Open AccessOX4 Is an NADPH-Dependent Dehydrogenase Catalyzing an Extended Michael Addition Reaction To Form the Six-Membered Ring in the Antifungal HSAF
Author(s) -
Xue Li,
Haoxin Wang,
Yuemao Shen,
Yaoyao Li,
Liangcheng Du
Publication year - 2019
Publication title -
biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.43
H-Index - 253
eISSN - 1520-4995
pISSN - 0006-2960
DOI - 10.1021/acs.biochem.9b00280
Subject(s) - polyene , chemistry , stereochemistry , ring (chemistry) , regioselectivity , hydride , stereoselectivity , enzyme , dehydrogenase , catalysis , biochemistry , organic chemistry , hydrogen
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 21 S 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.