Biochemical and structural insights of multifunctional flavin-dependent monooxygenase FlsO1-catalyzed unexpected xanthone formation

Abstract

Xanthone-containing natural products display diverse pharmacological properties. The biosynthetic mechanisms of the xanthone formation have not been well documented. Here we show that the flavoprotein monooxygenase FlsO1 in the biosynthesis of fluostatins not only functionally compensates for the monooxygenase FlsO2 in converting prejadomycin to dehydrorabelomycin, but also unexpectedly converts prejadomycin to xanthone-containing products by catalyzing three successive oxidations including hydroxylation, epoxidation and Baeyer-Villiger oxidation. We also provide biochemical evidence to support the physiological role of FlsO1 as the benzo[b]-fluorene C5-hydrolase by using nenestatin C as a substrate mimic. Finally, we resolve the crystal structure of FlsO1 in complex with the cofactor flavin adenine dinucleotide close to the “in” conformation to enable the construction of reactive substrate-docking models to understand the basis of a single enzyme-catalyzed multiple oxidations. This study highlights a mechanistic perspective for the enzymatic xanthone formation in actinomycetes and sets an example for the versatile functions of flavoproteins.

Insights into the Multisymmetric Tailoring of Julichrome Compounds by the Oxygenases JuiN and JuiO: Enzymatic Processing around the Biaryl C–C Axis

“…The overall structure is similar to the well-studied p -hydroxybenzoate hydroxylase ( p HBH) and other p HBH family members involved in the pathways of aromatic polyketides (Figure S2). − The dimeric interface of JuiN occupied by the FAD-binding domain consists of a series of hydrophobic/aromatic residues and two pairs of salt bridges (Arg172–Glu357) (Figure d,e). The electron density unambiguously delineates the binding of FAD and julichrome Q 6‑6 ( 3 ) in each monomer.…”

“…JuiN belongs to the class A flavin monooxygenases; many members of this family are involved in the biosynthetic pathways of aromatic polyketides, such as OxyS from the oxytetracycline pathway 19 and ROX from the rifamycin pathway, 20 as well as FlsO1 from the fluostatin pathway. 21 Specifically, JuiN is an iterative enzyme that catalyzes two sequential hydroxylations on each side of the dimeric substrate. Although both the substrate and product in this process are constitutionally symmetric, our crystal structure of JuiN in complex with the substrate shows that the enzyme interacts with both sides of julichrome Q 6-6 (3).…”

“…JuiN belongs to the class A flavin monooxygenases; many members of this family are involved in the biosynthetic pathways of aromatic polyketides, such as OxyS from the oxytetracycline pathway and ROX from the rifamycin pathway, as well as FlsO1 from the fluostatin pathway . Specifically, JuiN is an iterative enzyme that catalyzes two sequential hydroxylations on each side of the dimeric substrate.…”

Unravelling key enzymatic steps in C-ring cleavage during angucyclines biosynthesis

“…31 Interestingly, in vitro reactions utilizing the monooxygenase FlsO1 resulted in a C-ring cleavage and production of a xanthone compound from prejadomycin through the same mechanism. 51 FlsO1 is distantly related to LugOIII and LugOV, and acts in vivo as a C5 hydroxylase following the B-ring cleavage enzyme FlsG, in the biosynthesis of fluostatins. 52 Additionally, based on our data the epoxidation step is not catalyzed by the C-ring cleavage enzyme required for lugdunomycin (4) biosynthesis, making LugOV biochemically unique as compared to XanO4 or FlsO1.…”

Aromatic Polyketides from the Mangrove-Derived Streptomyces sp. SCSIO 40069

“…Marine-derived microorganisms, including mangrove sediment-associated bacteria and fungi, − have been documented as prolific sources for structurally diversified aromatic polyketides. , Our continuing efforts to explore marine actinobacteria have led to the discovery of a number of aromatic polyketides and the elucidation of their biosynthetic pathways. − Recently, the mangrove sediment-associated strain Streptomyces sp. SCSIO 40069 drew our attention due to its potential to produce bioactive secondary metabolites showing characteristic ultraviolet visible (UV–vis) absorptions of aromatic polyketides.…”