Literature summary for 6.1.1.4 extracted from

Yan, W.; Ye, Q.; Tan, M.; Chen, X.; Eriani, G.; Wang, E.D.
Modulation of aminoacylation and editing properties of leucyl-tRNA synthetase by a conserved structural module (2015), J. Biol. Chem., 290, 12256-12267 .

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of wild-type and mutant enzymes	Homo sapiens
recombinant expression of wild-type and mutant enzymes	Escherichia coli
recombinant expression of wild-type and mutant enzymes	Mesomycoplasma mobile
recombinant expression of wild-type and mutant enzymes	Aquifex aeolicus
recombinant expression of wild-type and mutant enzymes	Pyrococcus horikoshii

Protein Variants

Protein Variants	Comment	Organism
K452A	site-directed mutagenesis, the mutation has only a minimal effect on aminoacylation activity, the Km values is not significantly altered compared to wild-type	Mesomycoplasma mobile
K452E	site-directed mutagenesis, the mutation has only a minimal effect on aminoacylation activity, the Km values is not significantly altered compared to wild-type	Mesomycoplasma mobile
K671A	site-directed mutagenesis, the mutation does no affect the catalytic efificiency	Escherichia coli
K692A	site-directed mutagenesis, the mutation has no effect on tRNA charging activity	Pyrococcus horikoshii
K696A	site-directed mutagenesis, the mutant shows a highly reduced kcat value compared to wild-type, while the Km value is 3fold increased	Pyrococcus horikoshii
K699A	site-directed mutagenesis, the mutation has no effect on tRNA charging activity	Pyrococcus horikoshii
additional information	a helix alpha3-deletion mutant is inactive	Escherichia coli
R456A	site-directed mutagenesis, 75% reduced catalytic efficiency compared to wild-type, the Km values is not significantly altered	Mesomycoplasma mobile
R456E	site-directed mutagenesis, 79% reduced catalytic efficiency compared to wild-type, the Km values is not significantly altered	Mesomycoplasma mobile
R668A	site-directed mutagenesis, the mutant shows 77% reduced catalytic efficiency compared to wild-type, the rate of AMP formation is decreased compared to the wild-type	Escherichia coli
R668A/R672A	site-directed mutagenesis, the mutant shows 93.6% reduced catalytic efficiency compared to wild-type, the rate of AMP formation is decreased compared to the wild-type	Escherichia coli
R668E	site-directed mutagenesis, the mutant shows 95% reduced catalytic efficiency compared to wild-type, the rate of AMP formation is decreased compared to the wild-type	Escherichia coli
R668E/R672E	site-directed mutagenesis, the mutant shows 98.6% reduced catalytic efficiency compared to wild-type. But the almost inactive mutant exhibits intact Leu activation activity comparable with the wild-type enzyme	Escherichia coli
R672A	site-directed mutagenesis, the rate of AMP formation is decreased compared to the wild-type	Escherichia coli
R672E	site-directed mutagenesis, the rate of AMP formation is decreased compared to the wild-type	Escherichia coli
R698A	site-directed mutagenesis, the mutation has no effect on tRNA charging activity	Pyrococcus horikoshii
R703A	site-directed mutagenesis, kcat of mutant PhLeuRSR703A is much lower than that of wild-type PhLeuRS	Pyrococcus horikoshii
R766A	site-directed mutagenesis, the mutation decreases the kcat/Km value to less than 10% that of the wild-type enzyme hcLeuRS	Homo sapiens
R94A	site-directed mutagenesis, mutating Arg94 to Ala decreases kcat/Km values to 34% of that of wild-type AaLeuRS	Aquifex aeolicus
R94A/R98A	site-directed mutagenesis, 83% reduced catalytic efficiency compared to wild-type	Aquifex aeolicus
R94E	site-directed mutagenesis, mutating Arg94 to Glu decreases kcat/Km values to 22% of that of wild-type AaLeuRS	Aquifex aeolicus
R94E/R98E	site-directed mutagenesis, the rate of AMP formation is decreased compared to the wild-type	Aquifex aeolicus
R98A	site-directed mutagenesis, the mutation does not alter the catalytic efficiency	Aquifex aeolicus
R98E	site-directed mutagenesis, the rate of AMP formation is decreased compared to the wild-type	Aquifex aeolicus

