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Glutaminyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization, structure, and development as a screening platform.
Protein Science ( IF 8 ) Pub Date : 2019-12-24 , DOI: 10.1002/pro.3800
Yaritza Escamilla 1 , Casey A Hughes 1 , Jan Abendroth 2, 3 , David M Dranow 2, 3 , Samantha Balboa 1 , Frank B Dean 1 , James M Bullard 1
Affiliation  

Pseudomonas aeruginosa has a high potential for developing resistance to multiple antibiotics. The gene (glnS) encoding glutaminyl-tRNA synthetase (GlnRS) from P. aeruginosa was cloned and the resulting protein characterized. GlnRS was kinetically evaluated and the KM and kcat obs , governing interactions with tRNA, were 1.0 μM and 0.15 s-1 , respectively. The crystal structure of the α2 form of P. aeruginosa GlnRS was solved to 1.9 Å resolution. The amino acid sequence and structure of P. aeruginosa GlnRS were analyzed and compared to that of GlnRS from Escherichia coli. Amino acids that interact with ATP, glutamine, and tRNA are well conserved and structure overlays indicate that both GlnRS proteins conform to a similar three-dimensional structure. GlnRS was developed into a screening platform using scintillation proximity assay technology and used to screen ~2,000 chemical compounds. Three inhibitory compounds were identified and analyzed for enzymatic inhibition as well as minimum inhibitory concentrations against clinically relevant bacterial strains. Two of the compounds, BM02E04 and BM04H03, were selected for further studies. These compounds displayed broad-spectrum antibacterial activity and exhibited moderate inhibitory activity against mutant efflux deficient strains of P. aeruginosa and E. coli. Growth of wild-type strains was unaffected, indicating that efflux was likely responsible for the lack of sensitivity. The global mode of action was determined using time-kill kinetics. BM04H03 did not inhibit the growth of human cell cultures at any concentration and BM02E04 only inhibit cultures at the highest concentration tested (400 μg/ml). In conclusion, GlnRS from P. aeruginosa is shown to have a structure similar to that of E. coli GlnRS and two natural product compounds were identified as inhibitors of P. aeruginosa GlnRS with the potential for utility as lead candidates in antibacterial drug development in a time of increased antibiotic resistance.

中文翻译:

铜绿假单胞菌的谷氨酰胺基-tRNA合成酶:表征,结构和发展作为筛选平台。

铜绿假单胞菌对多种抗生素具有耐药性。克隆了来自铜绿假单胞菌的编码谷氨酰胺-tRNA合成酶(GlnRS)的基因(glnS),并鉴定了所得蛋白质。动力学评估了GlnRS,控制与tRNA相互作用的KM和kcat obs分别为1.0μM和0.15 s-1。铜绿假单胞菌GlnRS的α2形式的晶体结构解析为1.9Å分辨率。分析了铜绿假单胞菌GlnRS的氨基酸序列和结构,并将其与大肠杆菌中的GlnRS进行了比较。与ATP,谷氨酰胺和tRNA相互作用的氨基酸非常保守,结构重叠表明这两种GlnRS蛋白均符合相似的三维结构。GlnRS已通过闪烁邻近分析技术发展成为一个筛选平台,可用于筛选约2,000种化学化合物。鉴定了三种抑制性化合物,并针对临床相关细菌菌株分析了其酶促抑制作用以及最低抑制浓度。选择了其中两种化合物BM02E04和BM04H03进行进一步研究。这些化合物显示出广谱抗菌活性,并且对铜绿假单胞菌和大肠杆菌的突变体外排缺陷型菌株表现出中等的抑制活性。野生型菌株的生长不受影响,表明外排可能是缺乏敏感性的原因。使用时间杀灭动力学确定整体作用方式。BM04H03在任何浓度下均不抑制人细胞培养物的生长,而BM02E04仅抑制最高测试浓度(400μg/ ml)中的培养物。总之,铜绿假单胞菌的GlnRS被证明具有与大肠杆菌GlnRS类似的结构,并且两种天然产物化合物被鉴定为铜绿假单胞菌GlnRS的抑制剂,具有作为抗菌药物开发中的主要候选药物的潜力。抗生素耐药性增加的时间。
更新日期:2019-12-24
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