From the Department of Structural Biology, The Jacobs School of Medicine & Biomedical Sciences, State University of New York, Buffalo, New York 14203.
the Hauptman-Woodward Medical Research Institute, Buffalo, New York 14203, and.
J Biol Chem. 2018 May 18;293(20):7841-7852. doi: 10.1074/jbc.RA118.002798. Epub 2018 Apr 4.
Aerobactin, a citryl-hydroxamate siderophore, is produced by a number of pathogenic Gram-negative bacteria to aid in iron assimilation. Interest in this well-known siderophore was reignited by recent investigations suggesting that it plays a key role in mediating the enhanced virulence of a hypervirulent pathotype of (hvKP). In contrast to classical opportunistic strains of , hvKP causes serious life-threatening infections in previously healthy individuals in the community. Multiple contemporary reports have confirmed fears that the convergence of multidrug-resistant and hvKP pathotypes has led to the evolution of a highly transmissible, drug-resistant, and virulent "super bug." Despite hvKP harboring four distinct siderophore operons, knocking out production of aerobactin led to a significant attenuation of virulence. Herein, we continue our structural and functional studies on the biosynthesis of this crucial virulence factor. heterologous production and reconstitution of aerobactin biosynthesis from hvKP was carried out, demonstrating the specificity, stereoselectivity, and kinetic throughput of the complete pathway. Additionally, we present a steady-state kinetic analysis and the X-ray crystal structure of the second aerobactin synthetase IucC, as well as describe a surface entropy reduction strategy that was employed for structure determination. Finally, we show solution X-ray scattering data that support a unique dimeric quaternary structure for IucC. These new insights into aerobactin assembly will help inform potential antivirulence strategies and advance our understanding of siderophore biosynthesis.
铁载体 Aerobactin 是一种柠檬酸 - 羟肟酸盐类铁载体，由许多致病性革兰氏阴性菌产生，以帮助铁吸收。最近的研究表明，它在介导高毒力型 （hvKP） 的增强毒力方面发挥着关键作用，这重新激发了人们对这种著名铁载体的兴趣。与经典的机会性病原体不同，hvKP 会导致社区中原本健康的个体发生严重的危及生命的感染。多项当代报告证实了人们的担忧，即多药耐药和 hvKP 型的融合导致了一种高度可传播、耐药和毒力强的“超级细菌”的进化。尽管 hvKP 拥有四个不同的铁载体操纵子，但敲除 Aerobactin 的产生导致了毒力的显著减弱。在此，我们继续进行该关键毒力因子生物合成的结构和功能研究。我们进行了 heterologous 生产和从 hvKP 中重新构建 Aerobactin 生物合成，证明了完整途径的特异性、立体选择性和动力学通量。此外，我们还介绍了 Aerobactin 合成酶 IucC 的稳态动力学分析和 X 射线晶体结构，并描述了一种用于结构测定的表面熵降低策略。最后，我们展示了支持 IucC 独特二聚体四级结构的溶液 X 射线散射数据。这些对 Aerobactin 组装的新见解将有助于为潜在的抗毒力策略提供信息，并推进我们对铁载体生物合成的理解。
