Enzymatic spiroketal formation via oxidative rearrangement of pentangular polyketides

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.

Catalytic Control of Spiroketal Formation in Rubromycin Polyketide Biosynthesis

“…Actinobacteria produce aw ide range of bioactive aromatic polyketides,a se xemplified by the members of the rubromycin family that feature ac haracteristic bisbenzannulated [5,6]-spiroketal pharmacophore, [1][2][3] e.g., b-rubromycin (1)a nd griseorhodin A ( 2). Most rubromycins possess antibiotic or anti-cancer activities and act as protein inhibitors (e.g.,o fH IV reverse transcriptase), thus making them possible drug leads.…”

“…[4] Gene inactivation studies with the 2 biosynthetic gene cluster from Streptomyces sp.JP95 implied conventional early steps that generate ap olycyclica romatic backbone,b efore further tailoring reactions including ring hydroxylations afford the advanced intermediate collinone (3)a su niversal pentangular precursor for the ensuing spiroketal forming steps. [1,3,5,6] Recently,t wo group Af lavoprotein monooxygenases (FPMOs) (GrhO5 and GrhO6) and af lavoprotein oxidase (GrhO1) were shown to mediate the drastic remodeling of the 3 backbone and subsequent spiroketal pharmacophore formation. [6] First, GrhO5 generates a [ 6,6]-spiroketal moiety as part of the advanced intermediate dihydrolenticulone (4), which entails the rupture of three CÀCb onds and aC O 2 elimination step.T he NADPH-and dioxygen (O 2 )-dependent GrhO5-mediated reaction sequence is initiated by an unusual quinone reduction at ring Aof3 yielding dihydrocollinone (5).…”

“…[1,3,5,6] Recently,t wo group Af lavoprotein monooxygenases (FPMOs) (GrhO5 and GrhO6) and af lavoprotein oxidase (GrhO1) were shown to mediate the drastic remodeling of the 3 backbone and subsequent spiroketal pharmacophore formation. [6] First, GrhO5 generates a [ 6,6]-spiroketal moiety as part of the advanced intermediate dihydrolenticulone (4), which entails the rupture of three CÀCb onds and aC O 2 elimination step.T he NADPH-and dioxygen (O 2 )-dependent GrhO5-mediated reaction sequence is initiated by an unusual quinone reduction at ring Aof3 yielding dihydrocollinone (5). Then, orthohydroxylation of phenolic ring Et riggers two ring-cleaving retro-aldol condensations,c arbonyl hydration and several tautomerizations in addition to ap utative two-electron oxidation step that eventually re-install rings Ca nd Di nt he form of the [6,6]-spiroketal (Figure 1).…”

“…[6] First, GrhO5 generates a [ 6,6]-spiroketal moiety as part of the advanced intermediate dihydrolenticulone (4), which entails the rupture of three CÀCb onds and aC O 2 elimination step.T he NADPH-and dioxygen (O 2 )-dependent GrhO5-mediated reaction sequence is initiated by an unusual quinone reduction at ring Aof3 yielding dihydrocollinone (5). Then, orthohydroxylation of phenolic ring Et riggers two ring-cleaving retro-aldol condensations,c arbonyl hydration and several tautomerizations in addition to ap utative two-electron oxidation step that eventually re-install rings Ca nd Di nt he form of the [6,6]-spiroketal (Figure 1). [6] Thef unctional homolog RubL from rubromycin biosynthesis was shown to catalyze the same reaction sequence,p ointing toward au niversal strategy for spiroketal formation in the rubromycin family.The final product (6)proved to be highly unstable and spontaneously formed 4 or the shunt product 7.G rhO1 presumably mediates hydroquinone oxidation at ring Bo f6 and thereby boosts 4 formation while counteracting 7 formation.…”

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The medically important bacterial aromatic polyketide natural products typically feature ap lanar,p olycyclic core structure.A ne xception is found for the rubromycins, whose backbones are disrupted by ab isbenzannulated [5,6]spiroketal pharmacophore that was recently shown to be assembled by flavin-dependent enzymes.I np articular,aflavoprotein monooxygenase proved critical for the drastic oxidative rearrangement of ap entangular precursor and the installment of an intermediate [6,6]-spiroketal moiety.Here we provides tructural and mechanistic insights into the control of catalysis by this spiroketal synthase,w hich fulfills several important functions as reductase,m onooxygenase,a nd 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.

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