Rational protein engineering in action: The first crystal structure of a phenylalanine tRNA synthetase from Staphylococcus haemolyticus

Evdokimov, A.G.; Mekel, Marlene; Hutchings, Kim; Narasimhan, Lakshmi; Holler, Tod P.; McGrath, T.E.; Beattie, Bryan K.; Fauman, Eric B.; Yan, Chunhong; Heaslet, H.; Walter, R.L.; Finzel, B.C.; Ohren, Jeffrey F.; McConnell, Patrick I.; Braden, Timothy; Sun, Fei; Spessard, Cindy; Banotai, Craig; Al-Kassim, Loola; Ma, Weijun; Wengender, Paul; Kole, Denis; Garceau, Norman; Toogood, Peter L.; Liu, Jia

doi:10.1016/j.jsb.2007.11.002

Cited by 16 publications

(11 citation statements)

References 66 publications

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“…S2a). Comparisons to the liganded S. haemolyticus enzyme (27) showed nearly perfect superposition within the binding site. Although the phenyl-sulfonamide core occupies a similar position within the binding site as phenylalanine, the thiazolylurea component extends much deeper into an auxiliary hydrophobic pocket (Fig.…”

Section: Phenyl-thiazolylurea-sulfonamides Act Via Phers In Gram-mentioning

confidence: 91%

“…Protein Engineering-The rational protein engineering approach applied by Evdokimov et al (27) for Staphylococcus haemolyticus PheRS was utilized to engineer the P. aeruginosa PheRS complex for crystallographic studies. Briefly, a variety of full-length and N-terminal truncation constructs were designed to remove expected regions of disorder within PheS and to introduce a series of surface entropy reduction mutations within PheT to eliminate the formation of inefficient crystal contacts that would lead to low resolution and/or anisotropic diffraction and irreproducible crystallization.…”

Section: Methodsmentioning

confidence: 99%

“…The structure of apo PheRS from P. aeruginosa was solved at 2.79 Å resolution by molecular replacement using the program MOLREP with the coordinates of PheRS from S. haemolyticus (Protein Data Bank accession code 2RHQ) as the search model (27,32). The structures of all liganded PheRS complexes were subsequently determined using the coordinates of the apo structure.…”

Section: Methodsmentioning

confidence: 99%

“…Moreover, two PheRS structures from the Gram-positive pathogen S. haemolyticus with a phenyl-thiazolylurea-sulfonamide inhibitor (compound 1a) have been determined (27). To understand the structure-activity relationships of published inhibitors and to facilitate the structure-guided design of novel antibacterial compounds, the crystal structure of the PheRS complex from the Gram-negative pathogen P. aeruginosa was solved.…”

Section: Phenyl-thiazolylurea-sulfonamides Act Via Phers In Gram-mentioning

confidence: 99%

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The Role of a Novel Auxiliary Pocket in Bacterial Phenylalanyl-tRNA Synthetase Druggability

Abibi

Ferguson

Fleming

et al. 2014

Journal of Biological Chemistry

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Background: Phenylalanyl-tRNA synthetase inhibitors have been shown to be efficacious in animal models of infection. Results: Inhibitors occupy a newly identified hydrophobic auxiliary binding pocket. Conclusion: Compound binding in this pocket leads to high screening hit rates, resistance frequencies, and elevated plasma protein binding. Significance: New inhibitors may be identified by avoiding the auxiliary pocket.

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Section: Phenyl-thiazolylurea-sulfonamides Act Via Phers In Gram-mentioning

confidence: 91%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Phenyl-thiazolylurea-sulfonamides Act Via Phers In Gram-mentioning

confidence: 99%

See 2 more Smart Citations

The Role of a Novel Auxiliary Pocket in Bacterial Phenylalanyl-tRNA Synthetase Druggability

Abibi

Ferguson

Fleming

et al. 2014

Journal of Biological Chemistry

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“…The architecture of the PheRS enzymes has been extensively described, primarily from structural studies of PheRS from T. thermophilus [18,28-30] and more recently the structure of PheRS from E. coli [31], Staphylococcus haemolyticus [32] and P. aeruginosa [33] has been solved. The amino acid sequence conservation when comparing that of T. thermophilus PheRS with the corresponding enzymes from E. coli , P. aeruginosa , or S. pneumoniae is very similar (Table 1 ).…”

Section: Resultsmentioning

confidence: 99%

High Throughput Screen Identifies Natural Product Inhibitor of Phenylalanyl-tRNA Synthetase from Pseudomonas aeruginosa and Streptococcus pneumoniae

Hu¹,

Palmer²,

Muñoz³

et al. 2015

CDDT

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Pseudomonas aeruginosa and Streptococcus pneumoniae are causative agents in a wide range of infections. Genes encoding proteins corresponding to phenylalanyl-tRNA synthetase (PheRS) were cloned from both bacteria. The two forms of PheRS were kinetically evaluated and the Km’s for P. aeruginosa PheRS with its three substrates, phenylalanine, ATP and tRNAPhe were determined to be 48, 200, and 1.2 μM, respectively, while the Km’s for S. pneumoniae PheRS with respect to phenylalanine, ATP and tRNAPhe were 21, 225 and 0.94 μM, respectively. P. aeruginosa and S. pneumoniae PheRS were used to screen a natural compound library and a single compound was identified that inhibited the function of both enzymes. The compound inhibited P. aeruginosa and S. pneumoniae PheRS with IC50’s of 2.3 and 4.9 μM, respectively. The compound had a Ki of 0.83 and 0.98 μM against P. aeruginosa and S. pneumoniae PheRS, respectively. The minimum inhibitory concentration (MIC) of the compound was determined against a panel of Gram positive and negative bacteria including efflux pump mutants and hyper-sensitive strains. MICs against wild-type P. aeruginosa and S. pneumoniae cells in culture were determined to be 16 and 32 μg/ml, respectively. The mechanism of action of the compound was determined to be competitive with the amino acid, phenylalanine, and uncompetitive with AT P. There was no inhibition of cytoplasmic protein synthesis, however, partial inhibition of the human mitochondrial PheRS was observed.

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Making Proteins with Unnatural Amino Acids: The First Engineered Aminoacyl‐tRNA Synthetase Revisited

Kast

2011

ChemBioChem

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Rational protein engineering in action: The first crystal structure of a phenylalanine tRNA synthetase from Staphylococcus haemolyticus

Cited by 16 publications

References 66 publications

The Role of a Novel Auxiliary Pocket in Bacterial Phenylalanyl-tRNA Synthetase Druggability

The Role of a Novel Auxiliary Pocket in Bacterial Phenylalanyl-tRNA Synthetase Druggability

High Throughput Screen Identifies Natural Product Inhibitor of Phenylalanyl-tRNA Synthetase from Pseudomonas aeruginosa and Streptococcus pneumoniae

Making Proteins with Unnatural Amino Acids: The First Engineered Aminoacyl‐tRNA Synthetase Revisited

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