Abstract
Group A flavin-dependent monooxygenases catalyze the cleavage of the oxygen-oxygen bond of dioxygen, followed by the incorporation of one oxygen atom into the substrate molecule with the aid of NADPH and FAD. These flavoenzymes play an important role in many biological processes, and their most distinct structural feature is the choreographed motions of flavin, which typically adopts two distinct conformations (OUT and IN) to fulfill its function. Notably, these enzymes seem to have evolved a delicate control system to avoid the futile cycle of NADPH oxidation and FAD reduction in the absence of substrate, but the molecular basis of this system remains elusive. Using protein crystallography, size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), and small-angle X-ray scattering (SEC-SAXS) and activity assay, we report here a structural and biochemical characterization of PieE, a member of the Group A flavin-dependent monooxygenases involved in the biosynthesis of the antibiotic piericidin A1. This analysis revealed that PieE forms a unique hexamer. Moreover, we found, to the best of our knowledge for the first time, that in addition to the classical OUT and IN conformations, FAD possesses a "sliding" conformation that exists in between the OUT and IN conformations. This observation sheds light on the underlying mechanism of how the signal of substrate binding is transmitted to the FAD-binding site to efficiently initiate NADPH binding and FAD reduction. Our findings bridge a gap currently missing in the orchestrated order of chemical events catalyzed by this important class of enzymes.
Highlights
Group A flavin-dependent monooxygenases catalyze the cleavage of the oxygen– oxygen bond of dioxygen, followed by the incorporation of one oxygen atom into the substrate molecule with the aid of NADPH and FAD
The Group A enzymes seem to have evolved a delicate control system to avoid the futile cycle of NADPH oxidation and FAD reduction in the absence of substrate [5], but its molecular basis remains elusive
The two subunits in the dimer are organized through a “head-to-tail” style
Summary
Group A flavin-dependent monooxygenases catalyze the cleavage of the oxygen– oxygen bond of dioxygen, followed by the incorporation of one oxygen atom into the substrate molecule with the aid of NADPH and FAD These flavoenzymes play an important role in many biological processes, and their most distinct structural feature is the choreographed motions of flavin, which typically adopts two distinct conformations (OUT and IN) to fulfill its function. These enzymes seem to have evolved a delicate control system to avoid the futile cycle of NADPH oxidation and FAD reduction in the absence of substrate, but the molecular basis of this system remains elusive. These structures have revealed that most enzymes possess dissimilar substrate-binding sites to adapt to their specific substrates, which have distinct properties in terms of chemical structure, size, shape, and polarity, and may utilize different strategies/ paths to recruit their respective substrates
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