Literature summary for 6.1.1.3 extracted from

Hussain, T.; Kruparani, S.P.; Pal, B.; Dock-Bregeon, A.C.; Dwivedi, S.; Shekar, M.R.; Sureshbabu, K.; Sankaranarayanan, R.
Post-transfer editing mechanism of a D-aminoacyl-tRNA deacylase-like domain in threonyl-tRNA synthetase from archaea (2006), EMBO J., 25, 4152-4162.

Cloned(Commentary)

Cloned (Comment)	Organism
overexpression of His-tagged wild-type and mutant D-aminoacyl-tRNA deacylase-like domains in Escherichia coli strain BL21(DE3), mutant M129K is located in inclusion bodies	Pyrococcus abyssi

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant D-aminoacyl-tRNA deacylase-like domain with bound Ser3AA, hanging drop vapor diffusion method, mixing of equal volumes of protein, ligand and reservoir solution, crystallization from 0.2 M (NH4)2SO4, 0.1 M sodium cacodylate, pH 6.5, and 30% PEG 8000 and 0.1 M HEPES, pH 7.0, and 25% PEG 3350, purified recombinant D-aminoacyl-tRNA deacylase-like domain with bound SerAMS, using 0.1 M Bis-Tris, pH 6.5, and 25% PEG 3350, and the serine Pab-NTDL-serine cocrystals from 0.1 M HEPES pH 7.0 and 25% PEG 8000, X-ray diffraction structure determination and analysis at 1.86-2.25 A resolution, overview	Pyrococcus abyssi

Crystallization (Comment)

Organism

purified recombinant D-aminoacyl-tRNA deacylase-like domain with bound Ser3AA, hanging drop vapor diffusion method, mixing of equal volumes of protein, ligand and reservoir solution, crystallization from 0.2 M (NH4)2SO4, 0.1 M sodium cacodylate, pH 6.5, and 30% PEG 8000 and 0.1 M HEPES, pH 7.0, and 25% PEG 3350, purified recombinant D-aminoacyl-tRNA deacylase-like domain with bound SerAMS, using 0.1 M Bis-Tris, pH 6.5, and 25% PEG 3350, and the serine Pab-NTDL-serine cocrystals from 0.1 M HEPES pH 7.0 and 25% PEG 8000, X-ray diffraction structure determination and analysis at 1.86-2.25 A resolution, overview

Pyrococcus abyssi

Protein Variants

Protein Variants	Comment	Organism
E134A	site-directed mutagenesis, the mutation has no effect on the deacylation activity	Pyrococcus abyssi
H83A	site-directed mutagenesis, the mutant possesses the editing activity albeit with a slower rate compared to the wild-type enzyme	Pyrococcus abyssi
K121A	no expression for the alanine mutant	Pyrococcus abyssi
K121M	site-directed mutagenesis, substitution of Lys121 to serine does not abolish Ser-tRNAThr deacylation activity	Pyrococcus abyssi
K121S	site-directed mutagenesis, substitution of Lys121 to serine results in a complete abolition of Ser-tRNAThr deacylation activity	Pyrococcus abyssi
M129K	site-directed mutagenesis, the mutant shows binding not only to L-serine but also to a variety of other L-amino acids that are tested in addition to binding to various D-amino acids, overview	Pyrococcus abyssi
Y120A	site-directed mutagenesis, the mutation has no effect on the deacylation activity	Pyrococcus abyssi

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	-	Pyrococcus abyssi

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + L-threonine + tRNAThr	Pyrococcus abyssi	-	AMP + diphosphate + L-threonyl-tRNAThr	-	?

Organism

Organism	UniProt	Comment	Textmining
Pyrococcus abyssi	Q9UZ14	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant D-aminoacyl-tRNA deacylase-like domains from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration	Pyrococcus abyssi

Renatured (Commentary)

Renatured (Comment)	Organism
recombinant mutant M129K D-aminoacyl-tRNA deacylase-like domain from inclusion bodies after expression in Escherichia coli strain BL21(DE3) in 0.1 M phosphate buffer at pH 8.0 containing 6 M guanidinium hydrochloride and 10 mM Tris-HCl, followed by nickel affinity chromatography, the denatured protein is refolded stepwise by dialysis in 0.1 M phosphate buffer, pH 8.0, containing 10 mM Tris-HCl	Pyrococcus abyssi

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + L-threonine + tRNAThr	-	Pyrococcus abyssi	AMP + diphosphate + L-threonyl-tRNAThr	-	?
ATP + L-threonine + tRNAThr	the main chains atoms of Tyr119 and Tyr120 are sufficient to prevent the deacylation of Thr-tRNAThr	Pyrococcus abyssi	AMP + diphosphate + L-threonyl-tRNAThr	-	?
additional information	mischarging of the enzyme with noncognate amino acids, overview, post-transfer editing mechanism of the D-aminoacyl-tRNA deacylase-like domain in the archaeal threonyl-tRNA synthetase, mechanistic insights into the removal of noncognate L-serine from tRNAThr, M129 is responsible for enantiomeric selection in DTD, Glu134 is involved in fixing the seryl moiety in the active site, overview	Pyrococcus abyssi	?	-	?

Synonyms

Synonyms	Comment	Organism
Threonyl-tRNA synthetase	-	Pyrococcus abyssi
ThrRS	-	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
7.2	-	asay at	Pyrococcus abyssi

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Pyrococcus abyssi