts mutation for studying the essential gene. The trmD gene is essential for growth in bacteria, encoding the enzyme for converting G37 to m1G37 on the 3′ side of the tRNA anticodon. This conversion involves methyl transfer from S-adenosyl methionine and is critical to minimize tRNA frameshift errors on the ribosome. Using the ts-S88L mutation of Escherichia coli trmD as an example, we show that although the mutation confers thermal lability to the enzyme, the effect is relatively minor. In contrast, the mutation decreases the catalytic efficiency of the enzyme to 1% at the permissive temperature, and at the nonpermissive temperature, it renders further deterioration of activity to 0.1%. These changes are accompanied by losses of both the quantity and quality of tRNA methylation, leading to the potential of cellular pleiotropic effects. This work illustrates the principle that the ts phenotype of an essential gene mutation can be closely linked to the catalytic defect of the gene product and that such a mutation can provide a useful tool to study the mechanism of catalytic inactivation. Although temperature-sensitive (ts) mutations of TrmD exist, deficient in converting G37- to m1G37-tRNA, the basis of their phenotype is unknown. Results: The ts-S88L mutation, while conferring thermal lability, caused a stronger defect on catalysis. Conclusion: The catalytic defect of the ts-S88L mutation reduced the quantity and quality of tRNA methylation. Significance: ts mutations leading to catalytic defects are useful for studying enzyme mechanism. Conditional temperature-sensitive (ts) mutations are important reagents to study essential genes. Although it is commonly assumed that the ts phenotype of a specific mutation arises from thermal denaturation of the mutant enzyme, the possibility also exists that the mutation decreases the enzyme activity to a certain level at the permissive temperature and aggravates the negative effect further upon temperature upshifts. Resolving these possibilities is important for exploiting the ts mutation for studying the essential gene. The trmD gene is essential for growth in bacteria, encoding the enzyme for converting G37 to m1G37 on the 3′ side of the tRNA anticodon. This conversion involves methyl transfer from S-adenosyl methionine and is critical to minimize tRNA frameshift errors on the ribosome. Using the ts-S88L mutation of Escherichia coli trmD as an example, we show that although the mutation confers thermal lability to the enzyme, the effect is relatively minor. In contrast, the mutation decreases the catalytic efficiency of the enzyme to 1% at the permissive temperature, and at the nonpermissive temperature, it renders further deterioration of activity to 0.1%. These changes are accompanied by losses of both the quantity and quality of tRNA methylation, leading to the potential of cellular pleiotropic effects. This work illustrates the principle that the ts phenotype of an essential gene mutation can be closely linked to the catalytic defect of the gene product and that such a mutation can provide a useful tool to study the mechanism of catalytic inactivation. Essential genes encode critical cellular functions that are not supported by redundant pathways. Because of their indispensability, essential genes have been studied and functionally controlled by using temperature-sensitive (ts) 4The abbreviations used are: tstemperature-sensitiveAdoMetS-adenosyl methionineAdoHcyS-adenosyl homocysteineKankanamycin. alleles. Such alleles are typically mis-sense mutations, which preserve the gene function at permissive and low temperatures but inactivate the gene function upon temperature upshifts. The study of ts phenotypes is fundamental and important for insight into gene essentiality. However, the molecular basis of ts phenotypes of essential genes remains poorly understood; although it is generally assumed that such phenotypes result from thermal inactivation of gene products, the possibility that they arise from inherent functional defects that become lethal at higher temperatures is often overlooked. In the latter case, ts mutations giving rise to severe functional defects at the permissive temperature can be powerful tools to study essential genes and to correlate phenotypes with mechanisms of gene inactivation. temperature-sensitive S-adenosyl methionine S-adenosyl homocysteine kanamycin. In Escherichia coli, systematic exploration and profiling of single-gene knockouts has suggested that ∼7% of the ∼4,000 protein-coding genes are essential (1.Baba T. Ara T. Hasegawa M. Takai Y. Okumura Y. Baba M. Datsenko K.A. Tomita M. Wanner B.L. Mori H. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.Mol. Syst. Biol. 2006; 2: 2006-2008Crossref Scopus (5375) Google Scholar, 2.Nichols R.J. Sen S. Choo Y.J. Beltrao P. Zietek M. Chaba R. Lee S. Kazmierczak K.M. Lee K.J. Wong A. Shales M. Lovett S. Winkler M.E. Krogan N.J. Typas A. Gross C.A. Phenotypic landscape of a bacterial cell.Cell. 2011; 144: 143-156Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar). One example of an essential gene is trmD, broadly conserved in all bacterial species, encoding the tRNA methyltransferase for conversion of G37 to m1G37 necessary to prevent frameshift errors on the 3′ side of the anticodon (3.Byström A.S. Björk G.R. The structural gene (trmD) for the tRNA(m1G)methyltransferase is part of a four polypeptide operon in Escherichia coli K-12.Mol. Gen. Genet. 1982; 188: 447-454Crossref PubMed Scopus (24) Google Scholar, 4.Byström A.S. Björk G.R. Chromosomal location and cloning of the gene (trmD) responsible for the synthesis of tRNA (m1G) methyltransferase in Escherichia coli K-12.Mol. Gen. Genet. 1982; 188: 440-446Crossref PubMed Scopus (55) Google Scholar, 5.Björk G.R. Wikström P.M. Byström A.S. Prevention of translational frameshifting by the modified nucleoside 1-methylguanosine.Science. 1989; 244: 986-989Crossref PubMed Scopus (172) Google Scholar, 6.Hagervall T.G. Tuohy T.M. Atkins J.F. Björk G.R. Deficiency of 1-methylguanosine in tRNA from Salmonella typhimurium induces frameshifting by quadruplet translocation.J. Mol. Biol. 1993; 232: 756-765Crossref PubMed Scopus (69) Google Scholar). In addition to E. coli, the growth essentiality of trmD has also been shown for Streptococcus pneumoniae and Bacillus subtilis (7.O'Dwyer K. Watts J.M. Biswas S. Ambrad J. Barber M. Brulé H. Petit C. Holmes D.J. Zalacain M. Holmes W.M. Characterization of Streptococcus pneumoniae TrmD, a tRNA methyltransferase essential for growth.J. Bacteriol. 2004; 186: 2346-2354Crossref PubMed Scopus (36) Google Scholar, 8.Kobayashi K. Ehrlich S.D. Albertini A. Amati G. Andersen K.K. Arnaud M. Asai K. Ashikaga S. Aymerich S. Bessieres P. Boland F. Brignell S.C. Bron S. Bunai K. Chapuis J. Christiansen L.C. Danchin A. Débarbouille M. Dervyn E. Deuerling E. Devine K. Devine S.K. Dreesen O. Errington J. Fillinger S. Foster S.J. Fujita Y. Galizzi A. Gardan R. Eschevins C. Fukushima T. Haga K. Harwood C.R. Hecker M. Hosoya D. Hullo M.F. Kakeshita H. Karamata D. Kasahara Y. Kawamura F. Koga K. Koski P. Kuwana R. Imamura D. Ishimaru M. Ishikawa S. Ishio I. Le Coq D. Masson A. Mauël C. Meima R. Mellado R.P. Moir A. Moriya S. Nagakawa E. Nanamiya H. Nakai S. Nygaard P. Ogura M. Ohanan T. O'Reilly M. O'Rourke M. Pragai Z. Pooley H.M. Rapoport G. Rawlins J.P. Rivas L.A. Rivolta C. Sadaie A. Sadaie Y. Sarvas M. Sato T. Saxild H.H. Scanlan E. Schumann W. Seegers J.F. Sekiguchi J. Sekowska A. Seror S.J. Simon M. Stragier P. Studer R. Takamatsu H. Tanaka T. Takeuchi M. Thomaides H.B. Vagner V. van Dijl J.M. Watabe K. Wipat A. Yamamoto H. Yamamoto M. Yamamoto Y. Yamane K. Yata K. Yoshida K. Yoshikawa H. Zuber U. Ogasawara N. Essential Bacillus subtilis genes.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 4678-4683Crossref PubMed Scopus (1142) Google Scholar). In one genetic study of Salmonella typhimurium, several ts mutants of trmD were isolated for the deficiency in m1G37-tRNA synthesis, resulting in changes in the thiamine metabolism flux (9.Björk G.R. Nilsson K. 1-Methylguanosine-deficient tRNA of Salmonella enterica serovar Typhimurium affects thiamine metabolism.J. Bacteriol. 