Information on EC 6.1.1.7 - alanine-tRNA ligase

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

EC NUMBER
COMMENTARY hide
6.1.1.7
-
RECOMMENDED NAME
GeneOntology No.
alanine-tRNA ligase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + L-alanine + tRNAAla = AMP + diphosphate + L-alanyl-tRNAAla
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Aminoacylation
esterification
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
tRNA charging
-
-
alanine metabolism
-
-
Aminoacyl-tRNA biosynthesis
-
-
SYSTEMATIC NAME
IUBMB Comments
L-alanine:tRNAAla ligase (AMP-forming)
-
CAS REGISTRY NUMBER
COMMENTARY hide
9031-71-4
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
gene alaS
SwissProt
Manually annotated by BRENDA team
overproducing strain
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
strain OT3, gene PH0574
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strains 159, Pn16
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
G68BWM7, G8C0Z7
UniProt
Manually annotated by BRENDA team
Thermus thermophilus HB8 / ATCC 27634 / DSM 579
i.e. Kluyveromyces polysporus
UniProt
Manually annotated by BRENDA team
Vanderwaltozyma polyspora ATCC 22028 / DSM 70294
i.e. Kluyveromyces polysporus
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + alanine + tRNAAla
?
show the reaction diagram
ATP + glycine + tRNAAla
AMP + diphosphate + glycyl-tRNAAla
show the reaction diagram
-
-
-
?
ATP + L-alanine + dual specific tRNAPhe
AMP + diphosphate + L-alanyl-tRNAPhe
show the reaction diagram
-
effects of deoxynucleotide substitutions in the tRNAPhe substrate on the enzyme activity, overview
-
?
ATP + L-alanine + dual specific tRNAPhe(17)
AMP + diphosphate + L-alanyl-tRNAPhe(17)
show the reaction diagram
-
site-specific modification leads to deoxynucleotide substituted yeast tRNA substrate, the backbone is interrupted on the 3'-side of nucleotide 17
-
?
ATP + L-alanine + dual specific tRNAPhe(38)
AMP + diphosphate + L-alanyl-tRNAPhe(38)
show the reaction diagram
-
site-specific modification leads to deoxynucleotide substituted yeast tRNA substrate, the backbone is interrupted on the 3'-side of nucleotide 38
-
?
ATP + L-alanine + dual specific tRNAPhe(57)
AMP + diphosphate + L-alanyl-tRNAPhe(57)
show the reaction diagram
-
site-specific modification leads to deoxynucleotide substituted yeast tRNA substrate, the backbone is interrupted on the 3'-side of nucleotide 57
-
?
ATP + L-alanine + tmRNA
AMP + diphosphate + L-alanyl-tmRNA
show the reaction diagram
ATP + L-alanine + tRNAAla
AMP + diphosphate + L-alanyl-tRNAAl
show the reaction diagram
-
-
-
?
ATP + L-alanine + tRNAAla
AMP + diphosphate + L-alanyl-tRNAAla
show the reaction diagram
ATP + L-alanine + tRNAAla
AMP + diphosphate + L-alanyl-tRNAla
show the reaction diagram
ATP + L-alanine + tRNAPyl
AMP + diphosphate + L-alanyl-tRNAPyl
show the reaction diagram
-
-
-
-
?
ATP + L-serine + tRNAAla
AMP + diphosphate + L-seryl-tRNAAla
show the reaction diagram
-
-
-
?
ATP + lipid II L-alanine + tRNAAla
?
show the reaction diagram
-
-
-
-
?
ATP + lipid II L-serine + tRNAAla
?
show the reaction diagram
-
-
-
-
?
additional information
?
-
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 + alanine + tRNAAla
?
show the reaction diagram
ATP + L-alanine + tRNAAla
AMP + diphosphate + L-alanyl-tRNAAl
show the reaction diagram
Q5JTZ9
-
-
-
?
ATP + L-alanine + tRNAAla
AMP + diphosphate + L-alanyl-tRNAAla
show the reaction diagram
ATP + L-alanine + tRNAAla
AMP + diphosphate + L-alanyl-tRNAla
show the reaction diagram
additional information
?
