Information on EC 6.1.1.26 - pyrrolysine-tRNAPyl ligase and Organism(s) Methanosarcina mazei and UniProt Accession Q8PWY1

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Methanosarcina mazei
UNIPROT: Q8PWY1


The expected taxonomic range for this enzyme is: Bacteria, Archaea


The taxonomic range for the selected organisms is: Methanosarcina mazei

EC NUMBER
COMMENTARY hide
6.1.1.26
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RECOMMENDED NAME
GeneOntology No.
pyrrolysine-tRNAPyl ligase
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Aminoacyl-tRNA biosynthesis
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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].
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
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sequence alignment indicated that full-length PylRS contains a C-terminal class II AARS catalytic core and an N-terminal domain that apparently does not share sequence homology with any structurally known protein domains. The three dimensional organization of the PylRS catalytic core resembles that of other synthetases from the Class II AARS family
physiological function
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the genetic incorporation of the 22nd proteinogenic amino acid, pyrolysine (Pyl) at amber codon is achieved by the action of pyrrolysyl-tRNA synthetase (PylRS) together with its cognate tRNAPyl
additional information
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along with its special CUA anticodon for the recognition of amber codon, the pylT transcript, tRNAPyl, has a distinct anticodon stem of six base pairs instead of five base pairs as observed in most tRNAs, a single base between D and anticodon stems, a single base between D and acceptor stems, and a three-base small variable arm
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
show the reaction diagram
ATP + N-acetyl-L-lysine + tRNAPyl
AMP + diphosphate + N-acetyl-L-lysyl-tRNAPyl
show the reaction diagram
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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 + Nepsilon-(N-methylanthraniloyl)-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-(N-methylanthraniloyl)-L-lysyl-tRNAPyl
show the reaction diagram
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?
ATP + Nepsilon-(tert-butyloxycarbonyl)-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-(tert-butyloxycarbonyl)-L-lysyl-tRNAPyl
show the reaction diagram
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-
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?
ATP + Nepsilon-allyloxycarbonyl-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-allyloxycarbonyl-L-lysyl-tRNAPyl
show the reaction diagram
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?
ATP + Nepsilon-benzyloxycarbonyl-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-benzyloxycarbonyl-L-lysine-tRNAPyl
show the reaction diagram
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?
ATP + Nepsilon-nicotinoyl-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-nicotinoyl-L-lysyl-tRNAPyl
show the reaction diagram
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?
ATP + Boc-lysine + tRNAPyl
?
show the reaction diagram
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Nepsilon-tert-butyloxycarbonyl-L-lysine
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?
ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
show the reaction diagram
ATP + N-alpha-acetyl-L-lysine + tRNAPyl
?
show the reaction diagram
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?
ATP + N-alpha-benzyloxycarbonyl-L-lysine + tRNAPyl
?
show the reaction diagram
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
show the reaction diagram
ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
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required
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
7.8 - 35.3
N-acetyl-L-lysine
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00731 - 0.0323
N-acetyl-L-lysine
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
33000
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x * 51000, full-length enzyme, SDS-PAGE, x * 33000, recombinant N-terminally truncated enzyme form PylRS(c270), SDS-PAGE
51000
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x * 51000, full-length enzyme, SDS-PAGE, x * 33000, recombinant N-terminally truncated enzyme form PylRS(c270), SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
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x-ray crystallography
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x * 51000, full-length enzyme, SDS-PAGE, x * 33000, recombinant N-terminally truncated enzyme form PylRS(c270), SDS-PAGE
additional information
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PylRS is mainly composed of two domains: the N-terminal RNA-binding domain and the C-terminal aaRS catalytic domain
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
1.75 A X-ray crystal structure of the enzyme complexed with O-methyl-L-tyrosine and a non-hydrolyzable ATP analogue; mutant Y384F/A302T/N346V/C348W/V401L in complex with O-methyl-L-tyrosine, hanging drop vapor diffusion method, using
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apo-PylRS and PylRS complexes with different ligands, X-ray diffraction structure determination and analysis
