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Literature summary extracted from

  • Jaroensuk, J.; Wong, Y.H.; Zhong, W.; Liew, C.W.; Maenpuen, S.; Sahili, A.E.; Atichartpongkul, S.; Chionh, Y.H.; Nah, Q.; Thongdee, N.; McBee, M.E.; Prestwich, E.G.; DeMott, M.S.; Chaiyen, P.; Mongkolsuk, S.; Dedon, P.C.; Lescar, J.; Fuangthong, M.
    Crystal structure and catalytic mechanism of the essential m1G37 tRNA methyltransferase TrmD from Pseudomonas aeruginosa (2019), RNA, 25, 1481-1496 .
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
2.1.1.228 drug development the enzyme is a target for antibiotic development Pseudomonas aeruginosa

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.1.1.228 gene PA14_15990, sequence comparisons, recombinant expression of N-terminally His-tagged enzyme in Escherichia coli strain BL21(DE3) Rosetta T1R Pseudomonas aeruginosa

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.1.1.228 purified recombinant PaTrmD in complex with SAM, mixing equal volumes of protein and precipitant solution containing 0.1 M Tris-HCl, pH 8.4, 12.5% v/v MPD, 12.5% w/v PEG 1000, 12.5% w/v PEG 3350 and 5% w/v PEG 200, the enzyme inhibitor complex PaTrmD-sinefungin crystals are obtained by soaking the PaTrmD-SAM crystals in the precipitating solution supplemented with 2 mM sinefungin at 20°C for 2 h, for the complex with SFG and Mn2+, one crystal of PaTrmD-SAM is soaked in the precipitating solution supplemented with 10% (v/v) glycerol, 2 mM sinefungin and 5 mM MnCl2 at 20°C for 24 h, X-ray diffraction structure determination and analysis at 2.16 A resolution Pseudomonas aeruginosa

Protein Variants

EC Number Protein Variants Comment Organism
2.1.1.228 additional information attempts to construct a PA14_15990/trmD mutant in Pseudomonas aeruginosa strain PA14 using homologous recombination are not successful, due to the fact that the enzyme is essential in Pseudomonas aeruginosa. A temperature-sensitive allele of a Pseudomonas replicon, mSFts1 is used, to regulate expression of extra chromosomal trmD. Wild-type strain PA14 containing pBBR-trmD-mSFts1 and the trmD conditional knockout strain (trmD::Gm/pBBR-trmD-mSFts1) are both grown at a permissive temperature to maintain the stability of the plasmid (28 and 37°C) or at a non-permissive temperature to cause plasmid loss (46°C). The growth of the trmD conditional knockout strain at the permissive temperature is similar to wild-type PA14, while growth of the knockout strain is no longer observed at the non-permissive temperature Pseudomonas aeruginosa

Inhibitors

EC Number Inhibitors Comment Organism Structure
2.1.1.228 sinefungin and isosteric SAM analogue in which the methyl group of SAM is replaced by an amino group and the sulfur by a carbon atom, competitive inhibition with respect to SAM and uncompetitive for tRNA. 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. Crystal structure of PaTrmD bound to the SAM-competitive inhibitor sinefungin (SFG) is refined at a resolution of 2.45 A. In order to unambiguously locate the divalent ion, a third structure where crystals of PaTrmD are soaked with Mn2+ and SFG is determined. One Mn2+ near each of the bound sinefungin can be built. Mn2+ is coordinated by side-chain carbonyl group of E173', carboxylic groups of E121, D174' and D182' and the nitrogen atom of the sinefungin tail Pseudomonas aeruginosa

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.1.1.228 additional information
-
 additional information kinetics and thermodynamics analysis, overview. Steady-state kinetics with S-adenosyl-L-methionine (SAM) and tRNALeu(GAG) show that PaTrmD catalyzes the two-substrate reaction by way of a ternary-complex, while isothermal titration calorimetry revealed that SAM and tRNALeu(GAG) bind to PaTrmD independently, each with a dissociation constant of 0.014 mM Pseudomonas aeruginosa
2.1.1.228 0.0008
-
 guanine37 in tRNALeu(GAG) pH 7.5, 37°C, recombinant enzyme Pseudomonas aeruginosa
2.1.1.228 0.003
-
 S-adenosyl-L-methionine pH 7.5, 37°C, recombinant enzyme Pseudomonas aeruginosa

