Catalytic Control of Spiroketal Formation in Rubromycin Polyketide Biosynthesis

Abstract

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.

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

“…Interestingly, the FPMO GrhO5 was recently reported by the Teufel group to catalyze the conversion of a pentangular precursor to a spiroketal moiety-containing intermediate in rubromycin biosynthesis, in which an ortho –hydroxylation was critical for a ring-opening step (Supplementary Fig. 77 ) 42 , 43 . Inspired by the work from the Teufel group, we realized that an ortho –hydroxylation by FlsO1 was an alternative mechanism to generate C-ring-opened products 30 or 27 from 22 (Supplementary Fig.…”

“…Phylogenetic analysis reveals that FlsO1 is well clustered with type II group A FPMOs and thus is separated from the GrhO5-type I enzymes 43 (Supplementary Fig. 90 ).…”

An acetyltransferase controls the metabolic flux in rubromycin polyketide biosynthesis by direct modulation of redox tailoring enzymes

“…Therefore, GrhO5 and GrhO6 are likely responsible for the accelerated 5a/5b decomposition due to their ring A reductase activities. 15,16 Because GrhJ and HyalJ are predicted GNATs, acetyl-CoA (AcCoA) was next added to assay mixtures containing 3b, NADPH, GrhO5, GrhO1, GrhO6 together with HyalJ. Interestingly, supplementation of AcCoA resulted in an increase in 5a/ 5b formation in vitro (Fig.…”

“…Recently, the biosynthesis of the spiroketal could be elucidated in more detail by in vitro studies with enzymes encoded by the 1 BGC (grh). 15,16 Notably, these biosynthetic steps most likely proceed via ring A-reduced hydroquinonic intermediates (compounds introduced below denoted with a), while orthoquinonic intermediates and nal pathway products that are typically described in the literature are the result of autooxidation (compounds denoted as b). 16 Accordingly, the advanced pentangular pathway intermediate dihydrocollinone (3a) serves as a precursor for the generation of the [6,6]-spiroketal intermediate dihydrolenticulone (4a), which is subsequently converted to dihydro-7,8-dideoxy-6-oxo-griseorhodin C (5a) that features the mature [5,6]-spiroketal motif.…”

“…The formation of the spiroketal is facilitated by several avoenzymes, which are known to be mechanistically versatile. 10,11,13,[17][18][19][20][21][22][23][24][25] Accordingly, generation of 4a mainly depends on the group A avoprotein monooxygenase (FPMO) GrhO5, 15 which is assisted by avoprotein oxidase GrhO1. 16 The subsequent conversion of the [6,6]-spiroketal-containing 4a into 5a via oxygenation and succeeding decarboxylative ring contraction depends on a second group A FPMO, GrhO6, while further downstream steps require further investigation and presumably proceed via dihydro-7,8-dideoxygriseorhodin C (6a) to the mature rubromycins such as 1 and 2.…”