Cloned (Comment) | Organism |
---|---|
gene pylS, DNA and amino acid sequence determination and analysis, expression in Escherichia coli BL21(DE3) as His-tagged enzyme | Methanosarcina thermophila |
gene pylS, expression in Escherichia coli BL21(DE3) as His-tagged enzyme | Desulfitobacterium hafniense |
gene pylS, expression of His-tagged wild-type and mutant enzymes in Escherichia coli BL21(DE3) | Methanosarcina barkeri |
Protein Variants | Comment | Organism |
---|---|---|
D2A | site-directed mutagenesis, the mutant shows 95% reduced activity compared to the wild-type enzyme | Methanosarcina barkeri |
D2A/K4A | site-directed mutagenesis, the mutant shows almost completely reduced activity compared to the wild-type enzyme | Methanosarcina barkeri |
D7A | site-directed mutagenesis, the mutant shows only slightly reduced activity compared to the wild-type enzyme | Methanosarcina barkeri |
G21L | site-directed mutagenesis, the mutant shows only slightly reduced activity compared to the wild-type enzyme | Methanosarcina barkeri |
H24A | site-directed mutagenesis, the mutant shows 95% reduced activity compared to the wild-type enzyme | Methanosarcina barkeri |
I26G | site-directed mutagenesis, the mutant shows 80% reduced activity compared to the wild-type enzyme | Methanosarcina barkeri |
K3A | site-directed mutagenesis, the mutant shows 80% reduced activity compared to the wild-type enzyme | Methanosarcina barkeri |
K4A | site-directed mutagenesis, the mutant shows 95% reduced activity compared to the wild-type enzyme | Methanosarcina barkeri |
additional information | construction of several truncation mutants, which show 90-99% reduced activity compared to the wild-type enzyme, overview | Methanosarcina barkeri |
R19A | site-directed mutagenesis, the mutant shows only slightly reduced activity compared to the wild-type enzyme | Methanosarcina barkeri |
S11A | site-directed mutagenesis, the mutant shows 95% reduced activity compared to the wild-type enzyme | Methanosarcina barkeri |
S11A/T13A | site-directed mutagenesis, the mutant shows almost completely reduced activity compared to the wild-type enzyme | Methanosarcina barkeri |
S18A | site-directed mutagenesis, the mutant shows only slightly reduced activity compared to the wild-type enzyme | Methanosarcina barkeri |
T13A | site-directed mutagenesis, the mutant shows 95% reduced activity compared to the wild-type enzyme | Methanosarcina barkeri |
W16A | site-directed mutagenesis, the mutant shows 35% reduced activity compared to the wild-type enzyme | Methanosarcina barkeri |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.00015 | 0.00098 | additional information | equilibrium binding analysis, dissociation constants of the enzyme with tRNAPyl from different species, overview | Methanosarcina barkeri | |
0.00016 | 0.001 | additional information | equilibrium binding analysis, dissociation constants of the enzyme with tRNAPyl from different species, overview | Methanosarcina thermophila | |
0.0053 | 0.0069 | additional information | equilibrium binding analysis, dissociation constants of the enzyme with tRNAPyl from different species, overview | Desulfitobacterium hafniense |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | - |
Desulfitobacterium hafniense |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-pyrrolysine + tRNAPyl | Methanosarcina barkeri | - |
AMP + diphosphate + L-pyrrolysyl-tRNAPyl | - |
? | |
ATP + L-pyrrolysine + tRNAPyl | Desulfitobacterium hafniense | - |
AMP + diphosphate + L-pyrrolysyl-tRNAPyl | - |
? | |
ATP + L-pyrrolysine + tRNAPyl | Methanosarcina thermophila | - |
AMP + diphosphate + L-pyrrolysyl-tRNAPyl | - |
? | |
additional information | Methanosarcina thermophila | the amino-terminal extension present in archaeal PylRSs is dispensable for in vitro activity, but required for PylRS function in vivo | ? | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Desulfitobacterium hafniense | - |
