OX4 Is an NADPH-Dependent Dehydrogenase Catalyzing an Extended Michael Addition Reaction to Form the Six-Membered Ring in the Antifungal HSAF

Xue Li, Haoxin Wang, Yuemao Shen, Yaoyao Li, Liangcheng Du

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Abstract

The polycyclic tetramate macrolactam HSAF is an antifungal natural product isolated from Lysobacter enzymogenes. HSAF and its analogues have a distinct chemical structure and new mode of antifungal action. The mechanism by which the 5/5/6 tricycle of HSAF is formed from the polyene precursor is not totally clear. Here, we used purified OX4, a homologous enzyme of alcohol dehydrogenase/Zn-binding proteins, to show the enzymatic mechanism for six-membered ring formation. The results from the deuterium isotope incorporation demonstrated that OX4 selectively transfers the pro-R hydride of NADPH to C21 and one proton from water to C10 of 3-deOH alteramide C (1), resulting in 3-deOH HSAF (2) through a reductive cyclization of the polyene precursor by a mechanism consistent with an extended 1,6-Michael addition reaction. The regioselective incorporation of the NADPH hydride into C21 of 1 is also stereoselective, leading to the 21S configuration of 2. This work represents the first characterization of the activity and selectivity of the enzyme for six-membered ring formation in a group of distinct antifungal polycyclic tetramate macrolactams.

Original languageEnglish (US)
Pages (from-to)5245-5248
Number of pages4
JournalBiochemistry
Volume58
Issue number52
DOIs
Publication statusPublished - Dec 31 2019

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ASJC Scopus subject areas

  • Biochemistry

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