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首页> 外文期刊>Biochemistry >Functional Analysis, Overexpression, and Kinetic Characterization of Pyruvate Kinase from Methicillin-Resistant Staphylococcus aureus
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Functional Analysis, Overexpression, and Kinetic Characterization of Pyruvate Kinase from Methicillin-Resistant Staphylococcus aureus

机译：耐甲氧西林金黄色葡萄球菌丙酮酸激酶的功能分析，过表达和动力学表征

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摘要

Novel antimicrobial targets are urgently needed to overcome rising antibiotic resistance ofnimportant human pathogens including methicillin-resistant Staphylococcus aureus (MRSA). Here we reportnthe essentiality and kinetic properties of MRSA pyruvate kinase (PK). Targetron-mediated gene disruptionndemonstrated PKis essential for S. aureus growth and survival, suggesting that this proteinmay be a potentialndrug target. The presence of the pfk (6-phosphofructokinase)-pyk operon inMRSA252, and the nonessentialnnature of PFK shown by targetron, further emphasized the essential role of PK in cell viability. Thenimportance of PKin bacterial growth was confirmed by showing that its enzymatic activity peaked during thenlogarithmic phase of S. aureus growth. PK from Staphylococcus and several other species of bacteria have annextra C-terminal domain (CT) containing a phosphoenolpyruvate (PEP) binding motif. To elucidate thenpossible structure and function of this sequence, the quaternary structures and kinetic properties of the full-nlengthMRSA PK and truncatedMRSA PK lacking the CT domain were characterized. Our results showednthat (1) MRSA PK is an allosteric enzyme with homotetramer architecture activated by AMP or ribosen5-phosphate (R5P), but not by fructose 1,6-bisphosphate (FBP), which suggests a different mode of allostericnregulation when compared with human isozymes, (2) the CT domain is not required for the tetramerization ofnthe enzyme; homotetramerization occurred in a truncated PK lacking the domain, (3) truncated enzymenexhibited high affinity toward both PEP and ADP and exhibited hyperbolic kinetics toward PEP in thenpresence of activators (AMP andR5P) consistent with kinetic properties of full-length enzyme, indicating thatnthe CT domain is not required for substrate binding or allosteric regulation observed in the holoenzyme, (4)nthe kinetic efficiency (kcat/S0.5) of truncated enzyme was decreased by 24- and 16-fold, in ligand-free state,ntoward PEP and ADP, respectively, but was restored by 3-fold in AMP-bound state, suggesting that thensequence containing the CT domain (Gly473n-Leu585n) plays a substantial role in enzyme activity andncomformational stability, and (5) full-length MRSA PK activity was stimulated at low concentrations ofnATP (e.g., 1 mM) and inhibited by inorganic phosphate and high concentrations of FBP (10 mM) and ATPn(e.g., >2.5 mM), whereas for truncated enzyme, stimulation at low concentrations of ATP was lost. Thesenfindings suggest that the CT domain is involved in maintaining the specificity of allosteric regulation ofnMRSA PK by AMP, R5P, and ATP. The CT extension also encodes a protein domain with homology tonenzyme I of the Escherichia coli sugar-PTS system, suggesting that MRSA PK may also exert an importantnregulatory role in sugar transportmetabolism. These findings yield new insights intoMRSA PKfunction andnmode of allosteric regulation which may aid in the development of clinically important drugs targeting thisnenzyme and further define the role of the extra C-terminal domain in modulating the enzyme’s activity.
机译：迫切需要新的抗微生物目标，以克服重要的人类病原体（包括耐甲氧西林的金黄色葡萄球菌（MRSA））对抗生素的耐药性上升。在这里我们报告n MRSA丙酮酸激酶（PK）的必要性和动力学性质。 Targetron介导的基因破坏表明，PK对金黄色葡萄球菌的生长和存活至关重要，这表明该蛋白可能是潜在的靶向药物。 MRSA252中pfk（6-磷酸果糖激酶）-pyk操纵子的存在，以及靶标显示的PFK的非必需性质，进一步强调了PK在细胞生存力中的重要作用。然后通过显示其酶活性在金黄色葡萄球菌生长的对数生长期达到峰值来确认PKin在细菌生长中的重要性。来自葡萄球菌和其他几种细菌的PK具有含有磷酸烯醇丙酮酸（PEP）结合基序的Annextra C末端结构域（CT）。为了阐明该序列的可能结构和功能，表征了缺乏CT结构域的全长MRSA PK和截短的MRSA PK的四级结构和动力学性质。我们的结果表明（1）MRSA PK是一种具有同四聚体结构的变构酶，其被AMP或核糖5-磷酸（R5P）激活，但未被果糖1,6-二磷酸（FBP）激活，这表明与人相比，变构调控的方式不同（2）该酶的四聚化过程不需要CT结构域;在没有该结构域的截短的PK中发生同源四聚化，（3）截短的酶对PEP和ADP都具有高亲和力，并且对PEP表现出双曲线动力学，然后存在与全长酶动力学特性一致的激活剂（AMP和R5P），表明CT域不需要在全酶中观察到底物结合或变构调节，（4）在无配体状态下，截短酶的动力学效率（kcat / S0.5）降低了24倍和16倍，PEP和ADP向内分别，但恢复3倍的AMP绑定状态，这表明，包含CT结构域的序列（Gly473n-Leu585n）在酶活性和信息稳定性中起着重要作用，并且（5）全长MRSA PK活性受到刺激在低浓度的nATP（例如1 mM）下，并被无机磷酸盐和高浓度的FBP（10 mM）和ATPn（例如，> 2.5 mM）抑制，而对于截短的酶，在低浓度下进行刺激ATP丢失了。研究结果提示，CT域参与维持AMP，R5P和ATP对nMRSA PK的变构调节的特异性。 CT扩展还编码具有大肠杆菌糖-PTS系统同调酶I的蛋白质结构域，表明MRSA PK在糖转运代谢中也可能发挥重要的调节作用。这些发现为变构调节的MRSA PK功能和nmode提供了新的见解，这可能有助于开发针对该酶的临床重要药物，并进一步定义额外的C末端结构域在调节酶活性中的作用。

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