Literature summary for 6.1.1.27 extracted from

Fukunaga, R.; Yokoyama, S.
Structural insights into the first step of RNA-dependent cysteine biosynthesis in archaea (2007), Nat. Struct. Mol. Biol., 14, 272-279.

Application

Application	Comment	Organism
biotechnology	the mutant SepRS-tRNA pairs may be useful for translational incorporation of O-phosphoserine into proteins in response to the stop codons UGA and UAG, so that it could ligate O-phosphoserine to a suppressor tRNA for genetic-code expansion	Archaeoglobus fulgidus

Cloned(Commentary)

Cloned (Comment)	Organism
overexpression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), overexpression of SeMet-labeled SepRS in Escherichia coli strain B834(DE3)	Archaeoglobus fulgidus

Crystallization (Commentary)

Crystallization (Comment)	Organism
SepRS tetramer in complex with tRNACys and O-phosphoserine, selenomethionine SAD method, and SepRS-tRNACys binary complex, 0.001 ml of 6-8 mg/ml protein in 10 mM Tris-HCl buffer, pH 8.0, 5 mM MgCl2, 150 mM NaCl and 5 mM 2-mercaptoethanol, and 2 mM O-phospho-L-serine, mixed with 0.001 ml reservoir solution containing 8% w/v PEG 6000 and 1.2 M NaCl, 20°C, cryoprotection by 22% v/v glycerol, X-ray diffraction structure determination and analysis at 2.6 A and 2.8 A resolution, respectively, modeling, determination of crystal structures of SepRS(E418N/E420N)-tRNAOpal-O-phosphoserine and SepRS(E418N/E420N)-tRNAAmber-O-phosphoserine at 3.2 and 3.3 resolutions, respectively	Archaeoglobus fulgidus
tRNA-free SepRS, hanging drop vapor diffusion method, 0.001 ml of protein solution mixed with 0.001 ml reservoir solution containing 11.25% w/v PEG 4,000, 75 mM sodium citrate, 75 mM N-(2-acetamido)iminodiacetic acid-NaOH buffer, pH 6.7, versus 1 ml reservoir solution, 20°C, cryoprotection by 22% v/v glycerol, X-ray diffraction structure determination and analysis at 3.6 A resolution, modeling	Methanocaldococcus jannaschii

Crystallization (Comment)

Organism

SepRS tetramer in complex with tRNACys and O-phosphoserine, selenomethionine SAD method, and SepRS-tRNACys binary complex, 0.001 ml of 6-8 mg/ml protein in 10 mM Tris-HCl buffer, pH 8.0, 5 mM MgCl2, 150 mM NaCl and 5 mM 2-mercaptoethanol, and 2 mM O-phospho-L-serine, mixed with 0.001 ml reservoir solution containing 8% w/v PEG 6000 and 1.2 M NaCl, 20°C, cryoprotection by 22% v/v glycerol, X-ray diffraction structure determination and analysis at 2.6 A and 2.8 A resolution, respectively, modeling, determination of crystal structures of SepRS(E418N/E420N)-tRNAOpal-O-phosphoserine and SepRS(E418N/E420N)-tRNAAmber-O-phosphoserine at 3.2 and 3.3 resolutions, respectively

Archaeoglobus fulgidus

tRNA-free SepRS, hanging drop vapor diffusion method, 0.001 ml of protein solution mixed with 0.001 ml reservoir solution containing 11.25% w/v PEG 4,000, 75 mM sodium citrate, 75 mM N-(2-acetamido)iminodiacetic acid-NaOH buffer, pH 6.7, versus 1 ml reservoir solution, 20°C, cryoprotection by 22% v/v glycerol, X-ray diffraction structure determination and analysis at 3.6 A resolution, modeling

Methanocaldococcus jannaschii

Protein Variants

Protein Variants	Comment	Organism
E418D	site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme	Archaeoglobus fulgidus
E418D/E420D	site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme	Archaeoglobus fulgidus
E418D/E420Q	site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme	Archaeoglobus fulgidus
E418N	site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme	Archaeoglobus fulgidus
E418N/E420D	site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme	Archaeoglobus fulgidus
E418N/E420N	site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme	Archaeoglobus fulgidus
E418N/E420N/T423V	site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme	Archaeoglobus fulgidus
E418N/E420Q	site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme	Archaeoglobus fulgidus
E418Q	site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme	Archaeoglobus fulgidus
E418Q/E420D	site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme	Archaeoglobus fulgidus
E418Q/E420N	site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme	Archaeoglobus fulgidus
E418Q/E420Q	site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme	Archaeoglobus fulgidus
E420D	site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme	Archaeoglobus fulgidus
E420K	site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme	Archaeoglobus fulgidus
E420N	site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme	Archaeoglobus fulgidus
E420Q	site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme	Archaeoglobus fulgidus
E420R	site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme	Archaeoglobus fulgidus
additional information	engineering of SepRS to recognize tRNACys mutants with the anticodons UCA and CUA on the basis of the structure, phosphoserine ligation activity of the wild-type and mutant SepRSs for tRNACys, overview	Archaeoglobus fulgidus
additional information	mutation of the three anticodon nucleotides, G34, C35 and A36, as well as the next residue, G37, reduces the phosphoserylation activity	Methanocaldococcus jannaschii

