Literature summary for 2.1.1.228 extracted from

Wu, J.; Jia, Q.; Wu, S.; Zeng, H.; Sun, Y.; Wang, C.; Ge, R.; Xie, W.
The crystal structure of the Pyrococcus abyssi mono-functional methyltransferase PaTrm5b (2017), Biochem. Biophys. Res. Commun., 493, 240-245 .

Search for more literature for 2.1.1.228

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant overexpression of His6-tagged full-length PaTrm5b and PaTrm5b73-330 mutant in Escherichia coli strain BL21(DE3)	Pyrococcus abyssi

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant enzyme full-length PaTrm5b in the apo-form, sitting drop vapor diffusion method, 25°C, the sample and the well solution are mixed at a 1:1 volume ratio. The condition contains 10% w/v PEG 3350, 100 mM HEPES, pH 7.5, 100 mM Ca(OAc)2, and 100 mM KCl, iterative seeding is employed to obtain diffraction-quality crystals, X-ray diffraction structure determination and analysis at 3.3 A resolution, molecular replacement using the coordinates of the PaTrm5a structure (PDB 5HJM) as the search model, modeling. The removal or relocation of the His6-tag to the C-terminus fails to generate crystals, as does the removal of the D1 domain, although both constructs show comparable stability to the N-terminally His6-tagged PaTrm5b protein. Furthermore, the addition of SAM, SAH or other SAM analogues to the crystallization sample either generates tiny crystals unsuitable for data collection or no crystals altogether	Pyrococcus abyssi

Crystallization (Comment)

Organism

purified recombinant enzyme full-length PaTrm5b in the apo-form, sitting drop vapor diffusion method, 25°C, the sample and the well solution are mixed at a 1:1 volume ratio. The condition contains 10% w/v PEG 3350, 100 mM HEPES, pH 7.5, 100 mM Ca(OAc)2, and 100 mM KCl, iterative seeding is employed to obtain diffraction-quality crystals, X-ray diffraction structure determination and analysis at 3.3 A resolution, molecular replacement using the coordinates of the PaTrm5a structure (PDB 5HJM) as the search model, modeling. The removal or relocation of the His6-tag to the C-terminus fails to generate crystals, as does the removal of the D1 domain, although both constructs show comparable stability to the N-terminally His6-tagged PaTrm5b protein. Furthermore, the addition of SAM, SAH or other SAM analogues to the crystallization sample either generates tiny crystals unsuitable for data collection or no crystals altogether

Pyrococcus abyssi

Protein Variants

Protein Variants	Comment	Organism
additional information	construction of the D1-truncated PaTrm5b73-330 mutant using full-length Trm5b as the template	Pyrococcus abyssi

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
S-adenosyl-L-methionine + guanine37 in tRNAPhe	Pyrococcus abyssi	-	S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPhe	-	?

Organism

Organism	UniProt	Comment	Textmining
Pyrococcus abyssi	Q9V0Q0	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His6-tagged full-length PaTrm5b and PaTrm5b73-330 mutant fromEscherichia coli strain BL21(DE3) by nickel affinity and anion exchange chromatography, and dialysis	Pyrococcus abyssi

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
additional information	no activity of Trm5b with 7-[(3S)-(3-amino-3-carboxypropyl)]-4-demethylwyosine37 in tRNAPhe (cf. EC 2.1.1.282)	Pyrococcus abyssi	?	-	-
S-adenosyl-L-methionine + guanine37 in tRNAPhe	-	Pyrococcus abyssi	S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPhe	-	?

Subunits

Subunits	Comment	Organism
More	the PaTrm5b protein displays an open conformation	Pyrococcus abyssi

Synonyms

Synonyms	Comment	Organism
mono-functional methyltransferase	-	Pyrococcus abyssi
PAB_RS03940	-	Pyrococcus abyssi
PaTrm5b	-	Pyrococcus abyssi
trm5b	-	Pyrococcus abyssi

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Pyrococcus abyssi

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Pyrococcus abyssi

Cofactor

Cofactor	Comment	Organism	Structure
S-adenosyl-L-methionine	-	Pyrococcus abyssi

General Information

General Information	Comment	Organism
evolution	phylogenetic analyses revealed that the archaeal Trm5s can be grouped into three categories: Trm5a, Trm5b, and Trm5c, which all perform the N1-methylation of tRNAPhe G37. Trm5a exists in all crenarchaea. On the other hand, Trm5b is ubiquitously found in euryarchaeota, and is regarded as the original Trm5. Trm5c can be originated from euryarchaeota by horizontal gene transfer and exists in two crenarchaeal orders. In addition, both aTrm5b and aTrm5c contain an N-terminal domain named D1, which is responsible for the G19:C56 base pair recognition but may be absent from most aTrm5as. Despite the differences at the N-termini, all Trm5s have the Rossmann fold at the C-termini for catalysis, with the consensus NPPY motif located in the fourth beta-strand. The motif is known to position the nitrogen atom of G37 from the substrate, but specific sequences may vary from this consensus. Structure comparison of Methanococcus jannaschii MjTrm5b and PaTrm5b, overview	Pyrococcus abyssi
malfunction	Truncation of the N-terminal D1 domain leads to reduced tRNA binding as well as the methyltransfer activity of PaTrm5b	Pyrococcus abyssi
metabolism	the wyosine hypermodification found exclusively at G37 of tRNAPhe in eukaryotes and archaea is a very complicated process involving multiple steps and enzymes, and the derivatives are essential for the maintenance of the reading frame during translation. In the archaea Pyrococcus abyssi, two key enzymes from the Trm5 family, named PaTrm5a and PaTrm5b respectively, start the process by forming N1-methylated guanosine (m1G37). In addition, PaTrm5a catalyzes the further methylation of C7 on 4-demethylwyosine (imG-14) to produce isowyosine (imG2) at the same position (cf. EC 2.1.1.282)	Pyrococcus abyssi
physiological function	among various RNA types, tRNA is the most frequently modified type. One such modification in tRNAPhe is the methylation at N1 of G37 (m1G37), which is conserved among all three domains of life. The presence of m1G37 allows effective and rapid aminoacylation of certain archaeal tRNA species by cognate aminoacyl-tRNA synthetases, and prevents misacylation by noncognate aminoacyl-tRNA synthetases, as well as +1 frameshift during translation on the ribosome	Pyrococcus abyssi