-
-
analysis of the deacylation activities of the wild type and five different Escherichia coli AlaRS editing site substitution mutants using the free-standing Pyrococcus horikoshii AlaX editing domain complexed with serine as a model and both Ser-tRNAAla and Ala-tRNAAla as substrates, overview
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-
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
-
or Mg2+ required
KCl
-
optimum concentration: 50-100 mM
Zinc
-
thightly bound
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
-
-
2-mercaptoethylamine
-
-
5'-O-[N-(L-alanyl)-sulfamoyl]adenosine
-
-
5'-O-[N-(L-glycinyl)sulfamoyl]-adenosine
-
-
5,5'-dithiobis(2-nitrobenzoate)
elongation factor Tu
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strongly inhibits the aminoacylation of tmRNA, deviation from the mechanism of aminoacyl ester bond protection upon formation of the canonical ternary complx between the elongation gactor Tu, nucleotide cofactor and aminoacyl-tRNA
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guanidine hydrochloride
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at 0.8 M guanidine hydrochloride, the enzyme forms a molten globule like intermediate, which is enzymatically inactive
L-cysteine
-
-
L-glutathione
-
-
monobromobimane
-
-
p-Hydroxymercuriphenylsulfonic acid
-
-
p-Substituted mercuribenzoate
-
-
sulfhydryl group containing substances
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-
Urea
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denaturation at 3.3 M, the Zn2+-depleted enzyme is much more sensitive to denaturation by urea than the native enzyme
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2,6-dichlorophenol-indophenol
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enhances activity
Activator macromolecule
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260000-350000 MW, decreases Km values for ATP and Ala
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spermine
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stimulates only at low Mg2+ concentrations
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.23 - 1.41
alanine
0.067 - 1.7
ATP
26 - 493
glycine
0.05 - 502
L-alanine
31 - 600
L-serine
0.0331 - 0.04
lipid II L-Ala
0.125 - 0.146
lipid II L-Ser
0.00063
liver tRNA
-
Bombyx mori tRNA
-
0.024
tmRNA
-
pH 7.6, 37C, recombinant His-tagged enzyme
-
0.023
tRNA
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mutant AlaRS(K73E), aminoacylation
0.000104 - 0.028
tRNAAla
0.0021
tRNAPhe
-
native dual specific tRNAPhe derivative substrate from Saccharomyces cerevisiae, pH 7.5, 37C
0.0019
tRNAPhe(17)
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tRNAPhe mutant tRNAPhe(17), pH 7.5, 37C
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0.0031
tRNAPhe(38)
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tRNAPhe mutant tRNAPhe(38), pH 7.5, 37C
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0.017
tRNAPhe(57)
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tRNAPhe mutant tRNAPhe(57), pH 7.5, 37C
-
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
79 - 120
ATP
0.0035 - 3.7
glycine
0.0074 - 1.4
L-alanine
0.0028 - 2.4
L-serine
0.0635 - 0.071
lipid II L-Ala
0.0166
lipid II L-Ser
-
strain 159; strain Pn16
0.043 - 0.1
tmRNA
-
0.19 - 2.2
tRNAAla
0.25
tRNAPhe
-
native dual specific tRNAPhe substrate from Saccharomyces cerevisiae, pH 7.5, 37C
0.25
tRNAPhe(17)
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tRNAPhe mutant tRNAPhe(17), pH 7.5, 37C
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0.46
tRNAPhe(38)
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tRNAPhe mutant tRNAPhe(38), pH 7.5, 37C
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0.12
tRNAPhe(57)
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tRNAPhe mutant tRNAPhe(57), pH 7.5, 37C
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
-
the wild-type AlaRS editing domain deacylates Ser-tRNAAla with a kcat/Km of 66 mM/s, equivalent to a rate enhancement of 6000 over the rate of enzyme-independent deacylation but only 12.2fold greater than the rate with Ala-tRNAAla. While the E664A and T567G substitutions only minimally decrease kcat/Km, Q584H, I667E, and C666A AlaRS are more compromised in activity, with decreases in kcat/Km in the range of 6fold, 6.6fold, and 15fold. C666A AlaRS is 1.7fold more active on Ala-tRNAAla relative to Ser-tRNAAl. Deacylation rates of Ser-tRNAAla and Ala-tRNAAla in the absence of enzyme are determined by fitting the progress curves to equations describing a first-order decay
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0000002
5'-O-[N-(L-alanyl)-sulfamoyl]adenosine
-
in 20 mM Tris-HCl (pH 8.0), at 30C
0.000004
5'-O-[N-(L-glycinyl)sulfamoyl]-adenosine
