Literature summary for 6.1.1.4 extracted from

Dulic, M.; Cvetesic, N.; Zivkovic, I.; Palencia, A.; Cusack, S.; Bertosa, B.; Gruic-Sovulj, I.
Kinetic origin of substrate specificity in post-transfer editing by leucyl-tRNA synthetase (2018), J. Mol. Biol., 430, 1-16 .

Search for more literature for 6.1.1.4

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Escherichia coli

Crystallization (Commentary)

Crystallization (Comment)	Organism
enzyme LeuRS mutant T252A in a complex with tRNALeu and leucyl-adenylate sulphamoyl analogue (Leu-AMS), both positioned in the synthetic active site, and Leu2AA located in the editing domain, X-ray diffraction structure determination and analysis at resolution, replacement using structure PDB ID 4AQ7, modeling	Escherichia coli

Protein Variants

Protein Variants	Comment	Organism
D342A	site-directed mutagenesis, the mutant shows altered deacylation activity with amino acids norvaline, isoleucine, and leucine compared to the wild-type enzyme, overview	Escherichia coli
D345A	site-directed mutagenesis, the mutant shows altered deacylation activity with amino acids norvaline, isoleucine, and leucine compared to the wild-type enzyme, overview	Escherichia coli
additional information	in silico models of the wild-type and mutated LeuRS CP1 editing domain bound to the analogues with an ester linkage between the amino acid and adenosine as in real substrates [2'-L-leucyladenosine (Leu2A) and 2?-L-norvalyladenosine (Nva2A)] are constructed based on the structure of T252A LeuRS in a complex with tRNALeu and leucyl-adenylate sulphamoyl analogue (Leu-AMS), both positioned in the synthetic active site, and Leu2AA located in the editing domain. The tRNA body dominates the binding energetics of aa-tRNA:LeuRS complex formation	Escherichia coli
R344A	site-directed mutagenesis, the mutant shows altered deacylation activity with amino acids norvaline, isoleucine, and leucine compared to the wild-type enzyme, overview	Escherichia coli
T248A	site-directed mutagenesis, the mutant shows altered deacylation activity with amino acids norvaline, isoleucine, and leucine compared to the wild-type enzyme, overview	Escherichia coli
T252A	site-directed mutagenesis, the mutant shows altered deacylation activity with amino acids norvaline, isoleucine, and leucine compared to the wild-type enzyme, conformational changes associated with the binding of post-transfer editing analogues in the editing site of T252A LeuRS, overview	Escherichia coli

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	kinetic origin of substrate specificity in post-transfer editing by leucyl-tRNA synthetase, single-turnover measurements, overview	Escherichia coli

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + L-leucine + tRNALeu	Escherichia coli	-	AMP + diphosphate + L-leucyl-tRNALeu	-	?

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	P07813	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, co-purifying Leu-AMP is removed	Escherichia coli

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
ATP + L-leucine + tRNALeu	-	Escherichia coli	AMP + diphosphate + L-leucyl-tRNALeu	-	?
ATP + L-leucine + tRNALeu	substrate is tRNALeuTAA, overexpressed in and purified from Escherichia coli	Escherichia coli	AMP + diphosphate + L-leucyl-tRNALeu	-	?
additional information	kinetic origin of substrate specificity in post-transfer editing by leucyl-tRNA synthetase, overview. Binding and catalysis is analyzed independently using cognate leucyl- and non-cognate norvalyl-tRNALeu and their non-hydrolyzable analogues. The amino acid part (leucine versus norvaline) of (mis)aminoacyl-tRNAs can contribute approximately 10fold to ground-state discrimination at the editing site, while the rate of deacylation of leucyl- and norvalyl-tRNALeu differs by about 104fold. Critical role for the A76 3'-OH group of the tRNALeu in post-transfer editing. Molecular dynamics simulations reveals that the wild-type enzyme, but not the T252A mutant, enforces leucine to adopt the side-chain conformation that promotes the steric exclusion of a putative catalytic water. Editing can be distiguished from the synthetic site, which relies on ground-state discrimination in amino acid selection	Escherichia coli	?	-	?

Synonyms

Synonyms	Comment	Organism
Leucyl-tRNA synthetase	-	Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Escherichia coli

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Escherichia coli

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Escherichia coli

General Information

General Information	Comment	Organism
malfunction	abrogation of the LeuRS specificity determinant threonine 252 does not improve the affinity of the editing site for the cognate leucine as expected, but instead substantially enhances the rate of leucyl-tRNALeu hydrolysis. Molecular dynamics simulations reveals that the wild-type enzyme, but not the T252A mutant, enforces leucine to adopt the side-chain conformation that promotes the steric exclusion of a putative catalytic water	Escherichia coli
additional information	in silico models of the wild-type and mutated LeuRS CP1 editing domain bound to the analogues with an ester linkage between the amino acid and adenosine as in real substrates [2'-L-leucyladenosine (Leu2A) and 2?-L-norvalyladenosine (Nva2A)] are constructed based on the structure of T252A LeuRS in a complex with tRNALeu and leucyl-adenylate sulphamoyl analogue (Leu-AMS), both positioned in the synthetic active site, and Leu2AA located in the editing domain. The tRNA body dominates the binding energetics of aa-tRNA:LeuRS complex formation	Escherichia coli
physiological function	the ligation of amino acid to tRNA for purposes of protein synthesis proceeds in two steps, bothcatalyzed by a corresponding aminoacyl-tRNA synthetase(aaRS). The amino acid is first activated to anaminoacyl-adenylate (aa-AMP) intermediate at theexpense of ATP, followed by the transfer of aminoacylmoiety to the 2'- or 3'-OH groups at the terminal ribose of the cognate tRNA. Both steps occurwithin the same synthetic/aminoacylation active site located in thecatalytic aaRS domain. Based on the topology of the catalytic domains, the conserved recognition peptides and interaction with the tRNA, aaRSs can be divided into two classes, I and II. The mechanisms of aminoacylation and editing are basically conserved among the classes, although some class-specific features have been recognized. Editing aaRSs exercise specificity through a double-selection mechanism that uses structural/chemical differences between the cognate and non-cognate amino acids twice but in different ways. Leu-tRNALeu is excluded from proofreading basically at the level of catalysis, not binding. This is accomplished by the side chain of the cognate leucine, which adopts a conformation that sterically precludes the positioning of a water nucleophile near the tRNA-assisted hydrolytic machinery. The A76 3'-OH group is a crucial residue in the positioning and activation of the catalytic water. Deacylation mechanism of the enzyme, simulation and modeling, overview	Escherichia coli

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Release 2023.1 (January 2023)