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.1.1.228 Mg2+ required, one PaTrmD monomer can bind to one Mg2+ ion. Mg2+ ion does not bind tightly to PaTrmD Pseudomonas aeruginosa
2.1.1.228 additional information it is reported that TrmD-catalyzed methyl transfer from SAM to the N1 atom of G37 from the tRNA is strongly dependent on the presence of divalent metal ions, with Mg2+ as the most physiologically relevant Pseudomonas aeruginosa

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.1.1.228 S-adenosyl-L-methionine + guanine37 in tRNA Pseudomonas aeruginosa
-
 S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNA
-
 ?
2.1.1.228 S-adenosyl-L-methionine + guanine37 in tRNA Pseudomonas aeruginosa UCBPP-PA14
-
 S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNA
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.1.1.228 Pseudomonas aeruginosa Q02RL6
-
-
2.1.1.228 Pseudomonas aeruginosa UCBPP-PA14 Q02RL6
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.1.1.228 recombinant N-terminally His-tagged enzyme from Escherichia coli strain BL21(DE3) Rosetta T1R by nickel affinity chromatography and gel filtration Pseudomonas aeruginosa

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.1.1.228 additional information substrate specificity, mass spectrometric analysis confirms the G36G37-containing tRNAs Leu(GAG), Leu(CAG), Leu(UAG), Pro(GGG), Pro(UGG), Pro(CGG), and His(GUG) as PaTrmD substrates, overview. PaTrmD catalyzes m1G formation in synthetic tRNA substrates. PaTrmD catalyzes m1G at position 37 in the tRNA anticodon loop. Preparation of tRNA substrates by in vitro transcription, product determination by mass spectrometry Pseudomonas aeruginosa ?
-
-
2.1.1.228 additional information substrate specificity, mass spectrometric analysis confirms the G36G37-containing tRNAs Leu(GAG), Leu(CAG), Leu(UAG), Pro(GGG), Pro(UGG), Pro(CGG), and His(GUG) as PaTrmD substrates, overview. PaTrmD catalyzes m1G formation in synthetic tRNA substrates. PaTrmD catalyzes m1G at position 37 in the tRNA anticodon loop. Preparation of tRNA substrates by in vitro transcription, product determination by mass spectrometry Pseudomonas aeruginosa UCBPP-PA14 ?
-
-
2.1.1.228 S-adenosyl-L-methionine + guanine37 in tRNA
-
 Pseudomonas aeruginosa S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNA
-
 ?
2.1.1.228 S-adenosyl-L-methionine + guanine37 in tRNA
-
 Pseudomonas aeruginosa UCBPP-PA14 S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNA
-
 ?
2.1.1.228 S-adenosyl-L-methionine + guanine37 in tRNAHis(GUG)
-
 Pseudomonas aeruginosa S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAHis(GUG)
-
 ?
2.1.1.228 S-adenosyl-L-methionine + guanine37 in tRNAHis(GUG)
-
 Pseudomonas aeruginosa UCBPP-PA14 S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAHis(GUG)
-
 ?
2.1.1.228 S-adenosyl-L-methionine + guanine37 in tRNALeu(CAG)
-
 Pseudomonas aeruginosa S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNALeu(CAG)
-
 ?
2.1.1.228 S-adenosyl-L-methionine + guanine37 in tRNALeu(CAG)
-
 Pseudomonas aeruginosa UCBPP-PA14 S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNALeu(CAG)
-
 ?
2.1.1.228 S-adenosyl-L-methionine + guanine37 in tRNALeu(GAG)
-
 Pseudomonas aeruginosa S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNALeu(GAG)
-
 ?
2.1.1.228 S-adenosyl-L-methionine + guanine37 in tRNALeu(UAG)
-
 Pseudomonas aeruginosa S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNALeu(UAG)
-
 ?
2.1.1.228 S-adenosyl-L-methionine + guanine37 in tRNAPro(CGG)
-
 Pseudomonas aeruginosa S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPro(CGG)
-
 ?
2.1.1.228 S-adenosyl-L-methionine + guanine37 in tRNAPro(GGG)
-
 Pseudomonas aeruginosa S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPro(GGG)
-
 ?
2.1.1.228 S-adenosyl-L-methionine + guanine37 in tRNAPro(UGG)
-
 Pseudomonas aeruginosa S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPro(UGG)
-
 ?
2.1.1.228 S-adenosyl-L-methionine + guanine37 in tRNAPro(UGG)
-
 Pseudomonas aeruginosa UCBPP-PA14 S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPro(UGG)
-
 ?