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in 20 mM Tris-HCl (pH 8.0), at 30C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0007
-
substrate: lipid II L-Ser, strain 159
0.0009
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substrate: lipid II L-Ser, strain Pn16
0.0087
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substrate: lipid II L-Ala, strain Pn16
0.011
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substrate: lipid II L-Ala, strain 159
0.027
-
alanyl-tRNA synthesis
0.105
-
purified enzyme
0.114
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diphosphate-ATP exchange
0.168
-
alanyl-tRNA synthetase activity
2.65
-
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4 - 8.4
-
-
8.5
-
ATP-diphosphate exchange
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.4 - 7.5
-
6.4: about 60% of maximal activity, 7.5: optimum
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
aminoacylation
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10 - 37
-
independent of temperature in this range
25 - 75
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half-maximal activity at 25C and 75C with substrate tRNAAla, a sharp 345fold drop of activity occurs at 70C, temperature profile
35 - 65
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half-maximal activity at 35C and 65C with substrate tmRNA, a sharp 345fold drop of activity occurs at 40C, temperature profile
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
posterior
Manually annotated by BRENDA team
additional information
-
isozyme pattern of expression and localization, overview
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
PDB
SCOP
CATH
ORGANISM
UNIPROT
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126)
Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126)
Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126)
Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
18152
1 * 18152, crystal structure analysis, two-domain structure consisting of seven antiparallel beta-sheets and six helices
96000
-
1 * 96000, recombinant His-tagged enzyme, SDS-PAGE
101000 - 121000
-
sucrose density gradient centrifugation
110000
-
1 * 110000, SDS-PAGE
115000
-
1 * 115000, SDS-PAGE, gel filtration in 6 M guanidine-HCl
116000
-
gel filtration
118000
-
1 * 118000, denaturing electrophoresis
128000
-
1 * 128000, SDS-PAGE
131000
-
gel filtration
141000
-
gel filtration, cytoplasmic enzyme
151000
-
gel filtration
153000
-
gel filtration
162000
-
gel filtration, mitochondrial enzyme
179000
-
Zn2+-depleted enzyme, sedimentation coefficient and analytical ultracentrifugation
186000
195000
-
gel filtration
205000
size exclusion chromatography
245000
-
gel filtration
360000
-
gel filtration
380000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
-
2 * 96000, analytical ultracentrifugation
monomer
tetramer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
453 amino acid catalytic fragment Aa-AlaRS453
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cocrystal structures with Mg2+-ATP, L-alanine, glycine and L-serine with each separately
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AlaRS-DELTAC, comprising the aminoacylation, tRNA-recognition, and editing domains, and AlaRS-C, comprising the dimerization domain. The aminoacylation/tRNA-recognition domains contain an insertion incompatible with the class-specific tRNA-binding mode. The editing domain is fixed tightly via hydrophobic interactions to the aminoacylation/tRNA-recognition domains, on the side opposite from that in threonyl-tRNA synthetase. A groove formed between the aminoacylation/tRNA-recognition domains and the editing domain appears to be an alternative tRNA-binding site. The amino acid residues required for the G3:U70 base pair recognition are mapped in this groove. The dimerization domain consists of helical and globular subdomains. The helical subdomain mediates dimerization by forming a helixloophelix zipper. The globular subdomain, which is important for the aminoacylation and editing activities, has a positively-charged face suitable for tRNA binding
purified recombinant full-length enzyme, N-terminal domain, and C-terminal domain, hanging-drop vapour-diffusion method, mixing of 0.001 ml of protein and reservoir solution, and equilibration against 0.5 ml of reservoir solution at 20C, for crystallization of the AlaRS-FLtRNAAla complex, tRNAAla is heated at 80C for 5 min and is gradually cooled to room temperature for refolding, in presence of 1 mM AMP-PNP, different methods for the different protein samples are used, overview, X-ray diffraction structure determination and analysis at 3.2-3.5 A resolution
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molecular replacement with the apo-AlaRS structure from Aquifex aeolicus,Protein Data Bank accession code 1RIQ, as the initial model
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of small fragments
