In biosynthesis of the pancreatic cancer drug streptozotocin, the tridomain nonheme-iron oxygenase SznF hydroxylates N^δ and N^ω′ of N^ω-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.

在胰腺癌药物链脲佐菌素的生物合成中，三结构域非血红素铁加氧酶 SznF 羟基化N ^ω -甲基- L -精氨酸的N ^δ和N ^ω '，然后将三重修饰的胍氧化重排为N -甲基-N -亚硝基脲药效团。先前发表的结构可视化了该酶 C 端 cupin 结构域中的单铁辅助因子，该因子促进了最终的重排，但在N-羟基化血红素加氧酶 (HO -类似）中心域。我们利用最近的观察结果，即apo蛋白自组装其二铁(II/II)辅因子后， N-氧化μ-过氧化二铁(III/III)中间体可以在HO样结构域中形成，以解决SznF的结构它的铁辅助因子结合。这些经过生化验证的新兴血红素加氧酶类二铁氧化酶和加氧酶 (HDO) 超家族成员的结构，具有完整的二铁辅因子，揭示了组装 O ₂反应性 Fe ₂ (II/II) 所需的大规模构象变化。复杂性和辅因子不稳定的结构基础——这是其他经过验证的 HDO 所共有的特征。在辅因子（解）组装过程中，带有配体的核心螺旋动态（解）折叠。二铁辅因子还通过对接和分子动力学模拟协调由辅助螺旋贡献的意想不到的 Glu 配体，该辅助螺旋涉及底物结合。 额外的羧酸配体在另一个N-氧化 HDO 中保守，但在两个裂解碳-氢和碳-碳键以安装烯烃的 HDO 中不保守。在生物信息学上鉴定为新兴 HDO 超家族成员的约 9,600 个序列中，约 25% 保留了这一额外的羧酸残基，因此暂时指定为N-加氧酶。