2003; 185: 750-759Crossref PubMed Scopus (16) Google Scholar). It was suggested that the trmD deficiency at the restrictive temperature of these mutants reduced the m1G37-tRNA level, such that ribosome translation at codons normally read by the modified tRNA was slow, thus inactivating gene expression in the normal thiamine biosynthesis pathway while activating expression in an alternative pathway. This study showed that the deficiency of trmD had altered the profile of global gene expression, consistent with increasing evidence indicating that tRNA modifications often have regulatory and stress-response roles at genome-wide levels (10.Yi C. Pan T. Cellular dynamics of RNA modification.Acc. Chem. Res. 2011; 44: 1380-1388Crossref PubMed Scopus (87) Google Scholar). Although the isolated ts-trmD mutants had been confirmed for the deficiency in m1G37-tRNA synthesis (9.Björk G.R. Nilsson K. 1-Methylguanosine-deficient tRNA of Salmonella enterica serovar Typhimurium affects thiamine metabolism.J. Bacteriol. 2003; 185: 750-759Crossref PubMed Scopus (16) Google Scholar), none had been characterized at the structural level for locations, at the molecular level for thermal instability, or at the enzyme level for catalytic defect. To gain insight into these mutations, we have mapped their locations onto the known structure of E. coli TrmD (EcTrmD) in complex with S-adenosyl homocysteine (AdoHcy), the product after methyl transfer (11.Elkins P.A. Watts J.M. Zalacain M. van Thiel A. Vitazka P.R. Redlak M. Andraos-Selim C. Rastinejad F. Holmes W.M. Insights into catalysis by a knotted TrmD tRNA methyltransferase.J. Mol. Biol. 2003; 333: 931-949Crossref PubMed Scopus (106) Google Scholar). From this mapping, we selected for further analysis of the S88L mutation, which is placed closely adjacent to the S-adenosyl methionine (AdoMet)-binding site. Remarkably, TrmD binds AdoMet in a topologically knotted trefoil fold, involving the protein backbone making three passes in and out of a tightly folded loop (11.Elkins P.A. Watts J.M. Zalacain M. van Thiel A. Vitazka P.R. Redlak M. Andraos-Selim C. Rastinejad F. Holmes W.M. Insights into catalysis by a knotted TrmD tRNA methyltransferase.J. Mol. Biol. 2003; 333: 931-949Crossref PubMed Scopus (106) Google Scholar, 12.Ahn H.J. Kim H.W. Yoon H.J. Lee B.I. Suh S.W. Yang J.K. Crystal structure of tRNA(m1G37)methyltransferase. Insights into tRNA recognition.EMBO J. 2003; 22: 2593-2603Crossref PubMed Scopus (119) Google Scholar). The location of the S88L mutation within the trefoil knot is attractive for interrogating the molecular basis of its temperature sensitivity. We report here that although the S88L mutation indeed confers susceptibility to thermal denaturation, the effect is rather minor. Instead, the mutation markedly decreases the enzyme activity relative to the native sequence at the permissive temperature, and it causes further deterioration of activity upon temperature upshifts. We further show that the decrease in the enzyme activity is correlated with decrease in the level of tRNA methylation and the quality of tRNA recognition, suggesting the possibility of accumulating frameshift errors during protein synthesis and altering global gene expression. We suggest that it is the loss of the catalytic efficiency of TrmD by the S88L mutation, and consequently the increase of protein synthesis errors, that is the driving force for the lethal phenotype at the restrictive temperature. This work demonstrates that ts phenotypes can be closely correlated with catalytic defects of an essential gene product and that such correlation can provide unique insight into the function of the essential gene in vivo and the mechanism of action in vitro. The S88L-trmD mutation was isolated with a ts phenotype in Salmonella (25.Björk G.R. Jacobsson K. Nilsson K. Johansson M.J. Byström A.S. Persson O.P. A primordial tRNA modification required for the evolution of life?.EMBO J. 2001; 20: 231-239Crossref PubMed Scopus (199) Google Scholar). We introduced this mutation to the chromosomal trmD gene of E. coli Xac strain, using the λ Red recombinase-mediated gene disruption method (16.Datsenko K.A. Wanner