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	dissociation constants of LeuRS and mutants from Escherichia coli for their cognate tRNAs. Reaction kinetics of EcLeuRS for Mycoplasma mobile MmtRNAUAALeu and mutant variants, kinetic constants of EcLeuRS, chimeric LeuRS and their mutants for tRNALeu in aminoacylation reaction and for AMP formation in the presence of Nva and MmtRNACAA, detailed overview	Escherichia coli
additional information	-	additional information	dissociation constants of LeuRS and mutants from human cytoplasm for their cognate tRNAs	Homo sapiens
0.00012	-	tRNAGAGLeu	pH 7.8, 37°C, recombinant mutant R703A	Pyrococcus horikoshii
0.0003	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant wild-type enzyme	Aquifex aeolicus
0.00031	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant mutant R98A	Aquifex aeolicus
0.00039	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant mutant R98E	Aquifex aeolicus
0.00044	-	tRNAGAGLeu	pH 7.8, 37°C, recombinant mutant K692A	Pyrococcus horikoshii
0.00052	-	tRNAGAGLeu	pH 7.8, 37°C, recombinant mutant K698A	Pyrococcus horikoshii
0.0006	-	tRNAGAGLeu	pH 7.8, 37°C, recombinant mutant K699A	Pyrococcus horikoshii
0.00065	-	tRNAGAGLeu	pH 7.8, 37°C, recombinant mutant K696A	Pyrococcus horikoshii
0.00074	-	tRNACAGLeu	pH 7.8, 37°C, recombinant wild-type enzyme	Homo sapiens
0.00075	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant mutant R94A	Aquifex aeolicus
0.00081	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant mutant R94A/R98A	Aquifex aeolicus
0.00081	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant mutant R94E	Aquifex aeolicus
0.00088	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant mutant R94E/R98E	Aquifex aeolicus
0.0015	-	tRNAUAALeu	pH 7.8, 30°C, recombinant wild-type enzyme	Mesomycoplasma mobile
0.0016	-	tRNAUAALeu	pH 7.5, 37°C, recombinant wild-type enzyme	Escherichia coli
0.0017	-	tRNAUAALeu	pH 7.8, 30°C, recombinant mutant K452A	Mesomycoplasma mobile
0.0017	-	tRNACAGLeu	pH 7.8, 37°C, recombinant mutant R668A	Homo sapiens
0.0019	-	tRNAUAALeu	pH 7.8, 30°C, recombinant mutant K452E	Mesomycoplasma mobile
0.002	-	tRNAGAGLeu	pH 7.8, 37°C, recombinant wild-type enzyme	Pyrococcus horikoshii
0.0022	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant wild-type enzyme	Escherichia coli
0.0022	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant R668A/R672A	Escherichia coli
0.0023	-	tRNAUAALeu	pH 7.8, 30°C, recombinant mutant K456A	Mesomycoplasma mobile
0.0024	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant K671A	Escherichia coli
0.0024	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant R672A	Escherichia coli
0.0025	-	tRNAUAALeu	pH 7.8, 30°C, recombinant mutant K456E	Mesomycoplasma mobile
0.0028	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant R668A	Escherichia coli
0.0028	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant R672E	Escherichia coli
0.0054	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant R668E	Escherichia coli
0.0058	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant R668E/R672E	Escherichia coli

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
cytoplasm	-	Homo sapiens	5737	-

Metals/Ions

Metals/Ions	Comment	Organism
Mg2+	required	Homo sapiens
Mg2+	required	Escherichia coli
Mg2+	required	Mesomycoplasma mobile
Mg2+	required	Aquifex aeolicus
Mg2+	required	Pyrococcus horikoshii

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
ATP + L-leucine + tRNALeu	Homo sapiens	-	AMP + diphosphate + L-leucyl-tRNALeu	-	?
ATP + L-leucine + tRNALeu	Escherichia coli	-	AMP + diphosphate + L-leucyl-tRNALeu	-	?
ATP + L-leucine + tRNALeu	Mesomycoplasma mobile	-	AMP + diphosphate + L-leucyl-tRNALeu	-	?
ATP + L-leucine + tRNALeu	Aquifex aeolicus	-	AMP + diphosphate + L-leucyl-tRNALeu	-	?
ATP + L-leucine + tRNALeu	Pyrococcus horikoshii	-	AMP + diphosphate + L-leucyl-tRNALeu	-	?
ATP + L-leucine + tRNALeu	Mesomycoplasma mobile ATCC 43663 / 163K / NCTC 11711	-	AMP + diphosphate + L-leucyl-tRNALeu	-	?
ATP + L-leucine + tRNALeu	Pyrococcus horikoshii ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3	-	AMP + diphosphate + L-leucyl-tRNALeu	-	?

