Abstract：
Infections that are acquired due to a prolonged hospital stay and manifest 2 days following the admission of a patient to a health-care institution can be classified as hospital-acquired infections. Klebsiella pneumoniae (K. pneumoniae) has become a critical pathogen, posing serious concern globally due to the rising incidences of hypervirulent and carbapenem-resistant strains. Glutaredoxin is a redox protein that protects cells from oxidative stress as it associates with glutathione to reduce mixed disulfides. Protein adenylyltransferase (PrAT) is a pseudokinase with a proposed mechanism of transferring an AMP group from ATP to glutaredoxin. Inducing oxidative stress to the bacterium by inhibiting the activity of PrAT is a promising approach to combating its contribution to hospital-acquired infections. Thus, this study aims to overexpress, purify, and analyse the effects of ATP and Mg2+ binding to Klebsiella pneumoniae PrAT (KpPrAT). The pET expression system and nickel affinity chromatography were effective in expressing and purifying KpPrAT. Far-UV CD spectroscopy demonstrates that the protein is predominantly α-helical, even in the presence of Mg2+. Extrinsic fluorescence spectroscopy with ANS indicates the presence of a hydrophobic pocket in the presence of ATP and Mg2+, while mant-ATP studies allude to the potential nucleotide binding ability of KpPrAT. The presence of Mg2+ increases the thermostability of the protein. Isothermal titration calorimetry provides insight into the binding affinity and thermodynamic parameters associated with the binding of ATP to KpPrAT, with or without Mg2+. Conclusively, the presence of Mg2+ induces a conformation in KpPrAT that favours nucleotide binding.
摘要：
由于长期住院而获得的感染，并且在患者进入医疗机构2天后出现感染，可以归类为医院获得性感染。肺炎克雷伯菌(K.肺炎)已成为一种关键病原体，由于高毒力和耐碳青霉烯类菌株的发病率上升，在全球引起严重关注。谷氧还蛋白是一种氧化还原蛋白，可与谷胱甘肽结合以减少混合的二硫化物，从而保护细胞免受氧化应激。蛋白腺苷酰转移酶（PrAT）是一种假激酶，具有将AMP基团从ATP转移到谷氧还蛋白的机制。通过抑制PrAT的活性来诱导细菌的氧化应激是对抗其对医院获得性感染的贡献的有希望的方法。因此，这项研究旨在过度表达，净化，分析ATP和Mg2+与肺炎克雷伯菌PrAT(KpPrAT)的结合作用。pET表达系统和镍亲和层析可有效表达和纯化KpPrAT。远紫外CD光谱表明该蛋白质主要是α-螺旋，即使在Mg2+的存在下。用ANS的外在荧光光谱表明在ATP和Mg2+的存在下存在疏水口袋，而Mant-ATP研究暗示了KpPrAT的潜在核苷酸结合能力。Mg2+的存在增加了蛋白质的热稳定性。等温滴定量热法可深入了解与ATP与KpPrAT结合相关的结合亲和力和热力学参数，有或没有Mg2+。最后，Mg2+的存在诱导KpPrAT中有利于核苷酸结合的构象。