B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 6640-6645Crossref PubMed Scopus (11130) Google Scholar). We inserted the E. coli wt-trmD gene to the pQE-30 vector and introduced the ts-S88L mutation, by QuikChange site-directed mutagenesis (Stratagene). The modified coding sequence was amplified by PCR using the following primer set: 5′-ATGTGGATTGGCATAATTAGCCTGTTTCC-3′ (forward) and 5′-GGAACTTCGAAGCAGCTCCAGCCTACACTTACGCCATCCCATCATGTTTATG-3′ (reverse). The PCR product was purified and amplified again according to the megaprimer method (34.Chen J.R. Lü J.J. Wang H.F. Rapid and efficient gene splicing using megaprimer-based protocol.Mol. Biotechnol. 2008; 40: 224-230Crossref PubMed Scopus (9) Google Scholar) in a mixture containing the first PCR product, the kanamycin resistance cassette plasmid pKD4, and the third primer, 5′-ATCCTGGGTAAACTGATATCTCGGGGGCATGGGAATTAGCCATGGTCCATATG-3′. The final PCR product, together with pKD46 (the λ Red recombinase plasmid), was introduced to E. coli Xac strain and colonies were selected on LB-Kan plates. The mutation was verified by sequence analysis, and the temperature sensitivity and curing of pKD46 was confirmed. In parallel, a wt-trmD-Kan construct was made as a control. The quenching of the intrinsic tryptophan fluorescence of EcTrmD by AdoMet binding was determined as described (17.Christian T. Lahoud G. Liu C. Hou Y.M. Control of catalytic cycle by a pair of analogous tRNA modification enzymes.J. Mol. Biol. 2010; 400: 204-217Crossref PubMed Scopus (33) Google Scholar). The WT enzyme was titrated at 0.2 μm with AdoMet ranging from 0.2 to 4.5 μm, whereas the S88L mutant enzyme was titrated at 2.0 μm with AdoMet ranging from 1.5 to 22 μm. Enzyme fluorescence was excited at 280 nm in the standard buffer (100 mm Tris-HCl, pH 8.0, 4 mm DTT, 0.1 mm EDTA, 6 mm MgCl2, and 100 mm KCl), and emission was monitored at 320–340 nm at room temperature. Inner filter corrections were calculated by the equation: Fc/Fo = anti-log [(Aex + Aem)/2], where Fc is the dilution-corrected fluorescence, Fo is the observed fluorescence, and Aex and Aem are the absorbencies at the excitation and emission wavelengths, respectively. Nonspecific quenching was measured by titrating AdoMet against a solution of l-Trp. Data corrected after inner filter effects and nonspecific quenching were fit to a hyperbolic equation: y = A × S/(S + Kd), where y is the change in fluorescence from the reference point, A is the maximum change in amplitude, and S is the AdoMet concentration. E. coli cells harboring wt or S88L-trmD were grown in LB medium overnight at 30 °C. A fresh culture was inoculated with the overnight culture at 1:100-fold dilution and continued to grow for 3–4 h at 30 °C until A600 = ∼0.3. The culture was split into two, with one shifted to 43 °C by mixing with an equal volume of LB at 55 °C while the other was maintained at 30 °C by mixing with an equal volume of LB at 30 °C. Aliquots of each culture were sampled up to 24 h. Cell viability was determined by directly plating the cell culture at each time point with appropriate dilution. The plates were incubated at 30 or 43 °C, respectively, for additional 12–14 h. The number of colonies formed was normalized by A600 and shown as relative to the time of split. E. coli cells were grown as above. Each aliquot of cell culture was spun down, and the cells were disrupted by sonication to separate the lysate from debris. The lysate was directly used to measure enzyme activity in the TrmD reaction buffer (100 mm Tris-HCl, pH 8.0, 24 mm NH4Cl, 4 mm DTT, 100 μm EDTA, 6 mm MgCl2, 0.024 mg/ml BSA, 40 units/sample RNasin (Promega)) at 30 or 43 °C, using EctRNAPro as the substrate at 5-fold or lower concentration of Km. The A37 mutant of EctRNAPro, which was not a TrmD substrate, was used to measure methyl transfer to non-G37 positions. [3H-methyl]AdoMet (25 μm) was used, and the synthesis of [3H]m1G37-tRNA was monitored by acid precipitation (35.Christian T. Evilia C. Williams S. Hou Y.M. Distinct origins of tRNA(m1G37) methyltransferase.J. Mol. Biol. 2004; 339: 707-719Crossref PubMed Scopus (62) Google Scholar). The initial rate of m1G37-tRNA synthesis was normalized by A600 and shown as relative to the time of culture split. Because the endogenous level of TrmD is very low in E. coli (28.Wikström P.M. Björk G.R. Noncoordinate translation-level regulation of ribosomal and nonribosomal protein genes in the Escherichia coli trmD operon.J. Bacteriol. 