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Information on EC 6.1.1.26 - pyrrolysine-tRNAPyl ligase
for references in articles please use BRENDA:EC6.1.1.26
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EC Tree 6 Ligases
6.1 Forming carbon-oxygen bonds
6.1.1 Ligases forming aminoacyl-tRNA and related compounds
6.1.1.26 pyrrolysine-tRNAPyl ligase
IUBMB Comments
In organisms such as Methanosarcina barkeri that incorporate the modified amino acid pyrrolysine (Pyl) into certain methylamine methyltransferases, an unusual tRNAPyl, with a CUA anticodon, can be charged directly with pyrrolysine by this class II aminoacyl—tRNA ligase. The enzyme is specific for pyrrolysine as substrate as it cannot be replaced by lysine or any of the other natural amino acids .
The expected taxonomic range for this enzyme is: Bacteria, Archaea
- 6.1.1.26
- synthetases
- methanosarcina
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amber
- aminoacylation
- mazei
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ncaas
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anticodon
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aarss
- barkeri
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pylts
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histidyl-trnas
- hisrs
- hafniense
- desulfitobacterium
- methanosarcinaceae
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trnacua
- phers
- phenylalanyl-trna
- seryl-trna
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non-cognate
- glyrs
- molecular biology
- synthesis
showSynonyms
pylrs, pyrrolysyl-trna synthetase, class ii aminoacyl-trna synthetase, pylsn, lysz-rs, pylsc, more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
class II aminoacyl-tRNA synthetase
PylRS
PylS
PylSn
pyrrolysyl-tRNA synthetase
additional information
PylS
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in the Gram-positive bacterium Desulfitobacterium hafniense, PylS is divided into two separate proteins, PylSn and PylSc, corresponding to the N-terminal and C-terminal domains of the single PylS protein found in methanogens
pyrrolysyl-tRNA synthetase
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pyrrolysyl-tRNA synthetase
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pyrrolysyl-tRNA synthetase
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pyrrolysyl-tRNA synthetase
Methanosarcina mazei ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ATP + L-pyrrolysine + tRNAPyl = AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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PATHWAY SOURCE
PATHWAYS
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SYSTEMATIC NAME
IUBMB Comments
L-pyrrolysine:tRNAPyl ligase (AMP-forming)
In organisms such as Methanosarcina barkeri that incorporate the modified amino acid pyrrolysine (Pyl) into certain methylamine methyltransferases, an unusual tRNAPyl, with a CUA anticodon, can be charged directly with pyrrolysine by this class II aminoacyl---tRNA ligase. The enzyme is specific for pyrrolysine as substrate as it cannot be replaced by lysine or any of the other natural amino acids [1].
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid + tRNAPyl
AMP + diphosphate + 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoyl-tRNAPyl
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Substrates: catalytic efficiency (kcat/Km) is 9% of the catalytic efficiency for L-pyrrolysine
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ATP + 2-amino-6-(cyclopentanecarboxamido)hexanoic acid + tRNAPyl
AMP + diphosphate + 2-amino-6-(cyclopentanecarboxamido)hexanoyl-tRNAPyl
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Substrates: catalytic efficiency (kcat/Km) is 0.3% of the catalytic efficiency for L-pyrrolysine
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ATP + Boc-lysine + tRNAPyl
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Substrates: Nepsilon-tert-butyloxycarbonyl-L-lysine
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ATP + L-phenylalanine + tRNAPyl
AMP + diphosphate + L-phenylalanyl-tRNAPyl
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Substrates: mutant enzymes N346A/C348L, A302L/Y306M/N346S/C348L/Y384L, and A302F/Y306L/N346T/C348F/Y384L use L-phenylalanine as substrate
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ATP + N-alpha-benzyloxycarbonyl-L-lysine + tRNAPyl
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Substrates: -
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ATP + N-epsilon-cyclopentyloxycarbonyl-L-lysine + tRNAPyl
AMP + diphosphate + N-epsilon-cyclopentyloxycarbonyl-L-lysyl-tRNAPyl
ATP + N-epsilon-D-prolyl-L-lysine + tRNAPyl
AMP + diphosphate + N-epsilon-D-prolyl-L-lysyl-tRNAPyl
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Substrates: -
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ATP + Nepsilon-(N-methylanthraniloyl)-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-(N-methylanthraniloyl)-L-lysyl-tRNAPyl
ATP + Nepsilon-(tert-butyloxycarbonyl)-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-(tert-butyloxycarbonyl)-L-lysyl-tRNAPyl
