UniProt functional annotation for P0A8M3



	UniProt functional annotation for P0A8M3

UniProt code: P0A8M3.

Organism: Escherichia coli (strain K12).

Taxonomy: Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacterales; Enterobacteriaceae; Escherichia.

Function: Catalyzes the attachment of threonine to tRNA(Thr) in a two- step reaction: L-threonine is first activated by ATP to form Thr-AMP and then transferred to the acceptor end of tRNA(Thr) (PubMed:15079065, PubMed:10881191, PubMed:18997014). The rate-limiting step is amino acid activation in the presence of tRNA (PubMed:18997014). The 2'-OH of the acceptor base (adenine 76, A76) of tRNA(Thr) and His-309 collaborate to transfer L-Thr to the tRNA; substitution of 2'-OH of A76 with hydrogen or fluorine decreases transfer efficiency 760 and 100-fold respectively (PubMed:18997014). The zinc ion in the active site discriminates against charging of the isosteric amino acid valine (PubMed:10881191). Also activates L-serine, but does not detectably transfer it to tRNA(Thr) (PubMed:15079065). Edits incorrectly charged L-seryl- tRNA(Thr) via its editing domain (PubMed:15079065, PubMed:11136973, PubMed:15525511), in a post-transfer reaction probably via water- mediated hydrolysis (PubMed:15525511). {ECO:0000269|PubMed:10319817, ECO:0000269|PubMed:10881191, ECO:0000269|PubMed:11136973, ECO:0000269|PubMed:15079065, ECO:0000269|PubMed:15525511, ECO:0000269|PubMed:18997014}.

Function: ThrS is also a translational repressor protein, it controls binds its own mRNA in the operator region upstream of the start codon (PubMed:3086882). The mRNA region upstream of the start codon has a tRNA-like secondary structure; mRNA and tRNA compete for binding to ThrRS (PubMed:2254931). ThrRS represses translation by preventing the ribosome from to mRNA, and tRNA(Thr) acts as an antirepressor allowing fine level control of enzyme synthesis (PubMed:2254931). X-ray structures prove that operator mRNA and tRNA bind to overlapping sites in the protein (PubMed:10319817, PubMed:11953757). {ECO:0000269|PubMed:10319817, ECO:0000269|PubMed:11953757, ECO:0000269|PubMed:2254931, ECO:0000269|PubMed:3086882}.

Catalytic activity: Reaction=ATP + L-threonine + tRNA(Thr) = AMP + diphosphate + H(+) + L- threonyl-tRNA(Thr); Xref=Rhea:RHEA:24624, Rhea:RHEA-COMP:9670, Rhea:RHEA-COMP:9704, ChEBI:CHEBI:15378, ChEBI:CHEBI:30616, ChEBI:CHEBI:33019, ChEBI:CHEBI:57926, ChEBI:CHEBI:78442, ChEBI:CHEBI:78534, ChEBI:CHEBI:456215; EC=6.1.1.3; Evidence={ECO:0000255|HAMAP-Rule:MF_00184, ECO:0000269|PubMed:10319817, ECO:0000269|PubMed:10881191, ECO:0000269|PubMed:18997014};

Cofactor: Name=Zn(2+); Xref=ChEBI:CHEBI:29105; Evidence={ECO:0000255|HAMAP-Rule:MF_00184}; Note=Binds 1 zinc ion per subunit. It helps recognize and select the amino acid substrate, and thus has neither a purely catalytic or structural role (PubMed:10881191). {ECO:0000269|PubMed:10319817, ECO:0000269|PubMed:10881191, ECO:0000269|PubMed:11136973, ECO:0000269|PubMed:11953757, ECO:0000269|PubMed:23362938, ECO:0000269|PubMed:25824639};

Activity regulation: Inhibited non-competitively by borrelidin (BN, KI is 3.7 nM) which binds in a 1:1 stoichiometry, inhibiting L-thr activation (PubMed:15507440). BN binds to 4 distinct subsites in the protein, preventing binding of all 3 substrates; BN also inhibits human ThrRS, and thus it is not useful as an antibiotic (PubMed:25824639). {ECO:0000269|PubMed:15507440, ECO:0000269|PubMed:25824639}.