1988; 170: 3025-3031Crossref PubMed Google Scholar), we used E. coli strain SG13009 overexpressing the WT or S88L-TrmD from the plasmid pQE30. Cells were grown in LB medium at 30 °C for an hour and then split into three cultures, each at 30, 37, or 43 °C. After 8 h of growth, when the mutant TrmD was inactivated at the restrictive temperatures (see Fig. 5), cells were harvested and sonicated, the soluble cell lysates were collected, and protein concentrations were determined by the Bradford method. Total protein (10 μg) of each cell lysate was loaded to a 12% SDS-PAGE, transferred to Immobilon PVDF membrane (Millipore), and reacted with the primary antibody raised against StTrmD (given by Dr. Glenn Björk). The membrane was incubated with the anti-rabbit IgG secondary antibody conjugated with peroxidase (Sigma-Aldrich), and the signal was detected using SuperSignal West Pico (Thermo Scientific) and quantified by ImageQuant (GE Healthcare). Two independent experiments were performed to report the average signal of TrmD at 37 and 43 relative to 30 °C. Errors are standard deviations. E. coli wt- and S88L-trmD cells were grown at 30 °C to A600 of 0.3 and then shifted to 43 °C or maintained at 30 °C for 10 h. Total RNA was isolated from each cell culture, and the native E. coli tRNALeu/CAG was purified by a biotin-tagged oligonucleotide specific to the D loop region (36.Yokogawa T. Kitamura Y. Nakamura D. Ohno S. Nishikawa K. Optimization of the hybridization-based method for purification of thermostable tRNAs in the presence of tetraalkylammonium salts.Nucleic Acids Res. 2010; 38: e89Crossref PubMed Scopus (45) Google Scholar). The purified native tRNALeu/CAG was tested by the RNaseH assay (24.Hou Y.M. Li Z. Gamper H. Isolation of a site-specifically modified RNA from an unmodified transcript.Nucleic Acids Res. 2006; 34: e21Crossref PubMed Scopus (14) Google Scholar), using a DNA oligonucleotide complementary to the region of nucleotides from positions 31 to 46. RNaseH-treated tRNAs were separated on a 12% PAGE, 7 m urea. The fractions of intact tRNA and cleaved fragments were estimated based on ethidium bromide staining. Enzyme was purified by affinity resin and by FPLC, and concentration was determined by UV absorption for CD analysis. Spectra were measured on a JASCO spectrometer J-810 equipped with JASCO Peltier thermo controller. For the wavelength scan, a cuvette with 1-mm path length was used. Each sample (1 μm dimer) in the CD buffer (50 mm Tris-HCl, pH 8.0, 3 mm MgCl2, and 24 mm NH4Cl) was preincubated at 30 or 43 °C for 10 min, and then the spectrum was recorded from 260 to 200 nm at each temperature. Scan speed was 10 nm/min, response was 32 s, and bandwidth was 2 nm. Each spectrum was the average of four runs, and the average of two spectra for each sample was presented. For the temperature scan, a cuvette with 10-mm path length was used. The spectrum of each sample (0.1 μm) in the CD buffer was recorded from 15 to 75 °C at 222 nm. Temperature raise was 24 °C/hour, response was 32 s, and bandwidth was 1 nm. Calculation of α-helical content was based on the equation: % helix = (Δϵ220 − 0.25)/0.105, where Δϵ220 is the measured circular dichroism at 220 nm derived from [θ] = 3330 Δϵ as previously reported (37.Clark D.J. Hill C.S. Martin S.R. Thomas J.O. α-Helix in the carboxy-terminal domains of histones H1 and H5.EMBO J. 1988; 7: 69-75Crossref PubMed Scopus (137) Google Scholar). WT and the S88L mutant of EcTrmD enzymes, each with an N-His tag, were expressed from pQE30 in E. coli SG13009. Enzymes from one liter of culture were purified using HisLink (Promega). Steady-state kinetic assays were performed as described (35.Christian T. Evilia C. Williams S. Hou Y.M. Distinct origins of tRNA(m1G37) methyltransferase.J. Mol. Biol. 2004; 