Organism

Organism	UniProt	Comment	Textmining
Aquifex aeolicus	O66680 AND O67646	alpha- and beta-subunit	-
Escherichia coli	P07813	-	-
Homo sapiens	Q9P2J5	-	-
Mesomycoplasma mobile	Q6KHA5	-	-
Mesomycoplasma mobile ATCC 43663 / 163K / NCTC 11711	Q6KHA5	-	-
Pyrococcus horikoshii	O58698	-	-
Pyrococcus horikoshii ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3	O58698	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant wild-type and mutant enzymes	Homo sapiens
recombinant wild-type and mutant enzymes	Escherichia coli
recombinant wild-type and mutant enzymes	Mesomycoplasma mobile
recombinant wild-type and mutant enzymes	Aquifex aeolicus
recombinant wild-type and mutant enzymes	Pyrococcus horikoshii

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + L-leucine + tRNACAALeu	Mycoplasma mobile tRNACAALeu (MmtRNACAALeu) and mutat derivatives	Mesomycoplasma mobile	AMP + diphosphate + L-leucyl-tRNAUAALeu	-	?
ATP + L-leucine + tRNACAALeu	Mycoplasma mobile tRNACAALeu (MmtRNACAALeu) and mutat derivatives	Mesomycoplasma mobile ATCC 43663 / 163K / NCTC 11711	AMP + diphosphate + L-leucyl-tRNAUAALeu	-	?
ATP + L-leucine + tRNACAGLeu	human cytoplasmic tRNACAGLeu (hctRNACAG)	Homo sapiens	AMP + diphosphate + L-leucyl-tRNACAGLeu	-	?
ATP + L-leucine + tRNAGAGLeu	Aquifex aeolicus tRNAGAGLeu (AatRNAGAGLeu)	Aquifex aeolicus	AMP + diphosphate + L-leucyl-tRNAGAGLeu	-	?
ATP + L-leucine + tRNAGAGLeu	Escherichia coli tRNAGAGLeu (Ect-RNAGAGLeu)	Escherichia coli	AMP + diphosphate + L-leucyl-tRNAGAGLeu	-	?
ATP + L-leucine + tRNAGAGLeu	Pyrococcus horikoshii tRNAGAGLeu (PhtRNAGAGLeu)	Pyrococcus horikoshii	AMP + diphosphate + L-leucyl-tRNAGAGLeu	-	?
ATP + L-leucine + tRNAGAGLeu	Pyrococcus horikoshii tRNAGAGLeu (PhtRNAGAGLeu)	Pyrococcus horikoshii ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3	AMP + diphosphate + L-leucyl-tRNAGAGLeu	-	?
ATP + L-leucine + tRNALeu	-	Homo sapiens	AMP + diphosphate + L-leucyl-tRNALeu	-	?
ATP + L-leucine + tRNALeu	-	Escherichia coli	AMP + diphosphate + L-leucyl-tRNALeu	-	?
ATP + L-leucine + tRNALeu	-	Mesomycoplasma mobile	AMP + diphosphate + L-leucyl-tRNALeu	-	?
ATP + L-leucine + tRNALeu	-	Aquifex aeolicus	AMP + diphosphate + L-leucyl-tRNALeu	-	?
ATP + L-leucine + tRNALeu	-	Pyrococcus horikoshii	AMP + diphosphate + L-leucyl-tRNALeu	-	?
ATP + L-leucine + tRNALeu	-	Mesomycoplasma mobile ATCC 43663 / 163K / NCTC 11711	AMP + diphosphate + L-leucyl-tRNALeu	-	?
ATP + L-leucine + tRNALeu	-	Pyrococcus horikoshii ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3	AMP + diphosphate + L-leucyl-tRNALeu	-	?
ATP + L-leucine + tRNAUAALeu	Mycoplasma mobile tRNAUAALeu (MmtRNAUAALeu) and mutat derivatives	Mesomycoplasma mobile	AMP + diphosphate + L-leucyl-tRNACAALeu	-	?
ATP + L-leucine + tRNAUAALeu	Mycoplasma mobile tRNAUAALeu (MmtRNAUAALeu) and mutat derivatives	Mesomycoplasma mobile ATCC 43663 / 163K / NCTC 11711	AMP + diphosphate + L-leucyl-tRNACAALeu	-	?
ATP + L-leucine + tRNAUAALeu	Mycoplasma mobile MmtRNAUAALeu (Mmt-RNAUAALeu)	Escherichia coli	AMP + diphosphate + L-leucyl-tRNAUAALeu	-	?