ATP + Nepsilon-allyloxycarbonyl-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-allyloxycarbonyl-L-lysyl-tRNAPyl
ATP + Nepsilon-benzyloxycarbonyl-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-benzyloxycarbonyl-L-lysine-tRNAPyl
ATP + Nepsilon-cyclopentyloxycarbonyl-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-cyclopentyloxycarbonyl-L-lysyl-tRNAPyl
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Substrates: catalytic efficiency (kcat/Km) is 1.8% of the catalytic efficiency for L-pyrrolysine
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ATP + Nepsilon-nicotinoyl-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-nicotinoyl-L-lysyl-tRNAPyl
Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: pyrrolysyl-tRNA synthetase PylRS attaches L-pyrrolysine to its cognate tRNA, the special amber suppressor tRNAPyl, encoded by gene pylT
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: PylRS binds tRNA predominantly along the phosphate backbone of the T-loop, the D-stem and the acceptor stem, while no significant contacts with the anticodon arm occur, the tRNAPyl anticodon is not important for recognition by bacterial PylRS, overview
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: the bacterial PylRS displays a clear preference for the homologous cognate tRNA, substrate specificity, overview
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: the enzyme attaches L-pyrrolysine only to tRNA transcripts with a 3'-OH group at A76, whereas no aminoacylation at the 2'-OH group is detected
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: the enzyme is specific for L-pyrrolysine
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: the Methanosarcina acetivorans tRNAhis guanylyltranferase Thg1 gene contains an in-frame TAG codon. Its presence in Methanosarcina mRNA may lead to pyrrolysine incorporation achieved by Pyl-tRNAPyl, the product of pyrrolysyl-tRNA synthetase. Translation of Thg1 mRNA leads to a full-length, Pyl-containing, active enzyme as determined by immunoblotting, mass spectrometry, and biochemical analysis
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: direct charging of tRNA(CUA) with pyrrolysine in vitro and in vivo
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: Pyl-tRNAPyl insertion at UAG, a specialized mRNA motif is not essential for stopcodon recoding, unlike for selenocysteine incorporation
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: direct charging of tRNA(CUA), isolated from Methanosarcina acetivorans strain C2A, with pyrrolysine in vitro and in vivo, PylS activates pyrrolysine with ATP and ligates pyrrolysine to tRNACUA in vitro in reactions specific for pyrrolysine
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: 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
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: the enzyme uses a special amber suppressor tRNA, tRNAPyl, that presumably recognizes this UAG codon, and does not accept L-lysine or tRNALys as substrates, direct transfer of L-pyrroslysine to the UAG codon of tRNAPyl, overview
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: the enzyme attaches attaches L-pyrrolysine only to tRNA transcripts with a 3'-OH group at A76, whereas no aminoacylation at the 2'-OH group is detected
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Substrates: the N-terminal region of PylS influences complex formation with tRNAPyl
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Methanosarcina barkeri Fusaro / DSM 804
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Methanosarcina barkeri Fusaro / DSM 804
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Substrates: the enzyme uses a special amber suppressor tRNA, tRNAPyl, that presumably recognizes this UAG codon, and does not accept L-lysine or tRNALys as substrates, direct transfer of L-pyrroslysine to the UAG codon of tRNAPyl, overview
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Substrates: pyrrolysyl-tRNA synthetase PylRS attaches L-pyrrolysine to its cognate tRNA, the special amber suppressor tRNAPyl, encoded by gene pylT
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Substrates: substrate binding, PylRS utilizes a deep hydrophobic pocket for recognition of the Pyl side chain
Products: pyrrolysine-AMPbinds in a deep hydrophobic pocket, with its position coordinated by a hydrogen-bonding network with PylRS, binding structure, overview
Products: pyrrolysine-AMPbinds in a deep hydrophobic pocket, with its position coordinated by a hydrogen-bonding network with PylRS, binding structure, overview
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: the wild type enzyme recognizes the natural lysine derivative as well as many lysine analogs, including Nepsilon-(tert-butoxycarbonyl)-L-lysine (Boc-lysine), with diverse side chain sizes and structures
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Substrates: while the wild type enzyme has a negligible charging activity for N-acetyl-L-lysine, the mutant enzyme is able to acylate only N-acetyl-L-lysine (not natural amino acids) onto tRNAPyl