Subunits

EC Number Subunits Comment Organism
2.1.1.228 homodimer analysis of PaTrmD-SAM structure showing the dimer architecture and SAM-binding active site, overview Pseudomonas aeruginosa
2.1.1.228 More the PaTrmD monomer is formed by an N-terminal domain (NTD) that adopts a alpha/beta fold (residues 5-165) and a mostly helical C-terminal domain (CTD) spanning residues 178-250. These two domains are connected by a linker spanning residues 166-177 that acts as a lid covering up the active site Pseudomonas aeruginosa

Synonyms

EC Number Synonyms Comment Organism
2.1.1.228 m1G37 tRNA methyltransferase
-
 Pseudomonas aeruginosa
2.1.1.228 m1G37-forming tRNA methyltransferase
-
 Pseudomonas aeruginosa
2.1.1.228 PA14_15990
-
 Pseudomonas aeruginosa
2.1.1.228 PaTrmD
-
 Pseudomonas aeruginosa
2.1.1.228 TrmD
-
 Pseudomonas aeruginosa

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.1.1.228 37
-
 assay at Pseudomonas aeruginosa

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.1.1.228 0.217
-
 S-adenosyl-L-methionine pH 7.5, 37°C, recombinant enzyme Pseudomonas aeruginosa
2.1.1.228 0.217
-
 guanine37 in tRNALeu(GAG) pH 7.5, 37°C, recombinant enzyme Pseudomonas aeruginosa

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.1.1.228 7.5
-
 assay at Pseudomonas aeruginosa

Cofactor

EC Number Cofactor Comment Organism Structure
2.1.1.228 S-adenosyl-L-methionine binding structure analysis at 2.16 A resolution. PaTrmD shares functionally important amino acid residues involved in cofactor binding (Ser93-Gly96, Gly118, Ile123, Ser137, Gly145), tRNA binding (Gly60, Gly64, Ser203-His206) and catalytic activity (Asp54, Arg159, and Asp174) Pseudomonas aeruginosa

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
2.1.1.228 additional information
-
 additional information inhibition kinetics Pseudomonas aeruginosa
2.1.1.228 0.00041
-
 sinefungin versus S-adenosyl-L-methionine, pH 7.5, 37°C, recombinant enzyme Pseudomonas aeruginosa
2.1.1.228 0.0064
-
 sinefungin versus tRNA, pH 7.5, 37°C, recombinant enzyme Pseudomonas aeruginosa

General Information

EC Number General Information Comment Organism
2.1.1.228 additional information TrmD catalytic mechanism, overview. PaTrmD catalyzes the formation of m1G37 in tRNA by a ternary-complex mechanism in which tRNA and S-adenosyl-L-methionine can bind the protein independently. PaTrmD shares functionally important amino acid residues involved in cofactor binding (Ser93-Gly96, Gly118, Ile123, Ser137, Gly145), tRNA binding (Gly60, Gly64, Ser203-His206) and catalytic activity (Asp54, Arg159, and Asp174). Conformational changes are required to form a ternary complex with tRNA. PaTrmD catalyzes only the m1G modification in PA14 tRNAs that possess a G36G37 motif, the G36G37 motif is a substrate of PaTrmD. PaTrmD catalyzes m1G formation in synthetic tRNA substrates indicating that PaTrmD can use G36G37 containing tRNAs without other modifications as substrates. Enzyme structure modelling, overview Pseudomonas aeruginosa
2.1.1.228 physiological function m1G37 tRNA methyltransferase TrmD catalyzes m1G formation at position 37 in many tRNA isoacceptors and is essential Pseudomonas aeruginosa

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.1.1.228 72.2
-
 S-adenosyl-L-methionine pH 7.5, 37°C, recombinant enzyme Pseudomonas aeruginosa
2.1.1.228 270.8
-
 guanine37 in tRNALeu(GAG) pH 7.5, 37°C, recombinant enzyme Pseudomonas aeruginosa