Insights into Complex Oxidation during BE-7585A Biosynthesis: Structural Determination and Analysis of the Polyketide Monooxygenase BexE

Abstract

Cores of aromatic polyketides are essential for their biological activities. Most type II polyketide synthases (PKSs) biosynthesize these core structures involving the minimal PKS, a PKS-associated ketoreductase (KR) and aromatases/cyclases (ARO/CYCs). Oxygenases (OXYs) are rarely involved. BE-7585A is an anticancer polyketide with an angucyclic core.13C isotope labeling experiments suggest that its angucyclic core may arise from an oxidative rearrangement of a linear anthracyclinone. Here, we present the crystal structure and functional analysis of BexE, the oxygenase proposed to catalyze this key oxidative rearrangement step that generates the angucyclinone framework. Biochemical assays using various linear anthracyclinone model compounds combined with docking simulations narrowed down the substrate of BexE to be an immediate precursor of aklaviketone, possibly 12-deoxy-aklaviketone. The structural analysis, docking simulations, and biochemical assays provide insights into the role of BexE in BE-7585A biosynthesis and lay the groundwork for engineering such framework-modifying enzymes in type II PKSs.

Formation of an Angular Aromatic Polyketide from a Linear Anthrene Precursor via Oxidative Rearrangement

“…MrqO6 is an FAD-dependent monooxygenase with 29% identity and 38% similarity to BexE that has been proposed to catalyzed a Beayer-Villiger reaction in the BE-7585A biosynthesis and MrqO7 is an NAD(P)-dependent oxidoreductase. Unfortunately, when 6 was incubated with MrqO6 or MrqO7, or both MrqO6 and MrqO7, 6 was consumed and many tiny peaks were detected by HPLC (Figure S4), indicating unstable products, consistent with the findings for BexE and BexF (Jackson et al, 2016), although MrqO7 shows no homology to BexF. Thus, as proposed for BexE and BexF, 6 might be converted to a very unstable compound through MrqO6 and/or MrqO7 that is readily degraded or oxidized to generate several other compounds.…”

“…The mass of the product 7/7′ from MrqO6 is 16 Da greater than 6 , indicating a single oxygen atom to be inserted into 6 , consistent with a Baeyer-Villiger reaction, like those proposed for ChaZ (Xu et al, 2005) or BexE (Jackson et al, 2016). The mass of product 8 from MrqO7 is by 2 Da greater than 6 and NAD(P)H is necessary for its formation, indicating that 2 protons were added to 6 , and that the reaction catalyzed by MrqO7 is a reduction.…”

“…It was clearly demonstrated that the MrqO7-catalyzed hydrogenation on 6 is the first step, followed by the reactions of MrqO6 and MrqO3 to afford the final product 1 . Some remaining questions were also addressed by these experiments: (1) MrqO6 may not interact with MrqO7 and no complex should be formed, which is different from the previous speculation (Jackson et al, 2016); (2) The products of MrqO7 and MrqO6 seem to be stable enough in the reaction mixture to allow the follow-up conversion steps to 1 , but they readily decompose during the work-up process.…”

“…Sequence analysis suggested MrqO3 to be a putative cholesterol oxidase, MrqO6 to be a putative flavin adenine dinucleotide (FAD)-dependent monooxygenase homologous to BexE, and MrqO7 to be a putative NAD(P)-dependent oxidoreductase. Note that BexE was proposed to catalyze a Baeyer-Villiger reaction in the BE-7585A biosynthesis (Jackson et al, 2016). …”

“…Most recently, the crystal structure of BexE was resolved to 2.65 Å and used to perform docking stimulations to determine the key linear intermediate. However, unfortunately, the identification of BexE’s true substrate remains challenging (Jackson et al, 2016). In contrast, in this work on murayaquinone biosynthesis, we proved and confirmed the conversion of a linear polyphenolic intermediate to an angular aromatic polyketide.…”