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Methanosarcina mazei ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88
Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Substrates: while the wild type enzyme has a negligible charging activity for N-acetyl-L-lysine, the mutant enzyme is able to acylate only N-acetyl-L-lysine (not natural amino acids) onto tRNAPyl
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Substrates: the archaeal enzyme does not distinguish between archaeal and bacterial tRNAPyl species, substrate specificity, overview
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ATP + N-acetyl-L-lysine + tRNAPyl
AMP + diphosphate + N-acetyl-L-lysyl-tRNAPyl
Substrates: while the wild type enzyme has a negligible charging activity for N-acetyl-L-lysine, the mutant enzyme is able to acylate only N-acetyl-L-lysine (not natural amino acids) onto tRNAPyl
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ATP + N-acetyl-L-lysine + tRNAPyl
AMP + diphosphate + N-acetyl-L-lysyl-tRNAPyl
Substrates: while the wild type enzyme has a negligible charging activity for N-acetyl-L-lysine, the mutant enzyme is able to acylate only N-acetyl-L-lysine (not natural amino acids) onto tRNAPyl
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ATP + N-epsilon-cyclopentyloxycarbonyl-L-lysine + tRNAPyl
AMP + diphosphate + N-epsilon-cyclopentyloxycarbonyl-L-lysyl-tRNAPyl
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Substrates: substrate specificity, ability of 24 mutant tRNA species to be aminoacylated by the enzyme with the pyrrolysine analog N-epsilon-cyclopentyloxycarbonyl-L-lysine, overview, the discriminator base G73 and the first base pair G1-C72 in the acceptor stem are major identity elements
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ATP + N-epsilon-cyclopentyloxycarbonyl-L-lysine + tRNAPyl
AMP + diphosphate + N-epsilon-cyclopentyloxycarbonyl-L-lysyl-tRNAPyl
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Substrates: -
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ATP + Nepsilon-(N-methylanthraniloyl)-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-(N-methylanthraniloyl)-L-lysyl-tRNAPyl
Substrates: -
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ATP + Nepsilon-(N-methylanthraniloyl)-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-(N-methylanthraniloyl)-L-lysyl-tRNAPyl
Substrates: -
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ATP + Nepsilon-(tert-butyloxycarbonyl)-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-(tert-butyloxycarbonyl)-L-lysyl-tRNAPyl
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ATP + Nepsilon-(tert-butyloxycarbonyl)-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-(tert-butyloxycarbonyl)-L-lysyl-tRNAPyl
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ATP + Nepsilon-allyloxycarbonyl-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-allyloxycarbonyl-L-lysyl-tRNAPyl
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ATP + Nepsilon-allyloxycarbonyl-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-allyloxycarbonyl-L-lysyl-tRNAPyl
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ATP + Nepsilon-benzyloxycarbonyl-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-benzyloxycarbonyl-L-lysine-tRNAPyl
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ATP + Nepsilon-benzyloxycarbonyl-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-benzyloxycarbonyl-L-lysine-tRNAPyl
Substrates: -
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additional information
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Substrates: enzyme activity with wild-type and different mutant tRNAPyls, overview, 3'-labeled tRNAPyl footprinting with S1 and T1 nuclease digestions, overview
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additional information
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Substrates: molecular dynamics simulations of the structures of PylRS and its complexes with tRNAPyl and activated pyrrolysine, overview. Identification of key residues and interactions leading to shortest paths of communication in the structure networks of DhPylRS. Dimeric hPylRS in different states of ligation: Sys1 as native DhPylRS, Sys2 as DhPylRS-Pyl-AMP, and Sys3 as DhPylRS-Pyl-AMP-tRNA, detailed overview
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additional information
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Substrates: the wild type enzyme shows no activity with L-phenylalanine
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additional information
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Substrates: Methanosarcina cells have two pathways for acylating the suppressor tRNAPyl to ensure efficient translation of the in-frame UAG codon in case of pyrrolysine deficiency and safeguard the biosynthesis of the proteins whose genes contain this special codon, L-pyrrolysine is found in the Methanosarcina barkeri monomethylamine methyltransferase protein in a position that is encoded by an in-frame UAG stop codon in the mRNA, overview
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additional information
