In vitro biochemical characterization of catalytic reaction of two flavoproteins toward nocamycin derivatives

Abstract:

[Background] The tetramic acid derivatives, nocamycins and tirandamycins, possess C-10 ketone groups, the formation of which is catalyzed by two different enzymes: a short-chain dehydrogenase NcmD and a FAD-dependent dehydrogenase TrdL, respectively. In the biosynthetic pathway of nocamycins, whether the FAD-dependent oxidase NcmL can complement the function of NcmD remains unknown. Additionally, whether TrdL can catalyze the conversion of C-10 hydroxyl group to C-10 ketone group in nocamycins is also unknown. [Objective] To characterize the catalytic roles of NcmL and TrdL in the formation of C-10 ketone groups in nocamycins by using in vitro enzymatic assays. [Methods] The trdL and ncmL genes were respectively cloned into the vector pET-28a(+), and the recombinant vectors were then overexpressed in Escherichia coli BL21. TrdL and NcmL were purified and then used for the in vitro enzymatic assays. Nocamycins F and II were used as substrates and the products generated under the catalysis of NcmL and TrdL were determined by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometer (LC-MS). [Results] NcmL did not catalyze the dehydrogenation occurred on C-10 hydroxyl group. TrdL catalyzed the hydroxyl dehydrogenation at C-10 position in nocamycins II and F, leading to the generation of nocamycins I and G, respectively. [Conclusion] In vitro biochemical assays revealed that NcmL is not involved in formation of C-10 ketone group of nocamycins. TrdL shows a broad substrate spectrum and can catalyze the formation of C-10 ketone group in nocamycins.