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crystal structures of a catalytic fragment of the enzyme complexed with N3-(tert-butyloxycarbonyl)-L-lysine and an ATP analog and with Nepsilon-allyloxycarbonyl-L-lysine reveals that the enzyme requires an Nepsilon-carbonyl group bearing a substituent with a certain size
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crystal structures of the PylRS catalytic fragment in the substrate-free, ATP analogue (AMPPNP)-bound, and AMPPNP/pyrrolysine-bound forms, compared with other PylRS structures
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hanging-drop vapour-diffusion method at 21C, the triclinic form crystals contain two PylRS dimers (four monomer molecules) in the asymmetric unit, in which the two subunits in one dimer each bind Nepsilon-(tert-butyloxycarbonyl)-L-lysyladenylate and the two subunits in the other dimer each bind AMP
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purified recombinant His-tagged N-terminally truncated enzyme form PylRS(c270) in complex with an ATP analogue AMP-PNP, hanging drop vapour diffusion method, in 100 mM sodium cacodylate, pH 6.8, containing 0.25 M NaCl, 5 mM MgSO4 and 5% w/v PEG 4000, 20C, hexagonal crystals, X-ray diffraction structure determination and analysis at 1.9-2.6 A resolution
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purified recombinant N-terminally His-tagged catalytic domain of PylRS complexed with either AMP-PNP, pyrrolysine-AMP plus pyrophosphate, or the pyrrolysine analogue N-epsilon-[(cylopentyloxy)carbonyl]-L-lysine plus ATP, vapour diffusion method, 10 mg/ml protein in 100 mM Tris, pH 7.0-8.0, 8-14% PEG 2000 monomethyl ether, 10 mM pyrrolysine, and10 mM AMP-PNP or other ligands, overnight at 16C, stabilization and cryoprotection by 5 mM EDTA, 10 mM AMP-PNP, 5 mM MgCl2, 30% ethylen glycol, and additional 2% PEG, hexagonal-shaped crystals, X-ray diffraction structure determination and analysis at 1.8 A resolution
the crystal structures of the enzyme reveals that it has a unique, large pocket for amino acid binding
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
resource Q column chromatography
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recombinant His-tagged N-terminally truncated enzyme form PylRS(c270) from Escherichia coli strain BL21(DE3) to homogeneity by affinity chromatography, hydrophobic interaction chromatography, and adsorption chromatography
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recombinant N-terminally His-tagged catalytic domain of the enzyme from Escherichia coli by two different steps of affinity chromatography, and gel filtration
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli DH10beta cells; expression in Escherichia coli
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expression in Escherichia coli
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gene pylS, encoded in the pyl gene cluster
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expressed in HeLa cells
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expression in Escherichia coli
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expression of tRNAPyl in Chinese hamster ovary cells. ZLysRS-tRNAPyl pair and GRB2(Am111)-FLAG expressed in HEK293 c-18 cells
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expression of wild-type and mutant PylRS in Escherichia coli
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gene pylS, phylogenetic analysis and phylogeny of subclass IIc aaRSs, overview, expression of the N-terminally His-tagged catalytic domain of the enzyme in Escherichia coli
overexpression of the N-terminally truncated enzyme form PylRS(c270) as N-terminally His-tagged protein in Escherichia coli strain BL21(DE3), expression of selenomethionine-labeled enzyme in Escherichia coli strain B834(DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
L301M/Y306L/C348S/A315V
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while the wild type enzyme has a negligible charging activity for N-acetyl lysine, the mutant enzyme is able to acylate only N-acetyl lysine (not natural amino acids) onto tRNAPyl
L301M/Y306L/L309A/C348F
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while the wild type enzyme has a negligible charging activity for N-acetyl lysine, the mutant enzyme is able to acylate only N-acetyl lysine (not natural amino acids) onto tRNAPyl
Y306/Y384F
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together with tRNAPyl the mutant enzyme provides a good yield of the in vivo amber-suppression product containing Nepsilon-benzyloxycarbonyl-L-lysine
Y306A
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mutation of PylRS drastically increases the in vitro aminoacylation activity for Nepsilon-benzyloxycarbonyl-L-lysine
Y384F/A302T/N346V/C348W/V401L
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the mutant specifically incorporates the cognate unnatural amino acid O-methyl-L-tyrosine into proteins
L309A/C348V
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designated as ZLysRS has the L309A and C348V substitutions at the pyrrolysine binding pocket and three mutations at the other sites
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
molecular biology
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the pyrrolysyl-tRNA synthetase/tRNAPyl suppression system can be used for the in vitro synthesis of peptides with nonnatural backbones