Structure and assembly of the diiron cofactor in the heme-oxygenase-like domain of the N-nitrosourea-producing enzyme SznF.
Structure and assembly of the diiron cofactor in the heme-oxygenase-like domain of the N-nitrosourea-producing enzyme SznF.
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DOI：
10.1073/pnas.2015931118
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发表时间：
2021-01-26
影响因子：
11.1
通讯作者：
Boal AK
中科院分区：
文献类型：
--
作者：
McBride MJ;Pope SR;Hu K;Okafor CD;Balskus EP;Bollinger JM Jr;Boal AK
酶SznF组装药物链脲霉素的N-亚硝基脲药效团。其N-加氧酶结构域类似于血红素加氧酶（HO），属于一个新兴的HO样二铁酶（HDO）超家族，具有不稳定的金属辅因子，结构特征难以确定。深入了解辅因子动力学从我们以前的调查SznF的N-氧化反应提出了一种方法，已经产生了一个结构的功能分配的HDO与其二铁辅因子完整。伴随辅因子（解）组装的构象变化解释了其不稳定性，并且观察到仅在HDO序列的一个子集中保守的未预料到的谷氨酸配体为酶功能的顶级分配提供了潜在的基础。因此，我们的研究结果提供了一个路线图的结构和功能表征的新型HDO。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在氧化重排三重修饰的胍为N-甲基-N-亚硝基脲药效团之前羟基化Nω-甲基-L-精氨酸的Nδ和Nω′。以前发表的结构可视化的monoiron辅因子在酶的C-末端cupin域，这促进了最终的重排，但表现出的障碍和最小的金属占用率在网站的建议二铁辅因子在N-羟基化血红素加氧酶样（HO样）的中央域。我们利用了我们最近的观察结果，即在载脂蛋白自组装其二铁（II/II）辅因子后，N-氧化的µ-过氧二铁（III/III）中间体可以在HO样结构域中形成，以解析SznF与其两个铁辅因子结合的结构。这些结构的生物化学验证的新兴血红素加氧酶样二铁氧化酶和加氧酶（HDO）超家族与完整的二铁辅因子的成员揭示了大规模的构象变化所需的组装O2反应性Fe 2（II/II）复杂和结构基础的辅因子不稳定性的性状共享的其他验证HDO。在辅因子组装过程中，含有配体的核心螺旋动态地折叠。二铁辅因子还协调一个意想不到的Glu配体贡献的辅助螺旋参与底物结合的对接和分子动力学模拟。另外的羧酸根配体在另一个N-氧化HDO中是保守的，但在裂解碳-氢键和碳-碳键以安装烯烃的两个HDO中不是。在生物信息学鉴定为新兴HDO超家族成员的109，600个序列中，约25%保留了这一额外的羧酸残基，因此暂时被指定为N-加氧酶。
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.
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