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purified recombinant appended C-terminal domain (C-Ala), two different crystal forms, each of which is specific to a particular condition are achieved, one of these crystal forms harbors the monomer and is obtained using 0.1 M Tris, pH 8.5, and 25% w/v PEG 3350, whereas the other captures a dimer using 0.2 M ammonium acetate, 0.1 M Tris, pH 8.5, and 25% w/v PEG 3350, X-ray diffraction structure determination and analysis
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monomer structures of C-terminally truncated AlaRS, with both activation and editing domains in the apo form, in complex with an Ala-AMP analog, and in a high-resolution lysine-methylated form. Docking of the editing domain to the activation domain occurs opposite from the predicted tRNA-binding surface. The editing site is positioned more than 35 A from the activation site. Results suggest that tRNA translocation via a canonical CCA flipping is unlikely to occur in AlaRS. Zinc binds in the editing site, in which the specific coordination of zinc will be facilitated by a conserved GGQ motif
purified recombinant wild-type and selenomethionine-labeld AlaRS editing-domain homolog, PH0574, 37.6 mg/ml protein, 27.5%w/v PEG 4000, and 100 mM MES-Na, pH 6.3, using a full-automatic protein crystallization and observation system, X-ray diffraction structure determination and analysis at 1.45 A resolution
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
65
stable up to
85
denaturation at
additional information
-
the Zn2+-depleted enzyme shows reduced viscosity at 10C compared to 37C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
sensitive to repeated freezing and thawing
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Zn2+ stabilizes the enzyme
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 50% glycerol, stable for at least 1 year
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-80C, 3 months, retains approximately 90% of activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni-NTA agarose column chromatography, and gel filtration
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nickel-chelating Sepharose resin column chromatography
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partial, from a multienzyme complex containing at least 16 aminoacyl-tRNA synthetase activities
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purified by Ni-NTA chromatography and SDS gel electrophoresis
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recombinant full-length AlaRS and AlaRS domains from Escherichia coli strain BL21 by anion exchange chromatography followed by hydrophobic interaction and adsorption chromatography, respectively
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recombinant His-tagged wild-type and mutant enzmyes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
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recombinant His6-tagged full-length enzyme and C-terminal domain C-Ala from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and dialysis, followed by tag cleavage through TEV protease, tag and TEV protease are removed by another turn of nickel affinity chromatography, and ultrafiltration and dialysis
recombinant His6-tagged wild-type and chimeric fusion enzymes by nickel affinity chromatography and dialysis
recombinant His6-tagged wild-type and chimeric fusion enzymes by nickel affinity chromatography and dialysis; recombinant His6-tagged wild-type and chimeric fusion enzymes by nickel affinity chromatography and dialysis
recombinant PH0574, from Escherichia coli strain BL21 by two steps of anion exchange chromatography, hydroxyapatite chromatography, and gel filtration
Superose 12 column chromatography, Q Sepharose column chromatography and Sephacryl S200 gel filtration
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using Ni-Sepharose columns and anion-exchange chromatography. The yield of purified protein is 4-5 mg of protein per litre of culture, judged to be above 95% purity by SDS-PAGE
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
deletion mutants with reduced aminoacylation efficiency, expressed in Escherichia coli strain BL21
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expressed in Escherichia coli BL21(DE3) cells
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expressed in Escherichia coli BL21(deltaDE3) cells
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expressed in Escherichia coli BL21DE3star cells
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expressed in HEK-293T cells
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expression in Escherichia coli
expression in Escherichia coli strain BL21
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expression in Pichia sp.
