Information on EC 6.1.1.26 - pyrrolysine-tRNAPyl ligase

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

EC NUMBER
COMMENTARY hide
6.1.1.26
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RECOMMENDED NAME
GeneOntology No.
pyrrolysine-tRNAPyl ligase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + L-pyrrolysine + tRNAPyl = AMP + diphosphate + L-pyrrolysyl-tRNAPyl
show the reaction diagram
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-
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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
strain Fusaro, gene pylS
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Manually annotated by BRENDA team
gene pylS
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
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
show the reaction diagram
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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
show the reaction diagram
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catalytic efficiency (kcat/Km) is 0.3% of the catalytic efficiency for L-pyrrolysine
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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-phenylalanine + tRNAPyl
AMP + diphosphate + L-phenylalanyl-tRNAPyl
show the reaction diagram
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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 + 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
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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-
-
-
?
ATP + N-epsilon-cyclopentyloxycarbonyl-L-lysine + tRNAPyl
AMP + diphosphate + N-epsilon-cyclopentyloxycarbonyl-L-lysyl-tRNAPyl
show the reaction diagram
ATP + N-epsilon-D-prolyl-L-lysine + tRNAPyl
AMP + diphosphate + N-epsilon-D-prolyl-L-lysyl-tRNAPyl
show the reaction diagram
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-
-
-
?
ATP + Nepsilon-(N-methylanthraniloyl)-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-(N-methylanthraniloyl)-L-lysyl-tRNAPyl
show the reaction diagram
ATP + Nepsilon-(tert-butyloxycarbonyl)-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-(tert-butyloxycarbonyl)-L-lysyl-tRNAPyl
show the reaction diagram
ATP + Nepsilon-allyloxycarbonyl-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-allyloxycarbonyl-L-lysyl-tRNAPyl
show the reaction diagram
ATP + Nepsilon-benzyloxycarbonyl-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-benzyloxycarbonyl-L-lysine-tRNAPyl
show the reaction diagram
ATP + Nepsilon-cyclopentyloxycarbonyl-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-cyclopentyloxycarbonyl-L-lysyl-tRNAPyl
show the reaction diagram
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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
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
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.39
2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid
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pH 7.2, 37°C
5.6
2-amino-6-(cyclopentanecarboxamido)hexanoic acid
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pH 7.2, 37°C
1 - 22.8
L-phenylalanine
0.044 - 0.055
L-pyrrolysine
7.8 - 35.3
N-acetyl-L-lysine
0.67
N-epsilon-cyclopentyloxycarbonyl-L-lysine
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pH 7.2, 37°C, recombinant enzyme
0.5
N-epsilon-D-prolyl-L-lysine
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pH 7.2, 37°C, recombinant enzyme
0.55
Nepsilon-cyclopentyloxycarbonyl-L-lysine
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pH 7.2, 37°C
0.053
pyrrolysine
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pH 7.2, 37°C, recombinant enzyme
0.0029
tRNAPyl
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pH 7.2, 37°C, wild-type tRNAPyl
0.00015 - additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.062
2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid
Methanosarcina barkeri
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pH 7.2, 37°C
0.04
2-amino-6-(cyclopentanecarboxamido)hexanoic acid
Methanosarcina barkeri
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pH 7.2, 37°C
0.037 - 0.075
L-phenylalanine
0.1 - 0.325
L-pyrrolysine
0.00731 - 0.0323
N-acetyl-L-lysine
0.015
Nepsilon-cyclopentyloxycarbonyl-L-lysine
Methanosarcina barkeri
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pH 7.2, 37°C
0.19
tRNAPyl
Desulfitobacterium hafniense
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pH 7.2, 37°C, wild-type tRNAPyl
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.16
2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid
Methanosarcina barkeri
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pH 7.2, 37°C
194886
0.0071
2-amino-6-(cyclopentanecarboxamido)hexanoic acid
Methanosarcina barkeri
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pH 7.2, 37°C
194888
1.9
L-pyrrolysine
Methanosarcina barkeri
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pH 7.2, 37°C
63038
0.027
Nepsilon-cyclopentyloxycarbonyl-L-lysine
Methanosarcina barkeri
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pH 7.2, 37°C
194887
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)
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
?
