Literature summary for 2.1.1.228 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 .

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Application

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

Cloned(Commentary)

Cloned (Comment)	Organism
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)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
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

Inhibitors	Comment	Organism	Structure
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]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
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
0.0008	-	guanine37 in tRNALeu(GAG)	pH 7.5, 37°C, recombinant enzyme	Pseudomonas aeruginosa
0.003	-	S-adenosyl-L-methionine	pH 7.5, 37°C, recombinant enzyme	Pseudomonas aeruginosa

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required, one PaTrmD monomer can bind to one Mg2+ ion. Mg2+ ion does not bind tightly to PaTrmD	Pseudomonas aeruginosa
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)

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

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas aeruginosa	Q02RL6	-	-
Pseudomonas aeruginosa UCBPP-PA14	Q02RL6	-	-

Purification (Commentary)

Purification (Comment)	Organism
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)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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	?	-	-
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	?	-	-
S-adenosyl-L-methionine + guanine37 in tRNA	-	Pseudomonas aeruginosa	S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNA	-	?
S-adenosyl-L-methionine + guanine37 in tRNA	-	Pseudomonas aeruginosa UCBPP-PA14	S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNA	-	?
S-adenosyl-L-methionine + guanine37 in tRNAHis(GUG)	-	Pseudomonas aeruginosa	S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAHis(GUG)	-	?
S-adenosyl-L-methionine + guanine37 in tRNAHis(GUG)	-	Pseudomonas aeruginosa UCBPP-PA14	S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAHis(GUG)	-	?
S-adenosyl-L-methionine + guanine37 in tRNALeu(CAG)	-	Pseudomonas aeruginosa	S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNALeu(CAG)	-	?
S-adenosyl-L-methionine + guanine37 in tRNALeu(CAG)	-	Pseudomonas aeruginosa UCBPP-PA14	S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNALeu(CAG)	-	?
S-adenosyl-L-methionine + guanine37 in tRNALeu(GAG)	-	Pseudomonas aeruginosa	S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNALeu(GAG)	-	?
S-adenosyl-L-methionine + guanine37 in tRNALeu(UAG)	-	Pseudomonas aeruginosa	S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNALeu(UAG)	-	?
S-adenosyl-L-methionine + guanine37 in tRNAPro(CGG)	-	Pseudomonas aeruginosa	S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPro(CGG)	-	?
S-adenosyl-L-methionine + guanine37 in tRNAPro(GGG)	-	Pseudomonas aeruginosa	S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPro(GGG)	-	?
S-adenosyl-L-methionine + guanine37 in tRNAPro(UGG)	-	Pseudomonas aeruginosa	S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPro(UGG)	-	?
S-adenosyl-L-methionine + guanine37 in tRNAPro(UGG)	-	Pseudomonas aeruginosa UCBPP-PA14	S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPro(UGG)	-	?

Subunits

Subunits	Comment	Organism
homodimer	analysis of PaTrmD-SAM structure showing the dimer architecture and SAM-binding active site, overview	Pseudomonas aeruginosa
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

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

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

Cofactor

Cofactor	Comment	Organism	Structure
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]

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

General Information

General Information	Comment	Organism
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
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]

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