Biophysicochemical properties: Kinetic parameters: KM=94 uM for ATP {ECO:0000269|PubMed:15507440}; KM=110 uM for L-threonine activation {ECO:0000269|PubMed:10881191, ECO:0000269|PubMed:15507440}; KM=120 uM for L-threonine activation {ECO:0000269|PubMed:18997014}; KM=0.86 uM for L-threonine aminoacylation {ECO:0000269|PubMed:18997014}; KM=1.95 mM for beta-hydroxynorvaline activation {ECO:0000269|PubMed:10881191}; KM=81.5 mM for L-serine activation {ECO:0000269|PubMed:10881191}; Note=kcat is 36, 22 and 26 s(-1) for L-threonine, beta- hydroxynorvaline and L-serine respectively. {ECO:0000269|PubMed:10881191};

Subunit: Homodimer (PubMed:10319817, PubMed:11136973, PubMed:10881191, PubMed:11953757, PubMed:23362938, PubMed:25824639); binds 2 tRNA(Thr) per homodimer, each tRNA contacts both monomers and makes specific contacts with the anticodon and acceptor stems (PubMed:10319817). {ECO:0000269|PubMed:10319817, ECO:0000269|PubMed:10881191, ECO:0000269|PubMed:11136973, ECO:0000269|PubMed:11953757, ECO:0000269|PubMed:23362938, ECO:0000269|PubMed:25824639}.

Subcellular location: Cytoplasm {ECO:0000255|HAMAP-Rule:MF_00184}.

Domain: The protein structure shows 2 N-terminal domains, the central catalytic and C-terminal domain (PubMed:10319817). The C-terminal domain recognizes the anticodon region of the tRNA while the acceptor arm is sandwiched between the N-terminal domains and the catalytic domain (PubMed:10319817). The N-terminal also contributes to the precise recognition of tRNA(Thr) (PubMed:10319817). The editing domain encompasses approximately residues 62-224; when it is removed the protein mischarges tRNA(Thr) with L-serine (PubMed:15079065, PubMed:11136973). {ECO:0000269|PubMed:11136973, ECO:0000269|PubMed:15525511, ECO:0000305|PubMed:10319817, ECO:0000305|PubMed:15079065}.

Disruption phenotype: Essential, it cannot be deleted. {ECO:0000269|PubMed:15507440}.

Biotechnology: A number of inhibitors with high affinity for bacterial ThrRS and less affinity for human ThrRS have been identified that might make good antibiotics. {ECO:0000269|PubMed:23362938}.

Similarity: Belongs to the class-II aminoacyl-tRNA synthetase family. {ECO:0000255|HAMAP-Rule:MF_00184}.

Annotations taken from UniProtKB at the EBI.


Organism:		Escherichia coli (strain K12).
Taxonomy:		Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacterales; Enterobacteriaceae; Escherichia.



Function:		Catalyzes the attachment of threonine to tRNA(Thr) in a two- step reaction: L-threonine is first activated by ATP to form Thr-AMP and then transferred to the acceptor end of tRNA(Thr) (PubMed:15079065, PubMed:10881191, PubMed:18997014). The rate-limiting step is amino acid activation in the presence of tRNA (PubMed:18997014). The 2'-OH of the acceptor base (adenine 76, A76) of tRNA(Thr) and His-309 collaborate to transfer L-Thr to the tRNA; substitution of 2'-OH of A76 with hydrogen or fluorine decreases transfer efficiency 760 and 100-fold respectively (PubMed:18997014). The zinc ion in the active site discriminates against charging of the isosteric amino acid valine (PubMed:10881191). Also activates L-serine, but does not detectably transfer it to tRNA(Thr) (PubMed:15079065). Edits incorrectly charged L-seryl- tRNA(Thr) via its editing domain (PubMed:15079065, PubMed:11136973, PubMed:15525511), in a post-transfer reaction probably via water- mediated hydrolysis (PubMed:15525511). {ECO:0000269\|PubMed:10319817, ECO:0000269\|PubMed:10881191, ECO:0000269\|PubMed:11136973, ECO:0000269\|PubMed:15079065, ECO:0000269\|PubMed:15525511, ECO:0000269\|PubMed:18997014}.



Function:		ThrS is also a translational repressor protein, it controls binds its own mRNA in the operator region upstream of the start codon (PubMed:3086882). The mRNA region upstream of the start codon has a tRNA-like secondary structure; mRNA and tRNA compete for binding to ThrRS (PubMed:2254931). ThrRS represses translation by preventing the ribosome from to mRNA, and tRNA(Thr) acts as an antirepressor allowing fine level control of enzyme synthesis (PubMed:2254931). X-ray structures prove that operator mRNA and tRNA bind to overlapping sites in the protein (PubMed:10319817, PubMed:11953757). {ECO:0000269\|PubMed:10319817, ECO:0000269\|PubMed:11953757, ECO:0000269\|PubMed:2254931, ECO:0000269\|PubMed:3086882}.


