Oxidative cyclizations in orthosomycin biosynthesis expand the known chemistry of an oxygenase superfamily

Abstract

Orthosomycins are oligosaccharide antibiotics that include avilamycin, everninomicin, and hygromycin B and are hallmarked by a rigidifying interglycosidic spirocyclic ortho-δ-lactone (orthoester) linkage between at least one pair of carbohydrates. A subset of orthosomycins additionally contain a carbohydrate capped by a methylenedioxy bridge. The orthoester linkage is necessary for antibiotic activity but rarely observed in natural products. Orthoester linkage and methylenedioxy bridge biosynthesis require similar oxidative cyclizations adjacent to a sugar ring. We have identified a conserved group of nonheme iron, α-ketoglutarate-dependent oxygenases likely responsible for this chemistry. High-resolution crystal structures of the EvdO1 and EvdO2 oxygenases of everninomicin biosynthesis, the AviO1 oxygenase of avilamycin biosynthesis, and HygX of hygromycin B biosynthesis show how these enzymes accommodate large substrates, a challenge that requires a variation in metal coordination in HygX. Excitingly, the ternary complex of HygX with cosubstrate α-ketoglutarate and putative product hygromycin B identified an orientation of one glycosidic linkage of hygromycin B consistent with metal-catalyzed hydrogen atom abstraction from substrate. These structural results are complemented by gene disruption of the oxygenases evdO1 and evdMO1 from the everninomicin biosynthetic cluster, which demonstrate that functional oxygenase activity is critical for antibiotic production. Our data therefore support a role for these enzymes in the production of key features of the orthosomycin antibiotics.antibiotic biosynthesis | oxidative cyclization | crystal structure | nonheme iron α-ketoglutarate-dependent oxygenases

Divergent non-heme iron enzymes in the nogalamycin biosynthetic pathway

“…Related O-cyclizations have also been identified from biosynthetic pathways of orthosomycins (16). However, the only other proteins concluded to catalyze oxidative carbocyclizations at aliphatic carbons are nonrelated Rieske enzymes in the biosynthesis of streptorubin B and metacycloprodigiosin, where the function was inferred from molecular genetic data (17).…”

Novofumigatonin biosynthesis involves a non-heme iron-dependent endoperoxide isomerase for orthoester formation

“…Non-heme iron-utilizing enzymes are widespread in nature and perform versatile reactions 37 , but are mostly involved in oxidative reactions. Enzymatic orthoesterification can be performed by the non-heme iron-dependent enzymes for the orthosomycins pathways as well 4 , but their orthoester synthesis is an oxidative reaction without any structural rearrangement, thus completely differing from the NvfE-catalyzed reaction. Nevertheless, there are some examples in which isomerization reactions are catalyzed by NvfE homologs, such as CarC 38 , SnoN 39 , and AndA 16 , in the carbapenem, nogalamycin, and anditomin pathways, respectively.…”

“…Despite their intriguing structures and biological activities, little is known about the molecular basis for orthoester biogenesis. To the best of our knowledge, the Fe(II)/α-ketoglutarate (αKG)-dependent dioxygenases involved in the biosynthesis of orthosomycins are the only examples of orthoester-producing enzymes known to date 4 .…”

Hydrogen Donation but not Abstraction by a Tyrosine (Y68) during Endoperoxide Installation by Verruculogen Synthase (FtmOx1)

“…14 These enzymes share a largely conserved HX(D/E)X n H ligand motif located in close proximity to a substrate binding site, which typically presents to the iron cofactor an unactivated aliphatic C-H bond to be cleaved by HAT. 15,16 Although the best-studied Fe/2OG oxygenases mediate hydroxylation, a range of other outcomes [17][18][19][20][21][22][23][24][25][26][27][28][29][30] are known. In all Fe/2OG enzymes studied to date, addition of O 2 to the 2OGcoordinated Fe(II) cofactor yields CO 2 and a succinate-coordinated Fe(IV)-oxo (ferryl) intermediate.…”