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Substrates: pyrrolysine is required in e.g. methylamine methyltransferase MtmB, overview
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additional information
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Substrates: while pyrrolysine is the natural substrate of PylRS, lysine is not recognized by the enzyme
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additional information
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Substrates: structure of endogenous tRNAPyl, overview
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additional information
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Substrates: synthetic L-pyrrolysine is attached as a free molecule to tRNACUA by PylS, an archaeal class II aminoacyl-tRNA synthetase, inability of recombinant PylS-His6 to synthesize lysyltRNACUA
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additional information
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Substrates: unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate alpha-amine, and low selectivity toward the anticodon of tRNAPyl, overview. PylRS shows low selectivity toward the tRNA anticodon and low selectivity toward the Pyl alpha-amine
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additional information
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Substrates: the archaeal tRNAPyl features a secondary structure that significantly diverges from that of canonical tRNAs
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additional information
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Substrates: the recombinant pylS gene product charges tRNAPyl with Pyl, but the recombinant pylS gene product also succeeds in ligating [14C]Lys to tRNA. A mRNA secondary structure is definitely not necessary for the incorporation of Pyl at an amber codon. The binding of the side chain pyrroline of Pyl to the PylRS active site involves essentially van der Waals interactions. Replacing the side chain pyrroline with a similar size chemical component with a hydrophobic nature might retain the PylRS activity to aminoacylate tRNAPyl. PylRS weakly recognizes three non-canonical amino acids and mediates their incorporation into proteins at an amber codon in coordination with tRNAPyl, allowing the synthesis of proteins with site-specific lysine propionylation, butylation, and crotonylation
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additional information
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Methanosarcina barkeri Fusaro / DSM 804
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Substrates: Methanosarcina cells have two pathways for acylating the suppressor tRNAPyl to ensure efficient translation of the in-frame UAG codon in case of pyrrolysine deficiency and safeguard the biosynthesis of the proteins whose genes contain this special codon, L-pyrrolysine is found in the Methanosarcina barkeri monomethylamine methyltransferase protein in a position that is encoded by an in-frame UAG stop codon in the mRNA, overview
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additional information
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Methanosarcina barkeri Fusaro / DSM 804
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Substrates: structure of endogenous tRNAPyl, overview
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additional information
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Substrates: enzyme evolution study, PylRS can be placed in the aminoacyl-tRNA synthetase tree as the last known synthetase that evolved for genetic code expansion, pyrrolysine arose before the last universal common ancestral state
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additional information
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Substrates: enzyme evolution study, PylRS can be placed in the aminoacyl-tRNA synthetase tree as the last known synthetase that evolved for genetic code expansion, pyrrolysine arose before the last universal common ancestral state
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additional information
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Substrates: substrate-binding specificity of PylRS, overview
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additional information
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Substrates: substrate-binding specificity of PylRS, overview
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additional information
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Substrates: Nepsilon-(p-azidobenzoyl)-L-lysine, Nepsilon-biotinyl-L-lysine, and Nepsilon-(9-fluorenylmethoxycarbonyl)-L-lysine, which have even larger substituents at the N3-carbonyl group, are not detectably esterified to tRNAPyl. The enzyme exhibits no ligation activity for lysine derivatives without the Nepsilon-carbonyl group, such as Nepsilon-methyl-L-lysine, Nepsilon-dimethyl-L-lysine, Nepsilon-trimethyl-L-lysine, Nepsilon-isopropyl-L-lysine, Nepsilon-dansyl-L-lysine, Nepsilon-(o,p-dinitrophenyl)-L-lysine, Nepsilon-(p-toluenesulfonyl)-L-lysine, and Nepsilon-(DL-2-amino-2-carboxyethyl)-L-lysine, regardless of the size of the Nepsilon-substituent
