Structure and assembly of the diiron cofactor in the heme-oxygenase-like domain of the
| Autor: | Molly J, McBride, Sarah R, Pope, Kai, Hu, C Denise, Okafor, Emily P, Balskus, J Martin, Bollinger, Amie K, Boal |
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| Rok vydání: | 2021 |
| Předmět: |
Protein Conformation
bioinformatics Biological Sciences Crystallography X-Ray Ligands Biochemistry streptozotocin Catalysis Nitrosourea Compounds Nonheme Iron Proteins Streptozocin Oxygen Pancreatic Neoplasms Biophysics and Computational Biology Protein Domains N-oxygenase Heme Oxygenase (Decyclizing) Physical Sciences Oxygenases Humans Oxidation-Reduction X-ray crystallography |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America |
| ISSN: | 1091-6490 |
| Popis: | Significance The enzyme SznF assembles the N-nitrosourea pharmacophore of the drug streptozotocin. Its N-oxygenase domain resembles heme-oxygenase (HO) and belongs to an emerging superfamily of HO-like diiron enzymes (HDOs) with unstable metallocofactors that have resisted structural characterization. Insight into cofactor dynamics from our prior investigation of SznF’s N-oxygenation reactions suggested an approach that has yielded a structure of a functionally assigned HDO with its diiron cofactor intact. Conformational changes accompanying cofactor (dis)assembly explain its instability, and the observation of an unanticipated glutamate ligand that is conserved in only a subset of HDO sequences provides a potential basis for top-level assignment of enzymatic function. Our results thus provide a roadmap for structural and functional characterization of novel HDOs. In biosynthesis of the pancreatic cancer drug streptozotocin, the tridomain nonheme-iron oxygenase SznF hydroxylates Nδ and Nω′ of Nω-methyl-l-arginine before oxidatively rearranging the triply modified guanidine to the N-methyl-N-nitrosourea pharmacophore. A previously published structure visualized the monoiron cofactor in the enzyme’s C-terminal cupin domain, which promotes the final rearrangement, but exhibited disorder and minimal metal occupancy in the site of the proposed diiron cofactor in the N-hydroxylating heme-oxygenase–like (HO-like) central domain. We leveraged our recent observation that the N-oxygenating µ-peroxodiiron(III/III) intermediate can form in the HO-like domain after the apo protein self-assembles its diiron(II/II) cofactor to solve structures of SznF with both of its iron cofactors bound. These structures of a biochemically validated member of the emerging heme-oxygenase–like diiron oxidase and oxygenase (HDO) superfamily with intact diiron cofactor reveal both the large-scale conformational change required to assemble the O2-reactive Fe2(II/II) complex and the structural basis for cofactor instability—a trait shared by the other validated HDOs. During cofactor (dis)assembly, a ligand-harboring core helix dynamically (un)folds. The diiron cofactor also coordinates an unanticipated Glu ligand contributed by an auxiliary helix implicated in substrate binding by docking and molecular dynamics simulations. The additional carboxylate ligand is conserved in another N-oxygenating HDO but not in two HDOs that cleave carbon–hydrogen and carbon–carbon bonds to install olefins. Among ∼9,600 sequences identified bioinformatically as members of the emerging HDO superfamily, ∼25% conserve this additional carboxylate residue and are thus tentatively assigned as N-oxygenases. |
| Databáze: | OpenAIRE |
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