Subunits

Subunits	Comment	Organism
heterodimer	-	Aquifex aeolicus

Synonyms

Synonyms	Comment	Organism
AaLeuRS	-	Aquifex aeolicus
EcLeuRS	-	Escherichia coli
HcleuRS	-	Homo sapiens
Leucyl-tRNA synthetase	-	Homo sapiens
Leucyl-tRNA synthetase	-	Escherichia coli
Leucyl-tRNA synthetase	-	Mesomycoplasma mobile
Leucyl-tRNA synthetase	-	Aquifex aeolicus
Leucyl-tRNA synthetase	-	Pyrococcus horikoshii
LeuRS	-	Homo sapiens
LeuRS	-	Escherichia coli
LeuRS	-	Mesomycoplasma mobile
LeuRS	-	Aquifex aeolicus
LeuRS	-	Pyrococcus horikoshii
leuS	-	Mesomycoplasma mobile
leuS	-	Pyrococcus horikoshii
MmLeuRS	-	Mesomycoplasma mobile
PhLeuRS	-	Pyrococcus horikoshii

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Mesomycoplasma mobile
37	-	assay at	Homo sapiens
37	-	assay at	Escherichia coli
37	-	assay at	Pyrococcus horikoshii
65	-	assay at	Aquifex aeolicus

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.0055	-	tRNAGAGLeu	pH 7.8, 37°C, recombinant mutant R703A	Pyrococcus horikoshii
0.017	-	tRNAGAGLeu	pH 7.8, 37°C, recombinant mutant K696A	Pyrococcus horikoshii
0.02	-	tRNAGAGLeu	pH 7.8, 37°C, recombinant wild-type enzyme	Pyrococcus horikoshii
0.041	-	tRNAGAGLeu	pH 7.8, 37°C, recombinant mutant K692A	Pyrococcus horikoshii
0.05	-	tRNAGAGLeu	pH 7.8, 37°C, recombinant mutant K699A	Pyrococcus horikoshii
0.067	-	tRNAGAGLeu	pH 7.8, 37°C, recombinant mutant K698A	Pyrococcus horikoshii
0.18	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant R668E/R672E	Escherichia coli
0.19	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant mutant R94E/R98E	Aquifex aeolicus
0.31	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant R668A/R672A	Escherichia coli
0.53	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant R672E	Escherichia coli
0.56	-	tRNACAGLeu	pH 7.8, 37°C, recombinant mutant R668A	Homo sapiens
0.59	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant R668E	Escherichia coli
0.67	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant mutant R94A/R98A	Aquifex aeolicus
0.85	-	tRNAUAALeu	pH 7.8, 30°C, recombinant mutant K456E	Mesomycoplasma mobile
0.89	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant mutant R94E	Aquifex aeolicus
1	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant mutant R98E	Aquifex aeolicus
1.3	-	tRNAUAALeu	pH 7.8, 30°C, recombinant mutant K456A	Mesomycoplasma mobile
1.3	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant mutant R94A	Aquifex aeolicus
1.4	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant R668A	Escherichia coli
1.5	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant mutant R98A	Aquifex aeolicus
1.5	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant wild-type enzyme	Aquifex aeolicus
2.6	-	tRNACAGLeu	pH 7.8, 37°C, recombinant wild-type enzyme	Homo sapiens
2.8	-	tRNAUAALeu	pH 7.8, 30°C, recombinant mutant K452E	Mesomycoplasma mobile
3.2	-	tRNAUAALeu	pH 7.8, 30°C, recombinant mutant K452A	Mesomycoplasma mobile
3.3	-	tRNAUAALeu	pH 7.8, 30°C, recombinant wild-type enzyme	Mesomycoplasma mobile
4.2	-	tRNAUAALeu	pH 7.5, 37°C, recombinant wild-type enzyme	Escherichia coli
4.8	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant K671A	Escherichia coli
4.8	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant R672A	Escherichia coli
4.9	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant wild-type enzyme	Escherichia coli

