Kinetic Characterization and Phosphoregulation of the Francisella tularensis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)
Kinetic Characterization and Phosphoregulation of the Francisella tularensis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)
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DOI：
10.1371/journal.pone.0008288
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发表时间：
2009-12-14
期刊：
影响因子：
3.7
通讯作者：
Couch, Robin D.
中科院分区：
文献类型：
--
作者：
Jawaid, Safdar;Seidle, Heather;Couch, Robin D.
故意和自然爆发的传染病强调有效的疫苗和抗菌/抗病毒治疗的必要性。抗生素耐药菌株的流行和抗生素耐药细菌可以被有意地工程化的容易性进一步突出了针对新细菌靶标的新型抗生素的持续开发的需要。异戊二烯是一类基本上参与各种重要生物功能的分子。哺乳动物细胞利用甲羟戊酸途径进行异戊二烯生物合成，而许多细菌利用甲基戊二酸磷酸（MEP）途径，使后者成为抗生素开发的有吸引力的靶标。在这份报告中，我们描述了土拉热弗朗西斯MEP合酶，MEP途径酶和抗生素开发的潜在目标的克隆和表征。在体外生长抑制试验中使用磷霉素，MEP合成酶抑制剂，说明MEP途径抑制与F。土拉热。为了促进药物开发，F.克隆、表达、纯化和表征土拉热菌MEP合酶。酶测定产生表观动力学常数（K（M）（DXP）= 104 μ M，K（M）（NADPH）= 13 μ M，k（cat）（DXP）= 2 s（-1），k（cat）（NADPH）= 1.3 s（-1）），磷霉素的IC（50）为247 nM，磷霉素的K（i）为99 nM。该酶表现出对Mg（+2）作为二价阳离子的偏好。二氧化钛色谱-串联质谱鉴定Ser 177作为磷酸化位点。S177 D和S177 E定点突变体是失活的，提示通过F.土拉菌MEP途径。总之，我们的研究表明，MEP合酶是一个很好的目标，为开发新的抗生素对F。土拉热。
Deliberate and natural outbreaks of infectious disease underscore the necessity of effective vaccines and antimicrobial/antiviral therapeutics. The prevalence of antibiotic resistant strains and the ease by which antibiotic resistant bacteria can be intentionally engineered further highlights the need for continued development of novel antibiotics against new bacterial targets. Isoprenes are a class of molecules fundamentally involved in a variety of crucial biological functions. Mammalian cells utilize the mevalonic acid pathway for isoprene biosynthesis, whereas many bacteria utilize the methylerythritol phosphate (MEP) pathway, making the latter an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP synthase, a MEP pathway enzyme and potential target for antibiotic development. In vitro growth-inhibition assays using fosmidomycin, an inhibitor of MEP synthase, illustrates the effectiveness of MEP pathway inhibition with F. tularensis. To facilitate drug development, F. tularensis MEP synthase was cloned, expressed, purified, and characterized. Enzyme assays produced apparent kinetic constants (K(M)(DXP) = 104 mu M, K(M)(NADPH) = 13 mu M, k(cat)(DXP) = 2 s(-1), k(cat)(NADPH) = 1.3 s(-1)), an IC(50) for fosmidomycin of 247 nM, and a K(i) for fosmidomycin of 99 nM. The enzyme exhibits a preference for Mg(+2) as a divalent cation. Titanium dioxide chromatography-tandem mass spectrometry identified Ser177 as a site of phosphorylation. S177D and S177E site-directed mutants are inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP synthase is an excellent target for the development of novel antibiotics against F. tularensis.