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expression of the full-length AlaRS, of the N-terminal fragment lacking the C-terminal oligomerization domain, residues 1-739, and of the C-terminal oligomerization domain of AlaRS, residues 737-906, in Escherichia coli strain BL21
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gene ALA1, DNA and amino acid sequence determination and analysis, mitochondrial and cytoplasmic isozymes are encoded by a single nuclear gene ALA1, through alternative use of inframe successive ACG triplets and a downstream AUG triplet, overview, despite participation of the non-AUG-initiated leader peptide in mitochondrial localization, the leader peptide per se cannot target a cytoplasmic passenger protein into mitochondria under normal conditions, functional mapping
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gene ALA1, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis, recombinant expression of His6-tagged wild-type and chimeric fusion enzymes, recombinant expression of GFP-tagged isozyme ALA1 in Saccharomyces cerevisiae strain INVSc1; gene ALA2, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis, recombinant expression of His6-tagged wild-type and chimeric fusion enzymes, recombinant expression of GFP-tagged isozyme ALA2 in Saccharomyces cerevisiae strain INVSc1
gene ALA1, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis, recombinant expression of His6-tagged wild-type and chimeric fusion enzymes; gene ALA2, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis, recombinant expression of His6-tagged wild-type and chimeric fusion enzymes
G68BWM7, G8C0Z7
gene ALA2, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis, recombinant expression of His6-tagged wild-type and chimeric fusion enzymes
gene alaS, DNA and amino acid sequence determination and analysis, phylogenetic analysis, recombinant expression of His6-tagged full-length enzyme and C-terminal domain C-Ala in Escherichia coli strain BL21(DE3)
gene alaS, DNA sequence determination and analysis, phylogenetic origin
gene alaS, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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gene alaS, sequence comparisons, recombinant expression of a C-Ala construct consisting of the C-terminal 757-968 amino acids
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gene cdc64, DNA sequence determination and analysis, gene maps to a locus between met7 and prt1 on chromosome XV, gene derepresses gcn4 expression, the cdc64-1 mutant strain can be complemented by expression of the wild-type enzyme
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gene PH0574, expression in Escherichia coli strain BL21
recombinant MurN is expressed and purified as MBP fusion protein in Escherichia coli
-
temperature-sensitve mutant tsET12
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
differential regulation of gene expression of ALA genes in yeast species, overview
differential regulation of gene expression of ALA genes in yeast species, overview; differential regulation of gene expression of ALA genes in yeast species, overview
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C290S
-
mutant enzymes with replacement of Cys residues, Cys76Ser, Cys290Ser, Cys412Ser, Cys665Ser. Mutation of Cys665 to serine induces a 120-fold decrease in catalytic efficiency
C665S
-
mutant enzymes with replacement of Cys residues, Cys76Ser, Cys290Ser, Cys412Ser, Cys665Ser. Mutation of Cys665 to serine induces a 120-fold decrease in catalytic efficiency
C666A
-
site-directed mutagenesis, the mutant shows reduced deacylation rates of tRNAAla compared to the wild-type enzyme
C666A/Q584H
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aminoacylation activity is unchanged from that of wild-type. In contrast to the wild-type protein mutant protein mischarges Ser onto tRNAAla. Consistent with this mischarging, deacylation of Ser-tRNA Ala by the mutant protein is undetecable. Mutant protein is sensitive to high concentrations of serine; Serine toxicity, experienced by a strain harboring an C666A/Q584H editing-defective alanyl-tRNA synthetase mutant, is rescued by an AlaXp-encoding transgene from Methanosarcina mazei. AlaXp is a free-standing editing domain homolog of AlaRS. Rescue is dependent on amino acid residues in AlaXp that are needed for its in vitro catalytic activity
C76S
-
mutant enzymes with replacement of Cys residues, Cys76Ser, Cys290Ser, Cys412Ser, Cys665Ser. Mutation of Cys665 to serine induces a 120-fold decrease in catalytic efficiency
D235A
-
no improvement in the discrimination between alanine and serine
D235E
-
no improvement in the discrimination between alanine and serine
D235N
-
no improvement in the discrimination between alanine and serine
D235Q
-
no improvement in the discrimination between alanine and serine
DELTA1-437
-
mutant protein containing a deleted aminoacylation domain: mutant protein is fully active for clearance of Ser-tRNAAla but it is inactive deacylate Ser-tRNAAla
DELTA1-437/731-875
-
mutant protein containing a deleted aminoacylation domain: mutant protein is inactive for clearance of Ser-tRNAAla. Using RNA-binding assays, it is shown that the inactivity of the mutant correlates with a lack of binding of tRNAAla. However, at much higher concentrations, mutant is able of specifically deacylating misacylated tRNAAla. Thus, the catalytic site for editing is not disrupted instead, the reduction in editing activity results from a loss of affinity for tRNA
DELTA1-437/R693K