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additional information
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Substrates: unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate alpha-amine, and low selectivity toward the anticodon of tRNAPyl, overview. PylRS shows low selectivity toward the tRNA anticodon and low selectivity toward the Pyl alpha-amine
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additional information
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Substrates: the structure of Methanosarcina mazei PylRS catalytic core reveals a deep hydrophobic pocket for the binding of Pyl. Residues A302, L305, Y306, L309, N346, C348, and W417 form a bulky cavity for the binding of the side chain (4R,5R)-4-methyl-pyrroline-5-caboxylate of Pyl. The Pyl side chain also forms two hydrogen bonds at the PylRS active site, with one involving the side chain amide nitrogen of N346 and the Pyl side chain amide oxygen and the other involving the pyrroline nitrogen and the phenolic oxygen of Y384. Residue Y384 is at a flexible loop region that is random in the absence of Pyl but serves as a cap for the binding of Pyl to the active site. PylRS displays remarkably high tolerance toward variations of the substrate side chain, especially when a variation is at the pyrroline region. PylRS recognizes desmethyl-Pyl and is able to direct its incorporation at amber codon when in coordination with tRNAPyl. The binding of the side chain pyrroline of Pyl to the PylRS active site involves essentially van der Waals interactions. Replacing the side chain pyrroline with a similar size chemical component with a hydrophobic nature might retain the PylRS activity to aminoacylate tRNAPyl
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additional information
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Methanosarcina mazei ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88
Substrates: unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate alpha-amine, and low selectivity toward the anticodon of tRNAPyl, overview. PylRS shows low selectivity toward the tRNA anticodon and low selectivity toward the Pyl alpha-amine
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additional information
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Methanosarcina mazei ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88
Substrates: the structure of Methanosarcina mazei PylRS catalytic core reveals a deep hydrophobic pocket for the binding of Pyl. Residues A302, L305, Y306, L309, N346, C348, and W417 form a bulky cavity for the binding of the side chain (4R,5R)-4-methyl-pyrroline-5-caboxylate of Pyl. The Pyl side chain also forms two hydrogen bonds at the PylRS active site, with one involving the side chain amide nitrogen of N346 and the Pyl side chain amide oxygen and the other involving the pyrroline nitrogen and the phenolic oxygen of Y384. Residue Y384 is at a flexible loop region that is random in the absence of Pyl but serves as a cap for the binding of Pyl to the active site. PylRS displays remarkably high tolerance toward variations of the substrate side chain, especially when a variation is at the pyrroline region. PylRS recognizes desmethyl-Pyl and is able to direct its incorporation at amber codon when in coordination with tRNAPyl. The binding of the side chain pyrroline of Pyl to the PylRS active site involves essentially van der Waals interactions. Replacing the side chain pyrroline with a similar size chemical component with a hydrophobic nature might retain the PylRS activity to aminoacylate tRNAPyl
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additional information
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Substrates: Nepsilon-(p-azidobenzoyl)-L-lysine, Nepsilon-biotinyl-L-lysine, and Nepsilon-(9-fluorenylmethoxycarbonyl)-L-lysine, which have even larger substituents at the N3-carbonyl group, are not detectably esterified to tRNAPyl. The enzyme exhibits no ligation activity for lysine derivatives without the Nepsilon-carbonyl group, such as Nepsilon-methyl-L-lysine, Nepsilon-dimethyl-L-lysine, Nepsilon-trimethyl-L-lysine, Nepsilon-isopropyl-L-lysine, Nepsilon-dansyl-L-lysine, Nepsilon-(o,p-dinitrophenyl)-L-lysine, Nepsilon-(p-toluenesulfonyl)-L-lysine, and Nepsilon-(DL-2-amino-2-carboxyethyl)-L-lysine, regardless of the size of the Nepsilon-substituent
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additional information
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Substrates: Methanosarcina species pyrrolysyl-tRNA synthetase (PylRS) attaches Pyl to its cognate amber suppressor tRNA. The introduction of two mutations (Y384F and Y306A) into PylRS generates a mutant, designated LysZ-RS, that is able to attach N-benzyloxycarbonyl-L-lysine (LysZ) to its cognate tRNA
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additional information
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Substrates: the amino-terminal extension present in archaeal PylRSs is dispensable for in vitro activity, but required for PylRS function in vivo
