Catalytic Control of Spiroketal Formation in Rubromycin Polyketide Biosynthesis.

Marina Toplak,Robin Teufel,Jörn Piel,Raspudin Saleem‐Batcha

doi:10.1002/anie.202109384

Abstract

The medically important bacterial aromatic polyketide natural products typically feature a planar, polycyclic core structure. An exception is found for the rubromycins, whose backbones are disrupted by a bisbenzannulated [5,6]‐spiroketal pharmacophore that was recently shown to be assembled by flavin‐dependent enzymes. In particular, a flavoprotein monooxygenase proved critical for the drastic oxidative rearrangement of a pentangular precursor and the installment of an intermediate [6,6]‐spiroketal moiety. Here we provide structural and mechanistic insights into the control of catalysis by this spiroketal synthase, which fulfills several important functions as reductase, monooxygenase, and presumably oxidase. The enzyme hereby tightly controls the redox state of the substrate to counteract shunt product formation, while also steering the cleavage of three carbon‐carbon bonds. Our work illustrates an exceptional strategy for the biosynthesis of stable chroman spiroketals.

Highlights

Actinobacteria produce a wide range of bioactive aromatic polyketides, as exemplified by the members of the rubromycin family that feature a characteristic bisbenzannulated [5,6]-spiroketal pharmacophore,[1,2,3] e.g., b-rubromycin (1) and griseorhodin A (2)
GrhO5 features three domains similar to other group A flavoprotein monooxygenases (FPMOs) (Figure 2 A); domains I and II are essential for FAD- and substrate binding, respectively, whereas the C-terminal thioredoxin-like domain III lacks a catalytically important Cys residue that otherwise enables the degradation of H2O2 in the presence of dithiothreitol.[7]
Compared to other well characterized group A FPMOs (Figure 3), high sequence conservation for GrhO5 is only observed in the FAD-binding domain (Figure 2 A, gold; residues 1–77, 108– 188, and 287–386) including the GXGXXG, the GD and the DG (conserved Asp172/Asp180 and Gly173/Gly181 both involved in FAD and NAD(P)H binding) motives.[12]

Summary

Introduction

Hydroxylations afford the advanced intermediate collinone (3) as universal pentangular precursor for the ensuing spiroketal forming steps.[1,3,5,6] Recently, two group A flavoprotein monooxygenases (FPMOs) (GrhO5 and GrhO6) and a flavoprotein oxidase (GrhO1) were shown to mediate the drastic remodeling of the 3 backbone and subsequent spiroketal pharmacophore formation.[6]. Orthohydroxylation of phenolic ring E triggers two ring-cleaving retro-aldol condensations, carbonyl hydration and several tautomerizations in addition to a putative two-electron oxidation step that eventually re-install rings C and D in the form of the [6,6]-spiroketal (Figure 1).[6] The functional homolog RubL from rubromycin biosynthesis was shown to catalyze the same reaction sequence, pointing toward a universal strategy for spiroketal formation in the rubromycin family. GrhO6 hydroxylates phenolic ring B of 4, which facilitates the decarboxylative ring C opening and subsequent contraction to the mature [5,6]-spiroketal of 7,8dideoxy-6-oxo-griseorhodin C (8) (Figure 1). The spiroketal synthases were mechanistically and structurally investigated to understand how they orchestrate and control the distinct redox reactions and backbone rearrangements

Results and Discussion

Conclusion

Conflict of Interest