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a region important for tRNA-specificity is further localized to a predicted strand-loop-strand motif within the region 438-875. Arg 693 is highly conserved. Mutant R693K has relaxed specificity for tRNAThr, and deacylated Ser-tRNAThr. Thus, the AlaRS editing domain shares a second, independent way to recognize tRNAAla
E664A
-
site-directed mutagenesis, the mutant shows reduced deacylation rates of tRNAAla compared to the wild-type enzyme
G237A
-
mutation G237A introduces bulk into the alanine-binding pocket, with little other change in the pocket or the surrounding atoms. Shrinking of the alanine-binding pocket sharply raises the Km for alanine but does not greatly perturb the Km for serine
G674D
-
site-directed mutagenesis, a point mutation in the C-terminal domain, the mutation produces a monomeric variant with a fivefold reduced aminoacylation activity compared to the wild-type enzyme
I667E
-
site-directed mutagenesis, the mutant shows reduced deacylation rates of tRNAAla compared to the wild-type enzyme
L73A
-
mutant enzymes with replacement of Lys73 with Gln, Asn, Ala or Glu show reduction in catalytic efficiency in aminoacylation assay. Glu substitution causes a 5-fold decrease in affinity for alanine
L73E
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mutant enzymes with replacement of Lys73 with Gln, Asn, Ala or Glu show reduction in catalytic efficiency in aminoacylation assay. Glu substitution causes a 5-fold decrease in affinity for alanine
L73N
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mutant enzymes with replacement of Lys73 with Gln, Asn, Ala or Glu show reduction in catalytic efficiency in aminoacylation assay. Glu substitution causes a 5-fold decrease in affinity for alanine
L73Q
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mutant enzymes with replacement of Lys73 with Gln, Asn, Ala or Glu show reduction in catalytic efficiency in aminoacylation assay. Glu substitution causes a 5-fold decrease in affinity for alanine
Q584N
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site-directed mutagenesis, the mutant shows reduced deacylation rates of tRNAAla compared to the wild-type enzyme
T567G
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site-directed mutagenesis, the mutant shows reduced deacylation rates of tRNAAla compared to the wild-type enzyme
A77V
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naturally occuring mutation of a catalytic residue. the mutant likely affects alanine binding resulting in either totally inactive enzyme or with little aminoacylation activity due to decreased affinity to alanine
E405K
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naturally occuring mutation of a structural residue within the tRNA recognition subdomain of the aminoacylation domain, the mutation leads to a partly reduced rate of tRNA aminoacylation due to structural instability in the tRNA recognition fold
F50C
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naturally occuring mutation, leads to reduced rate of aminoacylation due to instability of alanine- and ATP-binding sites and impaired alanyl-adenylate formation
G965R
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naturally occuring mutation predicted to impair protein folding and stability resulting in loss of aminoacylation activity
L155R
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the mutation is associated with infantile mitochondrial cardiomyopathy
R199C
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naturally occuring mutation of a catalytic residue involved in ATP binding, the mutantion leads to reduced rate of tRNA aminoacylation due to affected ATP-binding and impaired alanyl-adenylate formation
R592W/A961V
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naturally occuring lethal mutation R592W in gene AARS2 causing infantile cardiomyopathy, mutation A961V is predicted to impair protein folding and stability resulting in loss of aminoacylation activity
R592W/C218L
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naturally occuring lethal mutation R592W in gene AARS2 causing infantile cardiomyopathy, truncated mutant
R592W/L155R
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naturally occuring lethal mutation R592W in gene AARS2 causing infantile cardiomyopathy, mutation L155R is predicted to impair protein folding and stability resulting in loss of aminoacylation activity
R592W/R329H
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naturally occuring lethal mutation R592W in gene AARS2 causing infantile cardiomyopathy, mutation R329H is predicted to impair protein folding and stability resulting in loss of aminoacylation activity
R592W/Y539C
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naturally occuring lethal mutation R592W in gene AARS2 causing infantile cardiomyopathy. The Y539C mutation causes a dramatic decrease of aminoacylation rate due to impaired tRNA binding and positioning of the 3'-end within the active site
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a complete reversibility of enzyme activity is observed on refolding by dilution from 5 to 0.5 M guanidine hydrochloride
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after refolding from denatured state, the dimeric full-length alaRS gets back similar activity as the native enzyme, while the monomeric, truncated or point mutants do not, secondary structure analysis, unfolding profiles, overview
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
molecular biology
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