Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 3335 Innovation Blvd, Richland, Washington 99354, United States.
Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195, United States.
J Am Chem Soc. 2024 Aug 21;146(33):22950-22958. doi: 10.1021/jacs.3c06537. Epub 2024 Jul 26.
The global manganese cycle relies on microbes to oxidize soluble Mn(II) to insoluble Mn(IV) oxides. Some microbes require peroxide or superoxide as oxidants, but others can use O directly, via multicopper oxidase (MCO) enzymes. One of these, MnxG from strain PL-12, was isolated in tight association with small accessory proteins, MnxE and MnxF. The protein complex, called Mnx, has eluded crystallization efforts, but we now report the 3D structure of a point mutant using cryo-EM single particle analysis, cross-linking mass spectrometry, and AlphaFold Multimer prediction. The β-sheet-rich complex features MnxG enzyme, capped by a heterohexameric ring of alternating MnxE and MnxF subunits, and a tunnel that runs through MnxG and its MnxEF cap. The tunnel dimensions and charges can accommodate the mechanistically inferred binuclear manganese intermediates. Comparison with the Fe(II)-oxidizing MCO, ceruloplasmin, identifies likely coordinating groups for the Mn(II) substrate, at the entrance to the tunnel. Thus, the 3D structure provides a rationale for the established manganese oxidase mechanism, and a platform for further experiments to elucidate mechanistic details of manganese biomineralization.
全球锰循环依赖于微生物将可溶性 Mn(II)氧化为不溶性 Mn(IV)氧化物。一些微生物需要过氧化物或超氧化物作为氧化剂，但其他微生物可以通过多铜氧化酶 (MCO) 酶直接利用 O 。其中一种是来自 菌株 PL-12 的 MnxG，它与小辅助蛋白 MnxE 和 MnxF 紧密结合被分离出来。该蛋白复合物称为 Mnx，尽管我们已经努力进行了结晶，但目前仍通过 cryo-EM 单颗粒分析、交联质谱和 AlphaFold 多聚体预测报告了一个点突变体的 3D 结构。该富含β-片层的复合物具有 MnxG 酶，由交替的 MnxE 和 MnxF 亚基组成的异六聚体环封顶，并且有一个贯穿 MnxG 和其 MnxEF 帽的隧道。隧道的尺寸和电荷可以容纳推断出的双核锰中间体。与 Fe(II)-氧化 MCO（铜蓝蛋白）的比较，确定了隧道入口处 Mn(II)底物的可能配位基团。因此，该 3D 结构为已建立的锰氧化酶机制提供了依据，并为进一步阐明锰生物矿化的机制细节提供了平台。