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Escherichia coli
7.8	-	assay at	Homo sapiens
7.8	-	assay at	Mesomycoplasma mobile
7.8	-	assay at	Aquifex aeolicus
7.8	-	assay at	Pyrococcus horikoshii

Cofactor

Cofactor	Comment	Organism
ATP	-	Homo sapiens
ATP	-	Escherichia coli
ATP	-	Mesomycoplasma mobile
ATP	-	Aquifex aeolicus
ATP	-	Pyrococcus horikoshii

General Information

General Information	Comment	Organism
evolution	based on sequence homology and the structures of the catalytic active sites, aaRSs are divided into two classes of 10 members each. Class I synthetases are further divided into three subclasses, a, b, and c, according to sequence homology. Leucyl-tRNA synthetase (LeuRS) belongs to class I aaRSs that include a typical Rossmann dinucleotide-binding fold active site architecture with the signature sequence modules HIGH and KMSKS. According to evolutionary models, the primitive catalytic core is extended by the insertion and/or fusion of additional domains (also called modules) in LeuRSs, most of which have inserted a large connective polypeptide 1 (CP1) domain that is responsible for amino acid editing. To ensure translation accuracy, LeuRSs have evolved a mechanism to remove aminoacyl AMP (aa-AMP, pre-transfer editing) and aa-tRNA (post-transfer editing). Sequence comparisons of the stem contact-fold domain (SC-fold) involved in editing, basic residues on helix alpha3 of the SC-fold are critical for catalytic efficiency, overview	Homo sapiens
evolution	based on sequence homology and the structures of the catalytic active sites, aaRSs are divided into two classes of 10 members each. Class I synthetases are further divided into three subclasses, a, b, and c, according to sequence homology. Leucyl-tRNA synthetase (LeuRS) belongs to class I aaRSs that include a typical Rossmann dinucleotide-binding fold active site architecture with the signature sequence modules HIGH and KMSKS. According to evolutionary models, the primitive catalytic core is extended by the insertion and/or fusion of additional domains (also called modules) in LeuRSs, most of which have inserted a large connective polypeptide 1 (CP1) domain that is responsible for amino acid editing. To ensure translation accuracy, LeuRSs have evolved a mechanism to remove aminoacyl AMP (aa-AMP, pre-transfer editing) and aa-tRNA (post-transfer editing). Sequence comparisons of the stem contact-fold domain (SC-fold) involved in editing, basic residues on helix alpha3 of the SC-fold are critical for catalytic efficiency, overview	Aquifex aeolicus
evolution	based on sequence homology and the structures of the catalytic active sites, aaRSs are divided into two classes of 10 members each. Class I synthetases are further divided into three subclasses, a, b, and c, according to sequence homology. Leucyl-tRNA synthetase (LeuRS) belongs to class I aaRSs that include a typical Rossmann dinucleotide-bindingfold active site architecture with the signature sequence modules HIGH and KMSKS. According to evolutionary models, the primitive catalytic core is extended by the insertion and/or fusion of additional domains (also called modules) in LeuRSs, most of which have inserted a large connective polypeptide 1 (CP1) domain that is responsible for amino acid editing. To ensure translation accuracy, LeuRSs have evolved a mechanism to remove aminoacyl AMP (aa-AMP, pre-transfer editing) and aa-tRNA (post-transfer editing). Although post-transfer editing is carried out by the CP1 domain in most LeuRSs, this domain has been naturally deleted in LeuRS from Mycoplasma mobile (MmLeuRS). Sequence comparisons of the stem contact-fold domain (SC-fold) involved in editing, basic residues on helix alpha3 of the SC-fold are critical for catalytic efficiency, overview	Mesomycoplasma mobile
evolution	based on sequence homology and the structures of the catalytic active sites, aaRSs are divided into two classes of 10 members each. Class I synthetases are further divided into three subclasses, a, b, and c, according to sequence homology. Leucyl-tRNA synthetase (LeuRS) belongs to class I aaRSs that include a typical Rossmann dinucleotide-bindingfold active site architecture with the signature sequence modules HIGH and KMSKS. According to evolutionary models, the primitive catalytic core is extended by the insertion and/or fusion of additional domains (also called modules) in LeuRSs, most of which have inserted a large connective polypeptide 1 (CP1) domain that is responsible for amino acid editing. To ensure translation accuracy, LeuRSs have evolved a mechanism to remove aminoacyl AMP (aa-AMP, pre-transfer editing) and aa-tRNA (post-transfer editing). Sequence comparison of the EcLeuRS stem contact-fold domain (SC-fold) with editing-deficient enzymes suggests that key residues of this module have evolved an adaptive strategy to follow the editing functions of LeuRS, basic residues on helix alpha3 of the SC-fold are critical for catalytic efficiency, overview	Escherichia coli