a strictly anaerobic bacterium | - |
Methanosarcina barkeri | - |
strains Fusaro and MS, gene pylS | - |
Methanosarcina thermophila | Q1L6A3 | gene pylS | - |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged enzyme from Escherichia coli BL21(DE3) | Desulfitobacterium hafniense |
recombinant His-tagged enzyme from Escherichia coli BL21(DE3) | Methanosarcina thermophila |
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli BL21(DE3) | Methanosarcina barkeri |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-pyrrolysine + tRNAPyl | - |
Methanosarcina barkeri | AMP + diphosphate + L-pyrrolysyl-tRNAPyl | - |
? | |
ATP + L-pyrrolysine + tRNAPyl | - |
Desulfitobacterium hafniense | AMP + diphosphate + L-pyrrolysyl-tRNAPyl | - |
? | |
ATP + L-pyrrolysine + tRNAPyl | - |
Methanosarcina thermophila | AMP + diphosphate + L-pyrrolysyl-tRNAPyl | - |
? | |
ATP + L-pyrrolysine + tRNAPyl | the archaeal enzyme does not distinguish between archaeal and bacterial tRNAPyl species, substrate specificity, overview | Methanosarcina thermophila | AMP + diphosphate + L-pyrrolysyl-tRNAPyl | - |
? | |
ATP + L-pyrrolysine + tRNAPyl | the archaeal enzyme does not distinguish between archaeal and bacterial tRNAPyl species, substrate specificity, overview, residues from the PylRS amino-terminal domain affect activity in vivo | Methanosarcina barkeri | AMP + diphosphate + L-pyrrolysyl-tRNAPyl | - |
? | |
ATP + L-pyrrolysine + tRNAPyl | the bacterial PylRS displays a clear preference for the homologous cognate tRNA, substrate specificity, overview | Desulfitobacterium hafniense | AMP + diphosphate + L-pyrrolysyl-tRNAPyl | - |
? | |
additional information | the amino-terminal extension present in archaeal PylRSs is dispensable for in vitro activity, but required for PylRS function in vivo | Methanosarcina thermophila | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | the PylRS sequence can be subdivided into three regions: the highly conserved class II aaRS catalytic core domain at the carboxy-terminal, the unique amino-terminal domain, and a highly variable region linking these two domains | Desulfitobacterium hafniense |
More | the PylRS sequence can be subdivided into three regions: the highly conserved class II aaRS catalytic core domain at the carboxy-terminal, the unique amino-terminal domain, and a highly variable region linking these two domains, residues from the PylRS amino-terminal domain affect activity in vivo | Methanosarcina barkeri |
More | the PylRS sequence can be subdivided into three regions: the highly conserved class II aaRS catalytic core domain at the carboxy-terminal, the unique amino-terminal domain, and a highly variable region linking these two domains, the amino-terminal extension present in archaeal PylRSs is dispensable for in vitro activity, but required for PylRS function in vivo | Methanosarcina thermophila |
Synonyms | Comment | Organism |
---|---|---|
PylRS | - |
Methanosarcina barkeri |
PylRS | - |
Desulfitobacterium hafniense |
PylRS | - |
Methanosarcina thermophila |
pyrrolysyl-tRNA synthetase | - |
Methanosarcina barkeri |
pyrrolysyl-tRNA synthetase | - |
Desulfitobacterium hafniense |
pyrrolysyl-tRNA synthetase | - |
Methanosarcina thermophila |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Methanosarcina barkeri |
37 | - |
assay at | Desulfitobacterium hafniense |
37 | - |
assay at | Methanosarcina thermophila |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.2 | - |
assay at | Methanosarcina barkeri |
7.2 | - |
assay at | Desulfitobacterium hafniense |
7.2 | - |
assay at | Methanosarcina thermophila |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Methanosarcina barkeri | |
ATP | - |
Desulfitobacterium hafniense | |
ATP | - |
Methanosarcina thermophila |