In vitro biochemical characterization of ikarugamycin oxidase CftA

Abstract:

[Background] Polycyclic tetramate macrolactams (PoTeMs), especially ikarugamycin and its analogs, display a wide range of antifungal, antibacterial, and cytotoxic activities. The cytochrome P450 CftA was shown to be an ikarugamycin oxidase according to the in vivo experiments, however, no in vitro evidence was available. [Objective] To study the biochemical function of ikarugamycin oxidase CftA. [Methods] CftA was purified by Ni-NTA affinity chromatography after the overexpression of synthetic gene cftA in Escherichia coli BL21(DE3), and was biochemically characterized by in vitro enzyme reaction and identification of reaction products through HPLC-HR-ESI-MS/MS. [Results] A new ikarugamycin derivative named hydroxyikarugamycin D and a known ikarugamycin derivative, clifednamide A, were observed after incubating CftA and ikarugamycin. [Conclusion] The in vitro evidence was provided to support that tandem oxidations on ikarugamycin was carried out by CftA at C29. This result paved the way for further researches to study the mechanism of ikarugamcin oxidases and the utilization to develop novel ikarugamycin type of PoTeMs.

Key words:ikarugamycin;polycyclic tetramate macrolactams;cytochrome P450;in vitro enzyme reaction