Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary 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.
    View publication on PubMed

Application

EC Number Application Comment Organism
6.1.1.27 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)

EC Number Cloned (Comment) Organism
6.1.1.27 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)

EC Number Crystallization (Comment) Organism
6.1.1.27 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
6.1.1.27 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

EC Number Protein Variants Comment Organism
6.1.1.27 E418D site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme Archaeoglobus fulgidus
6.1.1.27 E418D/E420D site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme Archaeoglobus fulgidus
6.1.1.27 E418D/E420Q site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme Archaeoglobus fulgidus
6.1.1.27 E418N site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme Archaeoglobus fulgidus
6.1.1.27 E418N/E420D site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme Archaeoglobus fulgidus
6.1.1.27 E418N/E420N site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme Archaeoglobus fulgidus
6.1.1.27 E418N/E420N/T423V site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme Archaeoglobus fulgidus
6.1.1.27 E418N/E420Q site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme Archaeoglobus fulgidus
6.1.1.27 E418Q site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme Archaeoglobus fulgidus
6.1.1.27 E418Q/E420D site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme Archaeoglobus fulgidus
6.1.1.27 E418Q/E420N site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme Archaeoglobus fulgidus
6.1.1.27 E418Q/E420Q site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme Archaeoglobus fulgidus
6.1.1.27 E420D site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme Archaeoglobus fulgidus
6.1.1.27 E420K site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme Archaeoglobus fulgidus
6.1.1.27 E420N site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme Archaeoglobus fulgidus
6.1.1.27 E420Q site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme Archaeoglobus fulgidus
6.1.1.27 E420R site-directed mutagenesis, the mutant shows reduced activity and altered tRNA substrate specificity, compared to the wild-type enzyme Archaeoglobus fulgidus
6.1.1.27 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
6.1.1.27 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)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
6.1.1.27 ATP + O-phospho-L-serine + tRNACys Methanocaldococcus jannaschii
-
 AMP + diphosphate + O-phospho-L-serine-tRNACys
-
 ?
6.1.1.27 ATP + O-phospho-L-serine + tRNACys Archaeoglobus fulgidus
-
 AMP + diphosphate + O-phospho-L-serine-tRNACys
-
 ?
6.1.1.27 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 ?
-
 ?
6.1.1.27 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

EC Number Organism UniProt Comment Textmining
6.1.1.16 no activity in Archaeoglobus fulgidus
-
-
-
6.1.1.16 no activity in Methanococcus jannaschii
-
-
-
6.1.1.27 Archaeoglobus fulgidus O30126
-
-
6.1.1.27 Methanocaldococcus jannaschii
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
6.1.1.27 recombinant Methanocaldococcus jannaschii
6.1.1.27 recombinant Archaeoglobus fulgidus

Reaction

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

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.1.1.27 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
-
 ?
6.1.1.27 ATP + O-phospho-L-serine + tRNACys
-
 Methanocaldococcus jannaschii AMP + diphosphate + O-phospho-L-serine-tRNACys
-
 ?
6.1.1.27 ATP + O-phospho-L-serine + tRNACys
-
 Archaeoglobus fulgidus AMP + diphosphate + O-phospho-L-serine-tRNACys
-
 ?
6.1.1.27 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
-
 ?
6.1.1.27 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
-
 ?
6.1.1.27 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 ?
-
 ?
6.1.1.27 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 ?
-
 ?
6.1.1.27 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

EC Number Subunits Comment Organism
6.1.1.27 tetramer
-
 Methanocaldococcus jannaschii
6.1.1.27 tetramer
-
 Archaeoglobus fulgidus

Synonyms

EC Number Synonyms Comment Organism
6.1.1.27 CysRS
-
 Methanocaldococcus jannaschii
6.1.1.27 CysRS
-
 Archaeoglobus fulgidus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
6.1.1.27 50
-
 assay at Archaeoglobus fulgidus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.1.1.27 7.6
-
 assay at Archaeoglobus fulgidus