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

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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 .
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Methanosarcina mazei
UNIPROT: Q8PWY1
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Word Map
The expected taxonomic range for this enzyme is: Bacteria, Archaea
The taxonomic range for the selected organisms is: Methanosarcina mazei
Synonyms
pylrs, pyrrolysyl-trna synthetase, class ii aminoacyl-trna synthetase, pylsn, lysz-rs, pylsc, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
PylRS
PylS
287176
gene name
pyrrolysyl-tRNA synthetase
PATHWAY SOURCE
PATHWAYS
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].
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
-
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
-
-
?
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-nicotinoyl-L-lysine + tRNAPyl
AMP + diphosphate + Nepsilon-nicotinoyl-L-lysyl-tRNAPyl
show the reaction diagram
-
-
-
-
?
ATP + Boc-lysine + tRNAPyl
?
show the reaction diagram
-
Nepsilon-tert-butyloxycarbonyl-L-lysine
-
-
?
ATP + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
show the reaction diagram
ATP + N-alpha-acetyl-L-lysine + tRNAPyl
?
show the reaction diagram
-
-
-
-
?
ATP + N-alpha-benzyloxycarbonyl-L-lysine + tRNAPyl
?
show the reaction diagram
-
-
-
-
?
additional information
?
-
NATURAL SUBSTRATE
NATURAL 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 + L-pyrrolysine + tRNAPyl
AMP + diphosphate + L-pyrrolysyl-tRNAPyl
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
-
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
-
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
-
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
-
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
-
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
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
33000
-
x * 51000, full-length enzyme, SDS-PAGE, x * 33000, recombinant N-terminally truncated enzyme form PylRS(c270), SDS-PAGE
51000
-
x * 51000, full-length enzyme, SDS-PAGE, x * 33000, recombinant N-terminally truncated enzyme form PylRS(c270), SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
homodimer
-
x-ray crystallography
?
-
x * 51000, full-length enzyme, SDS-PAGE, x * 33000, recombinant N-terminally truncated enzyme form PylRS(c270), SDS-PAGE
additional information
-
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
-
apo-PylRS and PylRS complexes with different ligands, X-ray diffraction structure determination and analysis
-
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
-
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
-
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
-
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
-
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
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
L301M/Y306L/C348S/A315V
-
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
-
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
Y306A
-
mutation of PylRS drastically increases the in vitro aminoacylation activity for Nepsilon-benzyloxycarbonyl-L-lysine
Y306/Y384F
-
together with tRNAPyl the mutant enzyme provides a good yield of the in vivo amber-suppression product containing Nepsilon-benzyloxycarbonyl-L-lysine
Y384F
Y384F/A302T/N346V/C348W/V401L
-
the mutant specifically incorporates the cognate unnatural amino acid O-methyl-L-tyrosine into proteins
L309A/C348V
-
designated as ZLysRS has the L309A and C348V substitutions at the pyrrolysine binding pocket and three mutations at the other sites
additional information
PURIFICATION/commentary
ORGANISM
UNIPROT
LITERATURE
resource Q column 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
-
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
-
expression in Escherichia coli
-
gene pylS, encoded in the pyl gene cluster
-
expressed in HeLa cells
-
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
-
expression of wild-type and mutant PylRS in Escherichia coli
-
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)
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
molecular biology
-
the pyrrolysyl-tRNA synthetase/tRNAPyl suppression system can be used for the in vitro synthesis of peptides with nonnatural backbones
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Yanagisawa, T.; Ishii, R.; Fukunaga, R.; Nureki, O.; Yokoyama, S.
Crystallization and preliminary X-ray crystallographic analysis of the catalytic domain of pyrrolysyl-tRNA synthetase from the methanogenic archaeon Methanosarcina mazei
Acta Crystallogr. Sect. F
62
1031-1033
2006
Methanosarcina mazei
Manually annotated by BRENDA team
Kavran, J.M.; Gundllapalli, S.; O'Donoghue, P.; Englert, M.; Soll, D.; Steitz, T.A.
