{"content"=>"Backbone resonance assignment for the N-terminal region of bacterial tRNA-(NG37) methyltransferase.", "sup"=>{"content"=>"1"}} (original) (raw)
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Backbone resonance assignment for the N-terminal region of bacterial tRNA-(N1G37) methyltransferase
Biomolecular Nmr Assignments, 2018
Bacterial tRNA (guanine37-N 1)-methyltransferase (TrmD) plays important roles in translation, making it an important target for the development of new antibacterial compounds. TrmD comprises two domains with the N-terminal domain binding to the S-adenosyl-L-methionine (SAM) cofactor and the C-terminal domain critical for tRNA binding. Bacterial TrmD is functional as a dimer. Here we report the backbone NMR resonance assignments for the full length TrmD protein of Pseudomonas aeruginosa. Most resonances were assigned and the secondary structure for each amino acid was determined according to the assigned backbone resonances. The availability of the assignment will be valuable for exploring molecular interactions of TrmD with ligands, inhibitors and tRNA. Keywords: TrmD · Pseudomonas aeruginosa · tRNA methyltransferase · epitranscriptome · drug discovery · antibacterial · protein dynamics · backbone assignment
Biomolecular NMR Assignments, 2019
Bacterial tRNA (guanine37-N 1)-methyltransferase (TrmD) plays important roles in translation, making it an important target for the development of new antibacterial compounds. TrmD comprises two domains with the N-terminal domain binding to the S-adenosyl-L-methionine (SAM) cofactor and the C-terminal domain critical for tRNA binding. Bacterial TrmD is functional as a dimer. Here we report the backbone NMR resonance assignments for the full length TrmD protein of Pseudomonas aeruginosa. Most resonances were assigned and the secondary structure for each amino acid was determined according to the assigned backbone resonances. The availability of the assignment will be valuable for exploring molecular interactions of TrmD with ligands, inhibitors and tRNA. Keywords: TrmD · Pseudomonas aeruginosa · tRNA methyltransferase · epitranscriptome · drug discovery · antibacterial · protein dynamics · backbone assignment
RNA, 2019
The tRNA (m 1 G37) methyltransferase TrmD catalyzes m 1 G formation at position 37 in many tRNA isoacceptors and is essential in most bacteria, which positions it as a target for antibiotic development. In spite of its crucial role, little is known about TrmD in Pseudomonas aeruginosa (PaTrmD), an important human pathogen. Here we present detailed structural, substrate, and kinetic properties of PaTrmD. The mass spectrometric analysis confirmed the G36G37-containing tRNAs Leu(GAG), Leu(CAG), Leu(UAG), Pro(GGG), Pro(UGG), Pro(CGG), and His(GUG) as PaTrmD substrates. Analysis of steady-state kinetics with S-adenosyl-L-methionine (SAM) and tRNA Leu(GAG) showed that PaTrmD catalyzes the two-substrate reaction by way of a ternary complex, while isothermal titration calorimetry revealed that SAM and tRNA Leu(GAG) bind to PaTrmD independently, each with a dissociation constant of 14 ± 3 µM. Inhibition by the SAM analog sinefungin was competitive with respect to SAM (K i = 0.41 ± 0.07 µM) and uncompetitive for tRNA (K i = 6.4 ± 0.8 µM). A set of crystal structures of the homodimeric PaTrmD protein bound to SAM and sinefungin provide the molecular basis for enzyme competitive inhibition and identify the location of the bound divalent ion. These results provide insights into PaTrmD as a potential target for the development of antibiotics.
2017
The tRNA (m 1 G37) methyltransferase TrmD catalyzes m 1 G formation at position 37 in many tRNA isoacceptors and is essential in most bacteria, which positions it as a target for antibiotic development. In spite of its crucial role, little is known about TrmD in Pseudomonas aeruginosa (PaTrmD), an important human pathogen. Here we present detailed structural, substrate, and kinetic properties of PaTrmD. The mass spectrometric analysis confirmed the G36G37-containing tRNAs Leu(GAG), Leu(CAG), Leu(UAG), Pro(GGG), Pro(UGG), Pro(CGG), and His(GUG) as PaTrmD substrates. Analysis of steady-state kinetics with S-adenosyl-L-methionine (SAM) and tRNA Leu(GAG) showed that PaTrmD catalyzes the two-substrate reaction by way of a ternary complex, while isothermal titration calorimetry revealed that SAM and tRNA Leu(GAG) bind to PaTrmD independently, each with a dissociation constant of 14 ± 3 µM. Inhibition by the SAM analog sinefungin was competitive with respect to SAM (K i = 0.41 ± 0.07 µM) and uncompetitive for tRNA (K i = 6.4 ± 0.8 µM). A set of crystal structures of the homodimeric PaTrmD protein bound to SAM and sinefungin provide the molecular basis for enzyme competitive inhibition and identify the location of the bound divalent ion. These results provide insights into PaTrmD as a potential target for the development of antibiotics.
Journal of Medicinal Chemistry
Among the >120 modified ribonucleosides in the prokaryotic epitranscriptome, many tRNA modifications are critical to bacterial survival, which makes their synthetic enzymes ideal targets for antibiotic development. Here we performed a structure-based design of inhibitors of tRNA-(N 1 G37) methyltransferase, TrmD, which is an essential enzyme in many bacterial pathogens. On the basis of crystal structures of TrmDs from Pseudomonas aeruginosa and Mycobacterium tuberculosis, we synthesized a series of thienopyrimidinone derivatives with nanomolar potency against TrmD in vitro and discovered a novel active site conformational change triggered by inhibitor binding. This tyrosine-flipping mechanism is uniquely found in P. aeruginosa TrmD and renders the enzyme inaccessible to the cofactor S-adenosyl-L-methionine (SAM) and probably to the substrate tRNA. Biophysical and biochemical structure−activity relationship studies provided insights into the mechanisms underlying the potency of thienopyrimidinones as TrmD inhibitors, with several derivatives found to be active against Grampositive and mycobacterial pathogens. These results lay a foundation for further development of TrmD inhibitors as antimicrobial agents.
