Enzymatic spiroketal formation via oxidative rearrangement of pentangular polyketides

Britta Frensch,Thorsten Lechtenberg,Bernd Kammerer,Martin Betschart,Michael Müller,Michel Kather,Jörn Piel,Steffen Lüdeke,Robin Teufel,Zeynep Yunt

doi:10.1038/s41467-021-21432-9

Abstract

The structural complexity and bioactivity of natural products often depend on enzymatic redox tailoring steps. This is exemplified by the generation of the bisbenzannulated [5,6]-spiroketal pharmacophore in the bacterial rubromycin family of aromatic polyketides, which exhibit a wide array of bioactivities such as the inhibition of HIV reverse transcriptase or DNA helicase. Here we elucidate the complex flavoenzyme-driven formation of the rubromycin pharmacophore that is markedly distinct from conventional (bio)synthetic strategies for spiroketal formation. Accordingly, a polycyclic aromatic precursor undergoes extensive enzymatic oxidative rearrangement catalyzed by two flavoprotein monooxygenases and a flavoprotein oxidase that ultimately results in a drastic distortion of the carbon skeleton. The one-pot in vitro reconstitution of the key enzymatic steps as well as the comprehensive characterization of reactive intermediates allow to unravel the intricate underlying reactions, during which four carbon-carbon bonds are broken and two CO2 become eliminated. This work provides detailed insight into perplexing redox tailoring enzymology that sets the stage for the (chemo)enzymatic production and bioengineering of bioactive spiroketal-containing polyketides.

Highlights

The structural complexity and bioactivity of natural products often depend on enzymatic redox tailoring steps
The benastatins, pradimicins, fredericamycins, xantholipins, as well as the rubromycin family belong to a growing group of biosynthetically related aromatic type II polyketide natural products with extended “pentangular” architecture that are produced by numerous actinobacterial species[1,2,3,4,5,6,7,8]
GrhO5 is predicted to function as flavoprotein monooxygenase based on the amino acid sequence[10] and is homologous to the NAD(P)H- and flavin adenine dinucleotide (FAD)-dependent class A flavoprotein monooxygenases with “glutathione reductase type” Rossmann fold[21]

Summary

Introduction

The structural complexity and bioactivity of natural products often depend on enzymatic redox tailoring steps. Following enzyme-catalyzed regioselective ketoreduction, cyclization, aromatization and ACP elimination, further tailoring reactions modify the polyketide backbone and lead to the advanced and highly oxidized intermediate collinone (3) (previously isolated from a heterologous producer expressing parts of the rubromycin biosynthetic gene cluster15), which may serve as a direct precursor for spiroketalization[10] This would necessitate an extensive oxidative backbone rearrangement as well as the elimination of two C1 units, which may be mediated by mechanistically versatile flavin-dependent enzymes[16,17,18,19,20,21,22] that often facilitate redox tailoring reactions in natural product biosynthesis (Fig. 1)[16,19]. We elucidate the conversion of 3 into the 1 kb (presumably GrhO10) converts 4 into 13. c Examples of mature rubromycins likely formed from 13

Methods

Results

Conclusion