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x * 51000, full-length enzyme, SDS-PAGE, x * 33000, recombinant N-terminally truncated enzyme form PylRS(c270), SDS-PAGE
homodimer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal strucuture of PylRS apoenzyme and in complex with tRNAPy, to 2.5 and 3.1 A resolution, respectively. The protein forms a dimer in the crystal and in solution
the apo 2.1 A resolution structure of the intact Desulfitobacterium hafniense PylSc protein and comparison to structures of the C-terminal truncated PylS from methanogenic species
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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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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 21°C, 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, 20°C, 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 16°C, 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
nickel-activated HiTrap column chromatography
purified by Ni-NTA chromatography and SDS gel electrophoresis
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recombinant His-tagged enzyme from Escherichia coli BL21(DE3)
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
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 His-tagged wild-type and mutant enzymes from Escherichia coli BL21(DE3)
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recombinant His6-tagged PylS from Escherichia coli strain BL21(DE3) by nickel affinity 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
resource Q column chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DhaPylSc transformed into Escherichia coli BL21 (DE3)
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expressed in Escherichia coli
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expressed in Escherichia coli BL21 cells
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expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli DH10beta cells; expression in Escherichia coli
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expressed in HeLa cells
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expression in Escherichia coli
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 variant pyrrolysyl-tRNA synthetase/tRNACUA Pyl pairs in Saccharomyces cerevisiae, tRNA mutant variants, overview
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expression of wild-type and mutant PylRS in Escherichia coli
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functional co-expression with tRNAPyl in Escherichia coli, the recombinant enzyme is active with substrate analogues N-epsilon-D-prolyl-L-lysine and N-epsilon-cyclopentyloxycarbonyl-L-lysine
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gene pylS, DNA and amino acid sequence determination and analysis, expression in Escherichia coli BL21(DE3) as His-tagged enzyme
gene pylS, expression as His6-tagged enzyme in Escherichia coli strain BL21 (DE3), addition of pyrrolysine to Escherichia coli cells expressing pylT, encoding tRNACUA, and pylS results in the translation of UAG in vivo as a sense codon, inability of PylS-His6 to synthesize lysyltRNACUA, co-expression with gene mtmB1
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gene pylS, expression in Escherichia coli BL21(DE3) as His-tagged enzyme
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gene pylS, expression of His-tagged wild-type and mutant enzymes in Escherichia coli BL21(DE3)
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gene pylS, expression of operon pylTSBCD in Escherichia coli strain BL21 Tuner(DE3)
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gene pylS, expression of the His-tagged enzyme in Escherichia coli strain BL21(DE3)
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
A302F/Y306L/N346T/C348F/Y384L
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the mutant acylates tRNAPyl with L-phenylalanine
A302L/Y306M/N346S/C348L/Y384L
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the mutant acylates tRNAPyl with L-phenylalanine
N346A/C348A
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the mutant displays specific recognition toward non-canonical amino acids and has a broad substrate spectrum with specific recognition of L-phenylalanine
N346A/C348L
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the mutant acylates tRNAPyl with L-phenylalanine
D2A
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site-directed mutagenesis, the mutant shows 95% reduced activity compared to the wild-type enzyme
D2A/K4A
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site-directed mutagenesis, the mutant shows almost completely reduced activity compared to the wild-type enzyme
D7A
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site-directed mutagenesis, the mutant shows only slightly reduced activity compared to the wild-type enzyme
G21L
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site-directed mutagenesis, the mutant shows only slightly reduced activity compared to the wild-type enzyme
H24A
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site-directed mutagenesis, the mutant shows 95% reduced activity compared to the wild-type enzyme
I26G
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site-directed mutagenesis, the mutant shows 80% reduced activity compared to the wild-type enzyme
K3A
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site-directed mutagenesis, the mutant shows 80% reduced activity compared to the wild-type enzyme
K4A
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site-directed mutagenesis, the mutant shows 95% reduced activity compared to the wild-type enzyme
R19A
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site-directed mutagenesis, the mutant shows only slightly reduced activity compared to the wild-type enzyme
S11A
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site-directed mutagenesis, the mutant shows 95% reduced activity compared to the wild-type enzyme
S11A/T13A
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site-directed mutagenesis, the mutant shows almost completely reduced activity compared to the wild-type enzyme
S18A
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site-directed mutagenesis, the mutant shows only slightly reduced activity compared to the wild-type enzyme
T13A
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site-directed mutagenesis, the mutant shows 95% reduced activity compared to the wild-type enzyme
W16A
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site-directed mutagenesis, the mutant shows 35% reduced activity compared to the wild-type enzyme
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
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
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
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
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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
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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
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Y306A
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mutation of PylRS drastically increases the in vitro aminoacylation activity for Nepsilon-benzyloxycarbonyl-L-lysine
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Y384F
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mutation increases the aminoacylation rate; the mutation increases the aminoacylation activity of the enzyme regardless of the amino acid substrate; the mutation increases the aminoacylation rate
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Y384F/A302T/N346V/C348W/V401L
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the mutant specifically incorporates the cognate unnatural amino acid O-methyl-L-tyrosine into proteins
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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
synthesis
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synthesis and genetic incorporation of aliphatic azides and alkynes into proteins using the natural pyrrolysyl tRNA synthetase/tRNACUA pair and the efficient bio-orthogonal labeling of these amino acids using [3+2] cycloaddition, i.e. click chemistry. Escherichia coli transformed with pBKPylS10 encoding pyrrolysyl tRNA synthetase and pMyo4TAGPylT-his610 encoding tRNACUA and a C-terminally hexahistidine tagged myoglobin gene with an amber codon at position 4 incorporates (2S)-2-amino-6-[[(prop-2-yn-1-yloxy)carbonyl]amino]hexanoic acid. The yield of protein containing (2S)-2-amino-6-[[(prop-2-yn-1-yloxy)carbonyl]amino]hexanoic acid is not improved by efforts to evolve the enzyme but is increased 5fold by increasing the concentration of (2S)-2-amino-6-[[(prop-2-yn-1-yloxy)carbonyl]amino]hexanoic acid 7.5fold