Catalytic activity:		Reaction=ATP + L-threonine + tRNA(Thr) = AMP + diphosphate + H(+) + L- threonyl-tRNA(Thr); Xref=Rhea:RHEA:24624, Rhea:RHEA-COMP:9670, Rhea:RHEA-COMP:9704, ChEBI:CHEBI:15378, ChEBI:CHEBI:30616, ChEBI:CHEBI:33019, ChEBI:CHEBI:57926, ChEBI:CHEBI:78442, ChEBI:CHEBI:78534, ChEBI:CHEBI:456215; EC=6.1.1.3; Evidence={ECO:0000255\|HAMAP-Rule:MF_00184, ECO:0000269\|PubMed:10319817, ECO:0000269\|PubMed:10881191, ECO:0000269\|PubMed:18997014};
Cofactor:		Name=Zn(2+); Xref=ChEBI:CHEBI:29105; Evidence={ECO:0000255\|HAMAP-Rule:MF_00184}; Note=Binds 1 zinc ion per subunit. It helps recognize and select the amino acid substrate, and thus has neither a purely catalytic or structural role (PubMed:10881191). {ECO:0000269\|PubMed:10319817, ECO:0000269\|PubMed:10881191, ECO:0000269\|PubMed:11136973, ECO:0000269\|PubMed:11953757, ECO:0000269\|PubMed:23362938, ECO:0000269\|PubMed:25824639};
Activity regulation:		Inhibited non-competitively by borrelidin (BN, KI is 3.7 nM) which binds in a 1:1 stoichiometry, inhibiting L-thr activation (PubMed:15507440). BN binds to 4 distinct subsites in the protein, preventing binding of all 3 substrates; BN also inhibits human ThrRS, and thus it is not useful as an antibiotic (PubMed:25824639). {ECO:0000269\|PubMed:15507440, ECO:0000269\|PubMed:25824639}.
Biophysicochemical properties:		Kinetic parameters: KM=94 uM for ATP {ECO:0000269\|PubMed:15507440}; KM=110 uM for L-threonine activation {ECO:0000269\|PubMed:10881191, ECO:0000269\|PubMed:15507440}; KM=120 uM for L-threonine activation {ECO:0000269\|PubMed:18997014}; KM=0.86 uM for L-threonine aminoacylation {ECO:0000269\|PubMed:18997014}; KM=1.95 mM for beta-hydroxynorvaline activation {ECO:0000269\|PubMed:10881191}; KM=81.5 mM for L-serine activation {ECO:0000269\|PubMed:10881191}; Note=kcat is 36, 22 and 26 s(-1) for L-threonine, beta- hydroxynorvaline and L-serine respectively. {ECO:0000269\|PubMed:10881191};
Subunit:		Homodimer (PubMed:10319817, PubMed:11136973, PubMed:10881191, PubMed:11953757, PubMed:23362938, PubMed:25824639); binds 2 tRNA(Thr) per homodimer, each tRNA contacts both monomers and makes specific contacts with the anticodon and acceptor stems (PubMed:10319817). {ECO:0000269\|PubMed:10319817, ECO:0000269\|PubMed:10881191, ECO:0000269\|PubMed:11136973, ECO:0000269\|PubMed:11953757, ECO:0000269\|PubMed:23362938, ECO:0000269\|PubMed:25824639}.
Subcellular location:		Cytoplasm {ECO:0000255\|HAMAP-Rule:MF_00184}.
Domain:		The protein structure shows 2 N-terminal domains, the central catalytic and C-terminal domain (PubMed:10319817). The C-terminal domain recognizes the anticodon region of the tRNA while the acceptor arm is sandwiched between the N-terminal domains and the catalytic domain (PubMed:10319817). The N-terminal also contributes to the precise recognition of tRNA(Thr) (PubMed:10319817). The editing domain encompasses approximately residues 62-224; when it is removed the protein mischarges tRNA(Thr) with L-serine (PubMed:15079065, PubMed:11136973). {ECO:0000269\|PubMed:11136973, ECO:0000269\|PubMed:15525511, ECO:0000305\|PubMed:10319817, ECO:0000305\|PubMed:15079065}.
Disruption phenotype:		Essential, it cannot be deleted. {ECO:0000269\|PubMed:15507440}.
Biotechnology:		A number of inhibitors with high affinity for bacterial ThrRS and less affinity for human ThrRS have been identified that might make good antibiotics. {ECO:0000269\|PubMed:23362938}.
Similarity:		Belongs to the class-II aminoacyl-tRNA synthetase family. {ECO:0000255\|HAMAP-Rule:MF_00184}.