Literature summary for 6.1.1.1 extracted from

Barros-Alvarez, X.; Kerchner, K.M.; Koh, C.Y.; Turley, S.; Pardon, E.; Steyaert, J.; Ranade, R.M.; Gillespie, J.R.; Zhang, Z.; Verlinde, C.L.M.J.; Fan, E.; Buckner, F.S.; Hol, W.G.J.
Leishmania donovani tyrosyl-tRNA synthetase structure in complex with a tyrosyl adenylate analog and comparisons with human and protozoan counterparts (2017), Biochimie, 138, 124-136 .

Application

Application	Comment	Organism
drug development	the LdTyrRS and its extra pocket structure can be a target for development of selective tyrosyl-tRNA synthetase inhibitors and drug design to treat leishmaniosis	Leishmania donovani

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of His-tagged enzyme in Escherichia coli from AVA0421 vector	Leishmania donovani

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified tyrosyl-tRNA synthetase in complex with a nanobody and inhibitory tyrosyl adenylate analogue TyrSA, purified LdTyrRS and NbA proteins are incubated on ice for 30 min at a 1:2 M ratio followed by buffer exchange to crystallization buffer. The protein complex is then incubated with 0.2 mM of TyrSA (5'-O-[N-(L-tyrosyl)sulfamoyl]adenosine) on ice for 30-60 min, sitting drop vapour diffsuion method, mixing of 0.001 ml of protein solution containing 5 mg/ml protein complex in 25 mM HEPES, pH 7.25, 100 mM NaCl, 1 mM TCEPHCl, 5% glycerol, and 0.025% NaN3, with 0.001 ml of reservoir solution containing 0.1 M sodium cacodylate, pH 5.7, and 22% PEG 4000, room temperature, 5-7 days, X-ray diffraction structure determination and analysis at 2.75 A resolution, modeling	Leishmania donovani

Crystallization (Comment)

Organism

purified tyrosyl-tRNA synthetase in complex with a nanobody and inhibitory tyrosyl adenylate analogue TyrSA, purified LdTyrRS and NbA proteins are incubated on ice for 30 min at a 1:2 M ratio followed by buffer exchange to crystallization buffer. The protein complex is then incubated with 0.2 mM of TyrSA (5'-O-[N-(L-tyrosyl)sulfamoyl]adenosine) on ice for 30-60 min, sitting drop vapour diffsuion method, mixing of 0.001 ml of protein solution containing 5 mg/ml protein complex in 25 mM HEPES, pH 7.25, 100 mM NaCl, 1 mM TCEPHCl, 5% glycerol, and 0.025% NaN3, with 0.001 ml of reservoir solution containing 0.1 M sodium cacodylate, pH 5.7, and 22% PEG 4000, room temperature, 5-7 days, X-ray diffraction structure determination and analysis at 2.75 A resolution, modeling

Leishmania donovani

Inhibitors

Inhibitors	Comment	Organism
5'-O-[N-(L-tyrosyl)sulfamoyl]adenosine	i.e. TyrSA, a tyrosyl adenylate analogue,. TyrSA is engaged in many interactions with active site residues occupying the tyrosine and adenine binding pockets. Residues making hydrogen bonds with the TyrSA tyrosyl group in the tyrosine binding pocket (YBP) are Y36, Y163, Q167, D170 and Q185. Residues G38, A72 and F75 are responsible for the hydrophobic interactions between enzyme and tyrosine moiety in the YBP	Leishmania donovani
fisetin	-	Leishmania donovani
additional information	LdTyrRS specific nanobodies are generated in Lama glama. Nanobodies are the variable domains of camelid heavy chain-only antibodies. The nanobody NbA makes numerous crystal contacts and in addition reduces the flexibility of a loop of LdTyrRS. NbA is cloned in the pMESy4 vector that carries the pelB sequence coding for the secretion signal peptide of PelB and is expressed as His-tagged protein in Escherichia coli for subsequent purification from the bacterial periplasm by affinity chromatography and gel filtration. 4 anti-LdTyrRS nanobodies are tested as crystallization chaperones, crystallization and structure analysis and modeling, overview	Leishmania donovani

Organism

Organism	UniProt	Comment	Textmining
Leishmania donovani	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged enzyme from Escherichia coli by nickel affinity chromatography, tag cleavage by 3C protease, and gel filtration	Leishmania donovani

Subunits

Subunits	Comment	Organism
More	the LdTyrRS polypeptide chain consists of two pseudo-monomers, each consisting of two domains	Leishmania donovani

Synonyms

Synonyms	Comment	Organism
LdTyrRS	-	Leishmania donovani
Tyrosyl-tRNA synthetase	-	Leishmania donovani

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
22	-	assay at room temperature	Leishmania donovani

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.6	-	assay at	Leishmania donovani

General Information

General Information	Comment	Organism
additional information	the LdTyrRS polypeptide chain consists of two pseudo-monomers, each consisting of two domains. Comparing the two independent chains in the asymmetric unit reveals that the two pseudo-monomers of LdTyrRS can bend with respect to each other essentially as rigid bodies. This flexibility might be useful in the positioning of tRNA for catalysis since both pseudo-monomers in the LdTyrRS chain are needed for charging tRNATyr. The LdTyrRS active site contains two critical pockets: the tyrosine binding pocket (YBP) where the tyrosyl group of inhibitor TyrSA is situated, and the adenine binding pocket (ABP) where the adenine moiety of TyrSA binds. Residues making hydrogen bonds with the TyrSA tyrosyl group in the tyrosine binding pocket (YBP) are Y36, Y163, Q167, D170 and Q185. Residues G38, A72 and F75 are responsible for the hydrophobic interactions between enzyme and tyrosine moiety in the YBP. An extra pocket (EP) appears to be present near the adenine binding. The extra pocket appears to be present near the adenine binding region of LdTyrRS, this pocket is absent in the two human homologous enzymes. Structure-based modelling, overview	Leishmania donovani