Journal of Molecular Biology, 2008
Methyltransferases (MTases) from the TrmI family catalyse the S-adenosyl-L-methionine (AdoMet)-dependent N 1 -methylation of tRNA adenosine 58. The crystal structure of Thermus thermophilus m 1 A 58 tRNA MTase (thTrmI) in complex with S-adenosyl-L-homocysteine was determined at 1.7 Å resolution. This structure is closely related to that of Mycobacterium tuberculosis TrmI (mycoTrmI), and their comparison enabled us to enlighten two grooves in the TrmI structure that are large enough and electrostatically compatible to accommodate one tRNA per face of TrmI tetramer. We have then conducted a biophysical study based on electrospray ionization mass spectrometry, site-directed mutagenesis and molecular docking. First, we confirmed the tetrameric oligomerisation state of TrmI and we showed that this protein remains tetrameric upon tRNA binding with formation of complexes involving one to two molecules of tRNA per TrmI tetramer. Secondly, three key residues for the methylation reaction were identified, the universally conserved D170, and two conserved aromatic residues Y78 and Y194. We then used molecular docking to position a N 9 -methyladenine in the active site of TrmI. The N 9 -methyladenine snugly fits in the catalytic cleft where the side chain of D170 acts as a bidentate ligand binding the amino moiety of AdoMet and the exocyclic amino group of the adenosine. Y194 interacts with the N 9 -methyladenine ring whereas Y78 can stabilize the sugar ring. From our results, we propose that the conserved residues that form the catalytic cavity (D170, Y78 and Y194) are essential to fashion an optimized shape of the catalytic pocket.
Proteins-structure Function and Bioinformatics, 2005
The Escherichia coli TrmB protein and its Saccharomyces cerevisiae ortholog Trm8p catalyze the S-adenosyl-L-methionine-dependent formation of 7-methylguanosine at position 46 (m7G46) in tRNA. To learn more about the sequence–structure–function relationships of these enzymes we carried out a thorough bioinformatics analysis of the tRNA:m7G methyltransferase (MTase) family to predict sequence regions and individual amino acid residues that may be important for the interactions between the MTase and the tRNA substrate, in particular the target guanosine 46. We used site-directed mutagenesis to construct a series of alanine substitutions and tested the activity of the mutants to elucidate the catalytic and tRNA-recognition mechanism of TrmB. The functional analysis of the mutants, together with the homology model of the TrmB structure and the results of the phylogenetic analysis, revealed the crucial residues for the formation of the substrate-binding site and the catalytic center in tRNA:m7G MTases. Proteins 2005. © 2005 Wiley-Liss, Inc.
Nucleic Acids Research, 2007
The Escherichia coli trmA gene encodes the tRNA(m 5 U54)methyltransferase, which catalyses the formation of m 5 U54 in tRNA. During the synthesis of m 5 U54, a covalent 62-kDa TrmA-tRNA intermediate is formed between the amino acid C324 of the enzyme and the 6-carbon of uracil. We have analysed the formation of this TrmA-tRNA intermediate and m 5 U54 in vivo, using mutants with altered TrmA. We show that the amino acids F188, Q190, G220, D299, R302, C324 and E358, conserved in the C-terminal catalytic domain of several RNA(m 5 U)methyltransferases of the COG2265 family, are important for the formation of the TrmA-tRNA intermediate and/or the enzymatic activity. These amino acids seem to have the same function as the ones present in the catalytic domain of RumA, whose structure is known, and which catalyses the formation of m 5 U in position 1939 of E. coli 23 S rRNA. We propose that the unusually high in vivo level of the TrmA-tRNA intermediate in wild-type cells may be due to a suboptimal cellular concentration of SAM, which is required to resolve this intermediate. Our results are consistent with the modular evolution of RNA(m 5 U)methyltransferases, in which the specificity of the enzymatic reaction is achieved by combining the conserved catalytic domain with different RNA-binding domains.
ACS Infectious Diseases, 2019
Bacterial tRNA modification synthesis pathways are critical to cell survival under stress and thus represent ideal mechanism-based targets for antibiotic development. One such target is the tRNA-(N 1 G37) methyltransferase (TrmD), which is conserved and essential in many bacterial pathogens. Here we developed and applied a widely applicable, radioactivity-free, bioluminescence-based highthroughput screen (HTS) against 116350 compounds from structurally diverse small-molecule libraries to identify inhibitors of Pseudomonas aeruginosa TrmD (PaTrmD). Of 285 compounds passing primary and secondary screens, a total of 61 TrmD inhibitors comprised of more than 12 different chemical scaffolds were identified, all showing submicromolar to low micromolar enzyme inhibitor constants, with binding affinity confirmed by thermal stability and surface plasmon resonance. S-Adenosyl-L-methionine (SAM) competition assays suggested that compounds in the pyridine-pyrazole-piperidine scaffold were substrate SAM-competitive inhibitors. This was confirmed in structural studies, with nuclear magnetic resonance analysis and crystal structures of PaTrmD showing pyridine-pyrazole-piperidine compounds bound in the SAM-binding pocket. Five hits showed cellular activities against Gram-positive bacteria, including mycobacteria, while one compound, a SAMnoncompetitive inhibitor, exhibited broad-spectrum antibacterial activity. The results of this HTS expand the repertoire of TrmD-inhibiting molecular scaffolds that show promise for antibiotic development.