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
-
Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: pyrrolysyl-tRNA synthetase PylRS attaches L-pyrrolysine to its cognate tRNA, the special amber suppressor tRNAPyl, encoded by gene pylT
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: the enzyme attaches L-pyrrolysine only to tRNA transcripts with a 3'-OH group at A76, whereas no aminoacylation at the 2'-OH group is detected
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
-
Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
-
Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
-
Substrates: -
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?
ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: direct charging of tRNA(CUA) with pyrrolysine in vitro and in vivo
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: Pyl-tRNAPyl insertion at UAG, a specialized mRNA motif is not essential for stopcodon recoding, unlike for selenocysteine incorporation
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
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Substrates: the enzyme attaches attaches L-pyrrolysine only to tRNA transcripts with a 3'-OH group at A76, whereas no aminoacylation at the 2'-OH group is detected
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Methanosarcina barkeri Fusaro / DSM 804
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Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
-
Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
-
Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Substrates: pyrrolysyl-tRNA synthetase PylRS attaches L-pyrrolysine to its cognate tRNA, the special amber suppressor tRNAPyl, encoded by gene pylT
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Methanosarcina mazei ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88
Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Substrates: -
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ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
Substrates: -
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?
additional information
?
-
-
Substrates: the wild type enzyme shows no activity with L-phenylalanine
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additional information
?
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Substrates: Methanosarcina cells have two pathways for acylating the suppressor tRNAPyl to ensure efficient translation of the in-frame UAG codon in case of pyrrolysine deficiency and safeguard the biosynthesis of the proteins whose genes contain this special codon, L-pyrrolysine is found in the Methanosarcina barkeri monomethylamine methyltransferase protein in a position that is encoded by an in-frame UAG stop codon in the mRNA, overview
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additional information
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Substrates: pyrrolysine is required in e.g. methylamine methyltransferase MtmB, overview
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additional information
?
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Substrates: while pyrrolysine is the natural substrate of PylRS, lysine is not recognized by the enzyme
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additional information
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Substrates: unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate alpha-amine, and low selectivity toward the anticodon of tRNAPyl, overview. PylRS shows low selectivity toward the tRNA anticodon and low selectivity toward the Pyl alpha-amine
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additional information
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Methanosarcina barkeri Fusaro / DSM 804
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Substrates: Methanosarcina cells have two pathways for acylating the suppressor tRNAPyl to ensure efficient translation of the in-frame UAG codon in case of pyrrolysine deficiency and safeguard the biosynthesis of the proteins whose genes contain this special codon, L-pyrrolysine is found in the Methanosarcina barkeri monomethylamine methyltransferase protein in a position that is encoded by an in-frame UAG stop codon in the mRNA, overview
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additional information
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Substrates: enzyme evolution study, PylRS can be placed in the aminoacyl-tRNA synthetase tree as the last known synthetase that evolved for genetic code expansion, pyrrolysine arose before the last universal common ancestral state
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additional information
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Substrates: enzyme evolution study, PylRS can be placed in the aminoacyl-tRNA synthetase tree as the last known synthetase that evolved for genetic code expansion, pyrrolysine arose before the last universal common ancestral state
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?
additional information
?
