Mechanistic Studies of the tRNA-Modifying Enzyme QueA: A Chemical Imperative for the Use of AdoMet as a “Ribosyl” Donor (original) (raw)
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tRNA Modification by S-Adenosylmethionine:tRNA Ribosyltransferase-Isomerase
Journal of Biological Chemistry, 2003
The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase catalyzes the penultimate step in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q), an unprecedented ribosyl transfer from the cofactor S-adenosylmethionine (AdoMet) to a modified-tRNA precursor to generate epoxyqueuosine (oQ). The complexity of the reaction makes it an especially interesting mechanistic problem, and as a foundation for detailed kinetic and mechanistic studies we have carried out the basic characterization of the enzyme. Importantly, to allow for the direct measurement of oQ formation, we have developed protocols for the preparation of homogeneous substrates; specifically, an overexpression system was constructed for tRNA Tyr in an E. coli queA deletion mutant to allow for the isolation of large quantities of substrate tRNA, and [U-ribosyl-14 C]AdoMet was synthesized. The enzyme shows optimal activity at pH 8.7 in buffers containing various oxyanions, including acetate, carbonate, EDTA, and phosphate. Unexpectedly, the enzyme was inhibited by Mg 2؉ and Mn 2؉ in millimolar concentrations. The steady-state kinetic parameters were determined to be K m AdoMet ؍ 101.4 M, K m tRNA ؍ 1.5 M, and k cat ؍ 2.5 min ؊1. A short minihelix RNA was synthesized and modified with the precursor 7-aminomethyl-7-deazaguanine, and this served as an efficient substrate for the enzyme (K m RNA ؍ 37.7 M and k cat ؍ 14.7 min ؊1), demonstrating that the anticodon stem-loop is sufficient for recognition and catalysis by QueA.
Crystal structure of Bacillus subtilis S-adenosylmethionine:tRNA ribosyltransferase-isomerase
Biochemical and Biophysical Research Communications, 2006
The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine. It is unprecedented in nature as it uses the cofactor S-adenosylmethionine as the donor of a ribosyl group. We have determined the crystal structure of Bacillus subtilis QueA at a resolution of 2.9 Å . The structure reveals two domains representing a 6-stranded b-barrel and an aba-sandwich, respectively. All amino acid residues invariant in the QueA enzymes of known sequence cluster at the interface of the two domains indicating the localization of the substrate binding region and active center. Comparison of the B. subtilis QueA structure with the structure of QueA from Thermotoga maritima suggests a high domain flexibility of this enzyme.
Chemistry & Biodiversity, 2005
tRNA is best known for its function as amino acid carrier in the translation process, using the anticodon loop in the recognition process with mRNA. However, the impact of tRNA on cell function is much wider, and mutations in tRNA can lead to a broad range of diseases. Although the cloverleaf structure of tRNA is wellknown based on X-ray-diffraction studies, little is known about the dynamics of this fold, the way structural dynamics of tRNA is influenced by the modified nucleotides present in tRNA, and their influence on the recognition of tRNA by synthetases, ribosomes, and other biomolecules. One of the reasons for this is the lack of good synthetic methods to incorporate modified nucleotides in tRNA so that larger amounts become available for NMR studies. Except of 2'-O-methylated nucleosides, only one other sugar-modified nucleoside is present in tRNA, i.e., 2'-O-b-d-ribofuranosyl nucleosides. The T loop of tRNA often contains charged modified nucleosides, of which 1-methyladenosine and phosphorylated disaccharide nucleosides are striking examples. A protecting-group strategy was developed to introduce 1-methyladenosine and 5''-O-phoshorylated 2'-O-(b-dribofuranosyl)-b-d-ribofuranosyladenine in the same RNA fragment. The phosphorylation of the disaccharide nucleoside was performed after the assembly of the RNA on solid support. The modified RNA was characterized by mass-spectrometry analysis from the RNase T1 digestion fragments. The successful synthesis of this T loop of the tRNA of Schizosaccharomyces pombe initiator tRNA Met will be followed by its structural analysis by NMR and by studies on the influence of these modified nucleotides on dynamic interactions within the complete tRNA.