Structure of pyrrolysyl-tRNA synthetase, an archaeal enzyme for genetic code innovation
Proc. Natl. Acad. Sci. USA
104
11268-11273
2007
Methanosarcina mazei, Methanosarcina mazei (Q8PWY1)
Manually annotated by BRENDA team
Mukai, T.; Kobayashi, T.; Hino, N.; Yanagisawa, T.; Sakamoto, K.; Yokoyama, S.
Adding l-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases
Biochem. Biophys. Res. Commun.
371
818-822
2008
Methanosarcina mazei
Manually annotated by BRENDA team
Yanagisawa, T.; Ishii, R.; Fukunaga, R.; Kobayashi, T.; Sakamoto, K.; Yokoyama, S.
Crystallographic studies on multiple conformational states of active-site loops in pyrrolysyl-tRNA synthetase
J. Mol. Biol.
378
634-652
2008
Methanosarcina mazei, Methanosarcina mazei (Q8PWY1)
Manually annotated by BRENDA team
Wang, Y.S.; Russell, W.K.; Wang, Z.; Wan, W.; Dodd, L.E.; Pai, P.J.; Russell, D.H.; Liu, W.R.
The de novo engineering of pyrrolysyl-tRNA synthetase for genetic incorporation of L-phenylalanine and its derivatives
Mol. Biosyst.
7
714-717
2011
Methanocaldococcus jannaschii, Methanosarcina mazei
Manually annotated by BRENDA team
Takimoto, J.K.; Dellas, N.; Noel, J.P.; Wang, L.
Stereochemical basis for engineered pyrrolysyl-tRNA synthetase and the efficient in vivo incorporation of structurally divergent non-native amino acids
ACS Chem. Biol.
6
733-743
2011
Methanosarcina mazei, Methanosarcina mazei (Q8PWY1), Methanosarcina mazei DSM 3647 (Q8PWY1)
Manually annotated by BRENDA team
Yanagisawa. T.; Sumida, T.; Ishii, R.; Yokoyama, S.
A novel crystal form of pyrrolysyl-tRNA synthetase reveals the pre- and post-aminoacyl-tRNA synthesis conformational states of the adenylate and aminoacyl moieties and an asparagine residue in the catalytic site
Acta Crystallogr. Sect. D
69
5-15
2013
Methanosarcina mazei, Methanosarcina mazei (Q8PWY1), Methanosarcina mazei DSM 3647 (Q8PWY1)
Manually annotated by BRENDA team
Yanagisawa, T.; Ishii, R.; Fukunaga, R.; Kobayashi, T.; Sakamoto, K.; Yokoyama, S.
Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification
Chem. Biol.
15
1187-1197
2008
Methanosarcina mazei (Q8PWY1), Methanosarcina mazei DSM 3647 (Q8PWY1)
Manually annotated by BRENDA team
Lacey, V.; Louie, G.; Noel, J.; Wang, L.
Expanding the library and substrate diversity of the pyrrolysyl-tRNA synthetase to incorporate unnatural amino acids containing conjugated rings
ChemBioChem
14
2100-2105
2013
Methanosarcina mazei
Manually annotated by BRENDA team
Umehara, T.; Kim, J.; Lee, S.; Guo, L.T.; Söll, D.; Park, H.S.
N-Acetyl lysyl-tRNA synthetases evolved by a CcdB-based selection possess N-acetyl lysine specificity in vitro and in vivo
FEBS Lett.
586
729-733
2012
Methanosarcina mazei, Methanosarcina mazei (Q8PWY1), Methanosarcina mazei DSM 3647 (Q8PWY1)
Manually annotated by BRENDA team
Kobayashi, T.; Yanagisawa, T.; Sakamoto, K.; Yokoyama, S.
Recognition of non-alpha-amino substrates by pyrrolysyl-tRNA synthetase
J. Mol. Biol.
385
1352-1360
2009
Methanosarcina mazei
Manually annotated by BRENDA team
Wan, W.; Tharp, J.M.; Liu, W.R.
Pyrrolysyl-tRNA synthetase an ordinary enzyme but an outstanding genetic code expansion tool
Biochim. Biophys. Acta
1844
1059-1070
2014
Methanosarcina barkeri (Q6WRH6), Methanosarcina mazei (Q8PWY1), Methanosarcina mazei ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88 (Q8PWY1)
Manually annotated by BRENDA team
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