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Substrates: unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate alpha-amine, and low selectivity toward the anticodon of tRNAPyl, overview. PylRS shows low selectivity toward the tRNA anticodon and low selectivity toward the Pyl alpha-amine
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additional information
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Methanosarcina mazei ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88
Substrates: unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate alpha-amine, and low selectivity toward the anticodon of tRNAPyl, overview. PylRS shows low selectivity toward the tRNA anticodon and low selectivity toward the Pyl alpha-amine
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additional information
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Substrates: the amino-terminal extension present in archaeal PylRSs is dispensable for in vitro activity, but required for PylRS function in vivo
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.39
2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid
-
pH 7.2, 37°C
0.67
N-epsilon-cyclopentyloxycarbonyl-L-lysine
-
pH 7.2, 37°C, recombinant enzyme
1 - 4
L-phenylalanine
-
mutant enzyme A302L/Y306M/N346S/C348L/Y384L, pH and temperature not specified in the publication
21
L-phenylalanine
-
mutant enzyme N346A/C348L, pH and temperature not specified in the publication
22.8
L-phenylalanine
-
mutant enzyme A302F/Y306L/N346T/C348F/Y384L, pH and temperature not specified in the publication
0.044
L-pyrrolysine
wild type enzyme, at 37°C in 50 mM KCl, 10 mM MgCl2, 5 mM ATP, and 5 mM dithiothreitol in 10 mM HEPES buffer, pH 7.2
0.044
L-pyrrolysine
-
wild type enzyme, at 37°C in 50 mM KCl, 10 mM MgCl2, 5 mM ATP, and 5 mM dithiothreitol in 10 mM HEPES buffer, pH 7.2
7.8
N-acetyl-L-lysine
pH and temperature not specified in the publication, mutant enzyme L301M/Y306L/C348S/A315V
35.3
N-acetyl-L-lysine
pH and temperature not specified in the publication, mutant enzyme L301M/Y306L/L309A/C348F
additional information
additional information
-
kinetics of pyrrolysylation of several mutant tRNAPyls, overview
-
0.00015 - 0.00098
additional information
-
equilibrium binding analysis, dissociation constants of the enzyme with tRNAPyl from different species, overview
-
0.00016 - 0.001
additional information
equilibrium binding analysis, dissociation constants of the enzyme with tRNAPyl from different species, overview
-
0.0053 - 0.0069
additional information
-
equilibrium binding analysis, dissociation constants of the enzyme with tRNAPyl from different species, overview
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.062
2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid
-
pH 7.2, 37°C
0.037
L-phenylalanine
-
mutant enzyme N346A/C348L, pH and temperature not specified in the publication
0.062
L-phenylalanine
-
mutant enzyme A302L/Y306M/N346S/C348L/Y384L, pH and temperature not specified in the publication
0.075
L-phenylalanine
-
mutant enzyme A302F/Y306L/N346T/C348F/Y384L, pH and temperature not specified in the publication
0.1
L-pyrrolysine
wild type enzyme, at 37°C in 50 mM KCl, 10 mM MgCl2, 5 mM ATP, and 5 mM dithiothreitol in 10 mM HEPES buffer, pH 7.2
0.325
L-pyrrolysine
wild type enzyme, at 37°C in 50 mM KCl, 10 mM MgCl2, 5 mM ATP, and 5 mM dithiothreitol in 10 mM HEPES buffer, pH 7.2
0.325
L-pyrrolysine
-
wild type enzyme, at 37°C in 50 mM KCl, 10 mM MgCl2, 5 mM ATP, and 5 mM dithiothreitol in 10 mM HEPES buffer, pH 7.2
0.00731
N-acetyl-L-lysine
pH and temperature not specified in the publication, mutant enzyme L301M/Y306L/C348S/A315V
0.0323
N-acetyl-L-lysine
pH and temperature not specified in the publication, mutant enzyme L301M/Y306L/L309A/C348F
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.16
2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid
-
pH 7.2, 37°C
0.0071
2-amino-6-(cyclopentanecarboxamido)hexanoic acid
-
pH 7.2, 37°C
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.2
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37