title: 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.
authors.affiliation: Department of Pharmacology, Vanderbilt University, Nashville, TN 37232;
authors.forename: Kathryn M
authors.initials: KM
authors.lastname: McCulloch
authors.affiliation: Department of Chemistry, Vanderbilt University, Nashville, TN 37235;
authors.forename: Emilianne K
authors.initials: EK
authors.lastname: McCranie
authors.affiliation: Department of Biochemistry, Vanderbilt University, Nashville, TN 37232.
authors.forename: Jarrod A
authors.initials: JA
authors.lastname: Smith
authors.affiliation: Department of Pharmacology, Vanderbilt University, Nashville, TN 37232;
authors.forename: Maruf
authors.initials: M
authors.lastname: Sarwar
authors.affiliation: Department of Pharmacology, Vanderbilt University, Nashville, TN 37232;
authors.forename: Jeannette L
authors.initials: JL
authors.lastname: Mathieu
authors.affiliation: Department of Pharmacology, Vanderbilt University, Nashville, TN 37232;
authors.forename: Bryan L
authors.initials: BL
authors.lastname: Gitschlag
authors.affiliation: Department of Chemistry, Vanderbilt University, Nashville, TN 37235;
authors.forename: Yu
authors.initials: Y
authors.lastname: Du
authors.affiliation: Department of Chemistry, Vanderbilt University, Nashville, TN 37235; Department of Biochemistry, Vanderbilt University, Nashville, TN 37232 brian.bachmann@vanderbilt.edu tina.iverson@vanderbilt.edu.
authors.forename: Brian O
authors.initials: BO
authors.lastname: Bachmann
authors.affiliation: Department of Pharmacology, Vanderbilt University, Nashville, TN 37232; Department of Biochemistry, Vanderbilt University, Nashville, TN 37232 brian.bachmann@vanderbilt.edu tina.iverson@vanderbilt.edu.
authors.forename: T M
authors.initials: TM
authors.lastname: Iverson
mesh_terms: D000617:Aminoglycosides / Q000737:chemistry*; D000900:Anti-Bacterial Agents / Q000737:chemistry*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003500:Cyclization; D006859:Hydrogen / Q000737:chemistry; D006921:Hygromycin B / Q000737:chemistry; D008670:Metals / Q000737:chemistry; D008847:Micromonospora / Q000201:enzymology / Q000235:genetics; D005810:Multigene Family; D009844:Oligosaccharides / Q000737:chemistry; D016366:Open Reading Frames; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D010105:Oxygenases / Q000737:chemistry*; D010802:Phylogeny; D011485:Protein Binding; D017433:Protein Structure, Secondary; D015203:Reproducibility of Results; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D008670:Metals; D009844:Oligosaccharides; D006921:Hygromycin B; C001202:evernimicin; C006653:avilamycin; D006859:Hydrogen; D010105:Oxygenases; D010100:Oxygen
keywords: antibiotic biosynthesis; crystal structure; nonheme iron α-ketoglutarate–dependent oxygenases; oxidative cyclization