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
ATP + O-phospho-L-serine + tRNACys	Methanocaldococcus jannaschii	-	AMP + diphosphate + O-phospho-L-serine-tRNACys	-	?
ATP + O-phospho-L-serine + tRNACys	Archaeoglobus fulgidus	-	AMP + diphosphate + O-phospho-L-serine-tRNACys	-	?
additional information	Archaeoglobus fulgidus	two-step Cys-tRNACys formation: in organisms like Archaeoglobus fulgidus lacking a canonical cysteinyl-tRNA synthetase for the direct Cys-tRNACys formation, Cys-tRNACys is produced by the indirect pathway, in which the noncanonical O-phosphoseryl-tRNA synthetase, SepRS, ligates the noncanonical amino acid O-phosphoserine, Sep, to tRNACys, and the Sep-tRNA:Cys-tRNA synthase converts the produced Sep-tRNACys to Cys-tRNACys, overview, the SepRS/SepCysS pathway is the sole route for cysteine biosynthesis in the organism	?	-	?
additional information	Methanocaldococcus jannaschii	two-step Cys-tRNACys formation: in organisms like Methanococcus jannaschii lacking a canonical cysteinyl-tRNA synthetase for the direct Cys-tRNACys formation, Cys-tRNACys is produced by the indirect pathway, in which the noncanonical O-phosphoseryl-tRNA synthetase, SepRS, ligates the noncanonical amino acid O-phosphoserine, Sep, to tRNACys, and the Sep-tRNA:Cys-tRNA synthase converts the produced Sep-tRNACys to Cys-tRNACys, overview, the SepRS/SepCysS pathway is the sole route for cysteine biosynthesis in the organism	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Archaeoglobus fulgidus	O30126	-	-
Methanocaldococcus jannaschii	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant	Methanocaldococcus jannaschii
recombinant	Archaeoglobus fulgidus

Reaction

Reaction	Comment	Organism	Reaction ID
ATP + O-phospho-L-serine + tRNACys = AMP + diphosphate + O-phospho-L-seryl-tRNACys	anticodon recognition mechanism	Archaeoglobus fulgidus	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + O-phospho-L-serine + tRNAAmber	recognition of U34 and C35 of tRNAAmber by mutant E418N/E420N, no activity with wild-type SepRS, overview	Archaeoglobus fulgidus	AMP + diphosphate + O-phospho-L-serine-tRNAAmber	-	?
ATP + O-phospho-L-serine + tRNACys	-	Methanocaldococcus jannaschii	AMP + diphosphate + O-phospho-L-serine-tRNACys	-	?
ATP + O-phospho-L-serine + tRNACys	-	Archaeoglobus fulgidus	AMP + diphosphate + O-phospho-L-serine-tRNACys	-	?
ATP + O-phospho-L-serine + tRNACys	tRNA substrate from Escherichia coli, wheat germ and Saccharomyces cerevisiae in a mixture, the catalytic domain of SepRS recognizes the negatively charged side chain of O-phosphoserine at a noncanonical site, using the dipole moment of a conserved alpha-helix, the unique C-terminal domain specifically recognizes the anticodon GCA of tRNACys, overview	Archaeoglobus fulgidus	AMP + diphosphate + O-phospho-L-serine-tRNACys	-	?
ATP + O-phospho-L-serine + tRNAOpal	recognition of U34 and C35 of tRNAOpal by mutant E418N/E420N, no activity with wild-type SepRS, overview	Archaeoglobus fulgidus	AMP + diphosphate + O-phospho-L-serine-tRNAOpal	-	?
additional information	two-step Cys-tRNACys formation: in organisms like Archaeoglobus fulgidus lacking a canonical cysteinyl-tRNA synthetase for the direct Cys-tRNACys formation, Cys-tRNACys is produced by the indirect pathway, in which the noncanonical O-phosphoseryl-tRNA synthetase, SepRS, ligates the noncanonical amino acid O-phosphoserine, Sep, to tRNACys, and the Sep-tRNA:Cys-tRNA synthase converts the produced Sep-tRNACys to Cys-tRNACys, overview, the SepRS/SepCysS pathway is the sole route for cysteine biosynthesis in the organism	Archaeoglobus fulgidus	?	-	?
additional information	two-step Cys-tRNACys formation: in organisms like Methanococcus jannaschii lacking a canonical cysteinyl-tRNA synthetase for the direct Cys-tRNACys formation, Cys-tRNACys is produced by the indirect pathway, in which the noncanonical O-phosphoseryl-tRNA synthetase, SepRS, ligates the noncanonical amino acid O-phosphoserine, Sep, to tRNACys, and the Sep-tRNA:Cys-tRNA synthase converts the produced Sep-tRNACys to Cys-tRNACys, overview, the SepRS/SepCysS pathway is the sole route for cysteine biosynthesis in the organism	Methanocaldococcus jannaschii	?	-	?
additional information	two-step Cys-tRNACys formation: in organisms like Archaeoglobus fulgidus lacking a canonical cysteinyl-tRNA synthetase for the direct Cys-tRNACys formation, Cys-tRNACys is produced by the indirect pathway, in which the noncanonical O-phosphoseryl-tRNA synthetase, SepRS, ligates the noncanonical amino acid O-phosphoserine, Sep, to tRNACys, and the Sep-tRNA:Cys-tRNA synthase converts the produced Sep-tRNACys to Cys-tRNACys, overview, the SepRS/SepCysS pathway is the sole route for cysteine biosynthesis in the organism. RNA substrate specificity of wild-type and mutant enzymes, overview, structural insights into the first step of RNA-dependent cysteine biosynthesis, a two-step mechanism, in archaea	Archaeoglobus fulgidus	?	-	?

Subunits

Subunits	Comment	Organism
tetramer	-	Methanocaldococcus jannaschii
tetramer	-	Archaeoglobus fulgidus

Synonyms

Synonyms	Comment	Organism
CysRS	-	Methanocaldococcus jannaschii
CysRS	-	Archaeoglobus fulgidus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
50	-	assay at	Archaeoglobus fulgidus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.6	-	assay at	Archaeoglobus fulgidus