evolution	based on sequence homology and the structures of the catalytic active sites, aaRSs are divided into two classes of 10 members each. Class I synthetases are further divided into three subclasses, a, b, and c, according to sequence homology. Leucyl-tRNA synthetase (LeuRS) belongs to class I aaRSs that include a typical Rossmann dinucleotide-bindingfold active site architecture with the signature sequence modules HIGH and KMSKS. According to evolutionary models, the primitive catalytic core is extended by the insertion and/or fusion of additional domains (also called modules) in LeuRSs, most of which have inserted a large connective polypeptide 1 (CP1) domain that is responsible for amino acid editing. To ensure translation accuracy, LeuRSs have evolved a mechanism to remove aminoacyl AMP (aa-AMP, pre-transfer editing) and aa-tRNA (post-transfer editing). Sequence comparisons of the stem contact-fold domain (SC-fold) involved in editing, basic residues on helix alpha3 of the SC-fold are critical for catalytic efficiency, overview	Pyrococcus horikoshii
malfunction	mutation of highly conserved basic residues on the third alpha-helix of the KMSKS catalytic loop domain impairs the affinity of LeuRS for the anticodon stem of tRNALeu, which decreases both aminoacylation and editing activities	Escherichia coli
additional information	the KMSKS catalytic loop exhibits alpha-alpha-beta-alpha topology in class Ia and Ib aminoacyl-tRNA synthetases, two glycine residues on the third alpha-helix contribute to flexibility, leucine activation, and editing of LeuRS from Escherichia coli (EcLeuRS), acidic residues on the beta-strand enhance the editing activity of EcLeuRS and sense the size of the tRNALeu D-loop. Incorporation of acidic residues on the beta-strand stimulates the tRNA-dependent editing activity of the chimeric minimalist enzyme Mycoplasma mobile LeuRS fused to the connective polypeptide 1 editing domain and leucine-specific domain from EcLeuRS. Sequence comparison of the EcLeuRS stem contact-fold domain with editing-deficient enzymes suggests that key residues of this module have evolved an adaptive strategy to follow the editing functions of LeuRS. Amino acid residues Arg668 or Arg672 are not involved in the amino acid activation step but rather the second tRNA transfer step	Escherichia coli
additional information	the KMSKS catalytic loop exhibits alpha-alpha-beta-alpha topology in class Ia and Ib aminoacyl-tRNA synthetases. Incorporation of acidic residues on the beta-strand stimulates the tRNA-dependent editing activity of the chimeric minimalist enzyme Mycoplasma mobile LeuRS fused to the connective polypeptide 1 editing domain and leucine-specific domain from EcLeuRS, acidic residues on the beta-strand enhance the editing activity of EcLeuRS and sense the size of the tRNALeu D-loop	Mesomycoplasma mobile
physiological function	aminoacyl-tRNA synthetases (aaRSs) are a large and diverse family of enzymes that catalyze the attachment of amino acids to their cognate tRNAs in a two-step aminoacylation reaction as follows: 1. amino acid activation by ATP hydrolysis to form an aminoacyl-adenylate intermediate, and 2. transfer of the aminoacyl moiety from the intermediate to the cognate tRNA isoacceptor to form aminoacyl-tRNA (aa-tRNA)	Homo sapiens
physiological function	aminoacyl-tRNA synthetases (aaRSs) are a large and diverse family of enzymes that catalyze the attachment of amino acids to their cognate tRNAs in a two-step aminoacylation reaction as follows: 1. amino acid activation by ATP hydrolysis to form an aminoacyl-adenylate intermediate, and 2. transfer of the aminoacyl moiety from the intermediate to the cognate tRNA isoacceptor to form aminoacyl-tRNA (aa-tRNA)	Escherichia coli
physiological function	aminoacyl-tRNA synthetases (aaRSs) are a large and diverse family of enzymes that catalyze the attachment of amino acids to their cognate tRNAs in a two-step aminoacylation reaction as follows: 1. amino acid activation by ATP hydrolysis to form an aminoacyl-adenylate intermediate, and 2. transfer of the aminoacyl moiety from the intermediate to the cognate tRNA isoacceptor to form aminoacyl-tRNA (aa-tRNA)	Mesomycoplasma mobile
physiological function	aminoacyl-tRNA synthetases (aaRSs) are a large and diverse family of enzymes that catalyze the attachment of amino acids to their cognate tRNAs in a two-step aminoacylation reaction as follows: 1. amino acid activation by ATP hydrolysis to form an aminoacyl-adenylate intermediate, and 2. transfer of the aminoacyl moiety from the intermediate to the cognate tRNA isoacceptor to form aminoacyl-tRNA (aa-tRNA)	Aquifex aeolicus
physiological function	aminoacyl-tRNA synthetases (aaRSs) are a large and diverse family of enzymes that catalyze the attachment of amino acids to their cognate tRNAs in a two-step aminoacylation reaction as follows: 1. amino acid activation by ATP hydrolysis to form an aminoacyl-adenylate intermediate, and 2. transfer of the aminoacyl moiety from the intermediate to the cognate tRNA isoacceptor to form aminoacyl-tRNA (aa-tRNA)	Pyrococcus horikoshii