339: 707-719Crossref PubMed Scopus (62) Google Scholar). Each tRNA sample (ranging in final concentration from 1.0 to 128.0 μm) was heat-denatured and annealed before use. Each reaction contained tRNA and saturating [3H-methyl]AdoMet (25 μm) in the standard buffer (0.1 m Tris-HCl, pH 8.0, 4 mm DTT, 0.1 mm EDTA, 6 mm MgCl2, 24 mm NH4Cl, and 0.024 mg/ml BSA) and was initiated by addition of the WT or mutant enzyme at final concentrations 0.025 to 1.5 μm. Aliquots were taken at every 2 min up to 10 min and precipitated by 5% (w/v) TCA on filter pads. Methyl transfer was determined by quantification of [3H] incorporation into acid precipitable counts. After correction of the filter quenching factor, the data of initial rate as a function of tRNA concentration were fit to the Michaelis-Menten equation using the KaleidaGraph software (Synergy software) to determine the Km, kcat, and kcat/Km values. Each value was the average of at least three independent measurements. Errors are standard deviations. Discrimination analysis between G36- and C36-EctRNALeu was performed in the same buffer. Data for the G36-tRNA was obtained from Table 1. For the C36-tRNA, the WT enzyme was assayed at 1.0 μm with 16.0 and 20.0 μm of tRNA at 30 and 43 °C, respectively, whereas the S88L mutant enzyme was assayed at 1.0 μm with 16 μm tRNA at 30 °C and at 2.6 μm with 50 μm tRNA at 43 °C. Each enzyme-tRNA condition was in the kcat/Km range. Aliquots were taken at every 5 min up to 25 min at 30 °C and at every 10 min up to 50 min at 43 °C. Samples were processed as above, and the initial rate of each reaction (pmol/s) was obtained. The value of kcat/Km (μm−1 s−1) was calculated by dividing the initial rate by the enzyme amount and the tRNA concentration. For all kinetic assays, each value was the average of at least two to three independent measurements, and standard deviations are reported.TABLE 1Steady-state kinetic parameters of TrmD on EctRNALeuWT-TrmDS88L-TrmD30 °C37 °C43 °C30 °C37 °C43 °CKm (μm)2.4 ± 0.43.1 ± 0.321.7 ± 4.313.0 ± 1.152.2 ± 4.662.5 ± 12.8kcat (s−1)(4.9 ± 0.2) × 10−2(9.3 ± 0.9) × 10−2(1.3 ± 0.1) × 10−1(3.2 ± 0.3) × 10−3(6.7 ± 0.4) × 10−3(1.5 ± 0.2) × 10−3kcat/Km (μm−1 s−1)(2.0 ± 0.4) × 10−2(3.0 ± 0.4) × 10−2(5.9 ± 1.2) × 10−3(2.4 ± 0.3) × 10−4(1.3 ± 0.1) × 10−4(2.5 ± 0.6) × 10−5kcat/Km ratio1.001.460.290.0120.00640.0012 Open table in a new tab A genetic study led by Björk and Nilsson (9.Björk G.R. Nilsson K. 1-Methylguanosine-deficient tRNA of Salmonella enterica serovar Typhimurium affects thiamine metabolism.J. Bacteriol. 2003; 185: 750-759Crossref PubMed Scopus (16) Google Scholar) isolated a group of ts mutations in S. typhimurium TrmD (StTrmD) that conferred altered thiamine metabolism at a restrictive temperature (P58L/L94F, S88L, G117S, G117N, G117Q, S165L, P184L, G199R, G214D, W217D, and E243K). A separate study by Li and Björk (13.Li J.N. Björk G.R. Structural alterations of the tRNA(m1G37)methyltransferase from Salmonella typhimurium affect tRNA substrate specificity.RNA. 1999; 5: 395-408Crossref PubMed Scopus (26) Google Scholar) isolated additional ts mutants (E243K, L94F, P184L, G140S, and A145T) that displayed elevated levels of frameshift errors caused by the deficiency in m1G37-tRNA. The two groups of mutants showed overlapping amino acid substitutions at the protein level and collectively occupied 12 positions in the StTrmD enzyme structure. Because these mutations were isolated before the structure of TrmD was available, nothing was known about their structural context. We now mapped these mutations onto the crystal structure of EcTrmD in complex with AdoHcy (11.Elkins P.A. Watts J.M. Zalacain M. van Thiel A. Vitazka P.R. Redlak M. Andraos-Selim C. Rastinejad F. Holmes W.M. Insights into catalysis by a knotted TrmD tRNA methyltransferase.J. Mol. Biol. 2003; 333: 931-949Crossref PubMed Scopus (106) Google Scholar), which was a logical model based on the over 92% homology in the primary sequence between the S. typhimurium and E. coli enzymes. Although TrmD exists as an obligated homodimer, with each subunit featuring an N-terminal domain (residues 1–159), a