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Separation of a phosphorylated histidine protein using phosphate affinity polyacrylamide gel electrophoresis

2006; Elsevier BV; Volume: 360; Issue: 1 Linguagem: Inglês

10.1016/j.ab.2006.10.005

1096-0309

Seiji Yamada, Hiro Nakamura, Eiji Kinoshita, Emiko Kinoshita‐Kikuta, Tohru Koike, Yoshitsugu Shiro,

RNA and protein synthesis mechanisms

Bacterial two-component systems (TCSs), which typically consist of a sensor histidine kinase (HK) and a response regulator (RR), have been investigated as attractive antibacterial drug targets. Unfortunately, current HK activity assays based on the quantification of autophosphorylated HKs are hampered by the instability of the phosphohistidine (pHis) product, rendering them ill-suited for high-throughput screenings. To address this challenge, we developed a simple HK activity assay using readily available reagents, which we have termed AUDECY (AUtophosphorylation-DEphosphorylation CYcle assay). Instead of trying to preserve the fragile pHis, we deliberately decomposed it with a pHis-specific phosphatase to constitute an ATPase-like cycle for convenient colorimetric measurements. This kinetic assay was successfully employed for the kinetic characterization of E. coli EnvZ and for high-throughput inhibitor screening of vancomycin-resistant Enterococcus faecium (VRE) VanS, of which histidine kinase activity was hardly detectable with conventional methods. Through the screening, we identified OSU-03012, a potent VanS HK inhibitor, which sensitized VRE toward vancomycin, highlighting the potential of AUDECY in HK inhibitor discovery.