Crystallographic Studies of Polyphosphate Kinase

描述(申请人提供)：聚磷酸盐(Poly(P))是一种由数百个正磷酸盐(PI)残基通过高能磷酸酸酐键连接而成的线性聚合物。许多证据表明，Poly(P)在对压力和营养缺乏的调节反应中发挥着关键作用。多聚磷酸酶(PPK)是一种由三磷酸腺苷(ATP)可逆合成聚(P)的酶。最近的研究表明，PPK对包括铜绿假单胞菌在内的许多病原微生物的生物被膜发育、群体感应、运动和毒力因子的释放都是必不可少的。PPK基因的敲除会导致病原微生物丧失生存能力。因此，PPK特异性抑制剂可能成为一类新的抗生素家族，在克服囊性纤维化和其他免疫缺陷患者的抗生素耐药性方面非常有用，并可能在打击生物恐怖主义方面发挥作用。PPK是一种膜相关酶，与其他蛋白质没有明显的序列同源性。PPK系列没有可用的三维结构信息。我们最近对全长的大肠杆菌PPK进行了纯化和结晶，并获得了用于X射线结晶学结构测定的初始相。这项建议的主要目的是确定PPK及其与各种底物和反应中间体的络合物的晶体结构。我们的结构研究将有助于确定PPK的催化机制，并为药物设计提供见解。

DESCRIPTION (provided by applicant): Polyphosphate (poly (P)) is a linear polymer of hundreds of orthophosphate (Pi) residues linked by high-energy phosphoanhydride bonds. Many lines of evidence indicate that poly (P) plays a critical role in regulatory responses to stresses and nutritional deficiencies. Polyphosphate kinase (PPK) is an enzyme responsible for the reversible synthesis of poly (P) from ATP. It has been shown recently that PPK is essential for biofilm development, quorum sensing, motility and release of virulence factors of many pathogenic microorganisms, including P. aeruginosa. Knockout of the PPK gene leads to the loss of viability of the pathogenic microorganism. Thus PPK specific inhibitors could become a novel family of antibiotics, which can be very useful to overcome antibiotic resistance in cystic fibrosis and other immunodeficient patients, and could play a role in combating bioterrorism. PPK is a membrane-associated enzyme that does not have apparent sequence homology with other proteins. No three-dimensional structural information is available for the PPK family. We have recently purified and crystallized the full-length E. coli PPK, and the initial phases for structure determination by x-ray crystallography have been obtained. The main goal of this proposal is the determination of crystal structures of PPK and its complexes with various substrates and reaction intermediate mimics. Our structural studies will aid in determining the PPK catalytic mechanism and provide insights into drug design.