flexible linker (residues 160–169), and a C-terminal domain (residues 170–250), the active site is built between the N-terminal domain of one subunit and the flexible linker and the C-terminal domain of the other. In this intriguing cross-subunit active site, AdoMet is bound to the trefoil knot fold in the N-terminal domain, whereas the target G37 is predicted to bind to the flexible linker (11.Elkins P.A. Watts J.M. Zalacain M. van Thiel A. Vitazka P.R. Redlak M. Andraos-Selim C. Rastinejad F. Holmes W.M. Insights into catalysis by a knotted TrmD tRNA methyltransferase.J. Mol. Biol. 2003; 333: 931-949Crossref PubMed Scopus (106) Google Scholar). However, the active site cannot be precisely depicted, because the flexible linker is disordered and invisible in structures that lack tRNA (12.Ahn H.J. Kim H.W. Yoon H.J. Lee B.I. Suh S.W. Yang J.K. Crystal structure of tRNA(m1G37)methyltransferase. Insights into tRNA recognition.EMBO J. 2003; 22: 2593-2603Crossref PubMed Scopus (119) Google Scholar), including the EcTrmD structure (11.Elkins P.A. Watts J.M. Zalacain M. van Thiel A. Vitazka P.R. Redlak M. Andraos-Selim C. Rastinejad F. Holmes W.M. Insights into catalysis by a knotted TrmD tRNA methyltransferase.J. Mol. Biol. 2003; 333: 931-949Crossref PubMed Scopus (106) Google Scholar). This prevented the mapping of the S165L mutation. To the rest of the mutations, we found that those localized to the N-terminal domain were near the AdoMet binding site, whereas those localized to the C-terminal domain were in helical regions (Fig. 1). We focused on mutations near the AdoMet binding site to provide a structural framework for interpreting mutational effects. In this framework, AdoMet is bound at the deep end of the trefoil knot fold, which is initiated with a β strand (β4) in the central β sheet structure of the N-terminal domain. The β4 curves around through α4 and β5 and turns into α5 and then into β6, which makes a circular insertion into the loop (Fig. 1A). Within this knot, AdoMet adopts a distinctively bent conformation that places the adenosine and methionine moieties facing each other, which is rare among AdoMet-dependent methyltransferases (12.Ahn H.J. Kim H.W. Yoon H.J. Lee B.I. Suh S.W. Yang J.K. Crystal structure of tRNA(m1G37)methyltransferase. Insights into tRNA recognition.EMBO J. 2003; 22: 2593-2603Crossref PubMed Scopus (119) Google Scholar, 14.Schubert H.L. Blumenthal R.M. Cheng X. Many paths to methyltransfer. A chronicle of convergence.Trends Biochem. Sci. 2003; 28: 329-335Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar). During methyl transfer, the AdoMet-binding site is presumably “capped” by the flexible linker provided by the C-terminal domain of the other monomer. Proteins with a knotted fold, such as the trefoil knot in TrmD, are rare but are known to have an inherent rigidity not present in unknotted proteins (15.King N.P. Jacobitz A.W. Sawaya M.R. Goldschmidt L. Yeates T.O. Structure and folding of a designed knotted protein.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 20732-20737Crossref PubMed Scopus (105) Google Scholar). Of the mutations localized to near the AdoMet site, some were discovered together in one mutant (e.g., P58L and L94F), whereas others were discovered in multiple mutants each with a distinct substitution (e.g., G117S, G117N, and G117Q). We chose to study the S88L mutation, which was discovered in one mutant at a single position. The natural S88 residue is at a position highly conserved among Gram-negative TrmD enzymes but not conserved in Gram-positive (Fig. 2A), indicating the possibility of differential roles in the two classes of bacteria. In the EcTrmD structure, S88 is placed in the beginning of the loop emanating from the central β4 that stabilizes the knot (Fig. 2B). The S88L mutation is therefore attractive for interrogating the molecular basis of its ts phenotypes, because on the one hand it is expected to interfere with AdoMet binding, because of the replacement of the smaller side chain" @default.
• W2006703609 created "2016-06-24" @default.
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