RNA, 2020
N 6-threonylcarbamoyl adenosine (t 6 A) is a nucleoside modification found in all kingdoms of life at position 37 of tRNAs decoding ANN codons, which functions in part to restrict translation initiation to AUG and suppress frameshifting at tandem ANN codons. In Bacteria the proteins TsaB, TsaC (or C2), TsaD, and TsaE, comprise the biosynthetic apparatus responsible for t 6 A formation. TsaC(C2) and TsaD harbor the relevant active sites, with TsaC(C2) catalyzing the formation of the intermediate threonylcarbamoyladenosine monophosphate (TC-AMP) from ATP, threonine, and CO 2 , and TsaD catalyzing the transfer of the threonylcarbamoyl moiety from TC-AMP to A 37 of substrate tRNAs. Several related modified nucleosides, including hydroxynorvalylcarbamoyl adenosine (hn 6 A), have been identified in select organisms, but nothing is known about their biosynthesis. To better understand the mechanism and structural constraints on t 6 A formation, and to determine if related modified nucleosides are formed via parallel biosynthetic pathways or the t 6 A pathway, we carried out biochemical and biophysical investigations of the t 6 A systems from E. coli and T. maritima to address these questions. Using kinetic assays of TsaC(C2), tRNA modification assays, and NMR, our data demonstrate that TsaC(C2) exhibit relaxed substrate specificity, producing a variety of TC-AMP analogs that can differ in both the identity of the amino acid and nucleotide component, whereas TsaD displays more stringent specificity, but efficiently produces hn 6 A in E. coli and T. maritima tRNA. Thus, in organisms that contain modifications such as hn 6 A in their tRNA, we conclude that their origin is due to formation via the t 6 A pathway.
Biophysical Journal, 1997
The conformations of MgATP and AMP bound to a monomeric tryptic fragment of methionyl tRNA synthetase have been investigated by two-dimensional proton transferred nuclear Overhauser effect spectroscopy (TRNOESY). The sample protocol was chosen to minimize contributions from adventitious binding of the nucleotides to the observed NOE. The experiments were performed at 500 MHz on three different complexes, E -MgATP, E * MgATP-L-methioninol, and E * AMP -L-methioninol. A starter set of distances obtained by fitting NOE build-up curves (not involving H5' and H5") were used to determine a CHARMm energy-minimized structure. The positioning of the H5' and H5" protons was determined on the basis of a conformational search of the torsion angle to obtain the best fit with the observed NOEs for their superposed resonance. Using this structure, a relaxation matrix was set up to calculate theoretical build-up curves for all of the NOEs and compare them with the observed curves. The final structures deduced for the adenosine moieties in the three complexes are very similar, and are described by a glycosidic torsion angle (X) of 560 ± 50 and a phase angle of pseudorotation (P) in the range of 470 to 520, describing a 3T-4E sugar pucker. The glycosidic torsion angle, X, deduced here for this adenylyl transfer enzyme and those determined previously for three phosphoryl transfer enzymes (creatine kinase, arginine kinase, and pyruvate kinase), and one pyrophosphoryl enzyme (PRibPP synthetase), are all in the range 520 ± 80. The narrow range of values suggests a possible common motif for the recognition and binding of the adenosine moiety at the active sites of ATP-utilizing enzymes, irrespective of the point of cleavage on the phosphate chain.
On the Specificity of Interactions between Transfer Ribonucleic Acids and Aminoacyl-tRNA Synthetases
European Journal of Biochemistry, 1973
= tRNAPhe containing ethidium instead of the Y base; tRNA?&, , , = NaBH,-reduced tRNAgg; tRNAE; = tRNASer containing ethidium at the three dihydrouracil positions; tRNA:&,, = NaBH,reduced tRNA:; containing about 1.5 moles ethidium/ mole tRNA; tRNA2 = tRNAser, the 3'-terminal adenosine of which is replaced by ethenoadenosine; tRNAg = tRNA*labeled with proflavine at the dihydrouracil positions. The pa-half and the CCA-half of tRNA*he comprise the nucleotides 1-36 and 38-76, respectively. The half molecules of tRNAser comprise the nucleotides 1-34 (PO-half) and 36-85 (CCA-half). Enzymes. Seryl-tRNA synthetase (EC 6.1.1.11); phenylalanyl-tRNA synthetase (EC 6.1.1.-); CC14-transferase (EC 2.7.7.25).
tRNA-dependent Pre-transfer Editing by Prokaryotic Leucyl-tRNA Synthetase
Journal of Biological Chemistry, 2010
To prevent genetic code ambiguity due to misincorporation of amino acids into proteins, aminoacyl-tRNA synthetases have evolved editing activities to eliminate intermediate or final noncognate products. In this work we studied the different editing pathways of class Ia leucyl-tRNA synthetase (LeuRS). Different mutations and experimental conditions were used to decipher the editing mechanism, including the recently developed compound AN2690 that targets the post-transfer editing site of LeuRS. The study emphasizes the crucial importance of tRNA for the pre-and post-transfer editing catalysis. Both reactions have comparable efficiencies in prokaryotic Aquifex aeolicus and Escherichia coli LeuRSs, although the E. coli enzyme favors post-transfer editing, whereas the A. aeolicus enzyme favors pre-transfer editing. Our results also indicate that the entry of the CCA-acceptor end of tRNA in the editing domain is strictly required for tRNA-dependent pre-transfer editing. Surprisingly, this editing reaction was resistant to AN2690, which inactivates the enzyme by forming a covalent adduct with tRNA Leu in the post-transfer editing site. Taken together, these data suggest that the binding of tRNA in the post-transfer editing conformation confers to the enzyme the capacity for pretransfer editing catalysis, regardless of its capacity to catalyze post-transfer editing.