Structural analyses of the Group A flavin-dependent monooxygenase PieE reveal a sliding FAD cofactor conformation bridging OUT and IN conformations

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.

Catalytic Control of Spiroketal Formation in Rubromycin Polyketide Biosynthesis

“…Moreover,t hese spiroketal synthases feature two distinct openings to the protein interior and the active site,d enoted by "substrate entry site" and "NADPH entry site" (Figure 2B-E). TheG rhO5 structure obtained in the absence of substrate adopts the acceptor state (= resting state) similar to three closely related group A FPMOs from bacterial secondary metabolism (RdmE, an-thracycline biosynthesis, [15] RebC,r ebeccamycin biosynthesis, [16] PieE, piericidin A1 biosynthesis [9] ). Notably,t he FAD-OUT conformation in GrhO5 seems to be stabilized by atryptophan residue (W281) that is properly positioned at the re-side to engage in aromatic p-p interactions with the isoalloxazine ring.…”

“…[7] Instead, domain III could be important for protein stability [8] or promote protein-protein interactions. [7,[9][10][11] It is noteworthy that the prototype of group AF PMOs, p-hydroxybenzoate hydroxylase (PHBH), is missing domain III entirely.C ompared to other well characterized group AFPMOs (Figure 3), high sequence conservation for GrhO5 is only observed in the FAD-binding domain (Figure 2A, gold;r esidues 1-77, 108-188, and 287-386) including the GXGXXG (part of the Rossmann fold important for binding of the ADP moiety; GXSXXG for GrhO5/RubL), the GD (contact of Gly300/ Gly308 and Asp301/Asp309 with the O3' of the ribose moiety) and the DG (conserved Asp172/Asp180 and Gly173/Gly181 both involved in FADand NAD(P)H binding) motives. [12] Group AFPMOs typically feature mobile,non-covalently bound FADc ofactors to prevent uncoupling (i.e.n onproductive NADPH oxidation and formation of H 2 O 2 from collapse of the FAD C4aOO(H) oxygenating species) in the absence of the substrates.…”

“…After the initial reduction of 3 to 5,G rhO5 is expected to catalyze an aromatic hydroxylation reaction, leading to the formation of unstable secocollinone (10,that required methylation prior to structuralc haracterization due to high innate reactivity [6] ). Compound 10 is further converted into 6,which may either autooxidize and decarboxylate to 4 (that once more autooxidizes to lenticulone (9), analogous to the spontaneous formation of 3 from 5), or decarboxylate and autooxidize to shunt product 7.F ormation of the shunt product is suppressed by GrhO1, which likely oxidizes ring Bo fcompound 6 to boost 4 formation.F inally,G rhO6 converts 4 into the [5,6]-spiroketal containing compound 7,8-dideoxy-6-oxo-griseorhodin C( 8)t hat is further processed by pathway-specific enzymes (not shown) to the mature rubromycins (red box). The numbered compounds were previously identified and (partially) structurally characterized( other intermediates are postulated).…”

A dedicated flavin-dependent monooxygenase catalyzes the hydroxylation of demethoxyubiquinone into ubiquinone (coenzyme Q) in Arabidopsis

“…The function of this domain, which is found in a number of group A FAD-dependent monooxygenases, is uncertain. Structural studies of some bacterial monooxygenases indicate that this domain is localized at the interface of dimers of these enzymes and therefore could take part in their oligomerization ( 38 , 39 ). However, other group A monooxygenases that harbor a similar C-terminal domain have been shown to be active as monomers ( 40 , 41 ).…”

“…However, other group A monooxygenases that harbor a similar C-terminal domain have been shown to be active as monomers ( 40 , 41 ). The sole consensus seems to be that this C-terminal extension does not contribute directly to the binding of substrates or flavin cofactor ( 39 , 40 , 42 ).…”