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
30	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant R668E/R672E	Escherichia coli
30	-	tRNAGAGLeu	pH 7.8, 37°C, recombinant mutant K696A	Pyrococcus horikoshii
50	-	tRNAGAGLeu	pH 7.8, 37°C, recombinant mutant R703A	Pyrococcus horikoshii
80	-	tRNAGAGLeu	pH 7.8, 37°C, recombinant mutant K699A	Pyrococcus horikoshii
90	-	tRNAGAGLeu	pH 7.8, 37°C, recombinant mutant K692A	Pyrococcus horikoshii
110	-	tRNAGAGLeu	pH 7.8, 37°C, recombinant wild-type enzyme	Pyrococcus horikoshii
110	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant R668E	Escherichia coli
130	-	tRNAGAGLeu	pH 7.8, 37°C, recombinant mutant K698A	Pyrococcus horikoshii
140	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant R668A/R672A	Escherichia coli
190	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant R672E	Escherichia coli
220	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant mutant R94E/R98E	Aquifex aeolicus
330	-	tRNACAGLeu	pH 7.8, 37°C, recombinant mutant R668A	Homo sapiens
340	-	tRNAUAALeu	pH 7.8, 30°C, recombinant mutant K456E	Mesomycoplasma mobile
500	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant R668A	Escherichia coli
600	-	tRNAUAALeu	pH 7.8, 30°C, recombinant mutant K456A	Mesomycoplasma mobile
830	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant mutant R94A/R98A	Aquifex aeolicus
1100	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant mutant R94E	Aquifex aeolicus
1500	-	tRNAUAALeu	pH 7.8, 30°C, recombinant mutant K452E	Mesomycoplasma mobile
1700	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant mutant R94A	Aquifex aeolicus
1900	-	tRNAUAALeu	pH 7.8, 30°C, recombinant mutant K452A	Mesomycoplasma mobile
2000	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant K671A	Escherichia coli
2000	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant mutant R672A	Escherichia coli
2200	-	tRNAUAALeu	pH 7.8, 30°C, recombinant wild-type enzyme	Mesomycoplasma mobile
2230	-	tRNAGAGLeu	pH 7.5, 37°C, recombinant wild-type enzyme	Escherichia coli
2600	-	tRNAUAALeu	pH 7.5, 37°C, recombinant wild-type enzyme	Escherichia coli
2600	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant mutant R98E	Aquifex aeolicus
3500	-	tRNACAGLeu	pH 7.8, 37°C, recombinant wild-type enzyme	Homo sapiens
4800	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant mutant R98A	Aquifex aeolicus
5000	-	tRNAGAGLeu	pH 7.8, 65°C, recombinant wild-type enzyme	Aquifex aeolicus