Mikulska-Ruminska Karolina, Licht Matthew, Ertem Mehmed Z, Shanklin John, Liu Qun, Bahar Ivet
Institute of Physics, Faculty of Physics, Astronomy and Informatics, Nicolaus Copernicus University in Torun, PL87100 Torun, Poland.
Department of Pharmacological Sciences, Renaissance School of Medicine, Stony Brook University, Stony Brook, New York 11794, USA.
bioRxiv. 2025 Jun 27:2025.06.23.661152. doi: 10.1101/2025.06.23.661152.
The transmembrane alkane monooxygenase AlkB and rubredoxin AlkG form an electron transfer complex that hydroxylates terminal alkanes to produce alcohols. The recent cryoEM study of AlkB-AlkG complex (FtAlkBG) revealed its architecture, including a dodecane (D12) substrate in the diiron active site. However, the molecular mechanism of action of FtAlkBG remains unknown. Here, we examined the FtAlkBG dynamics and interactions by multiscale computations, including molecular dynamics (MD) simulations, elastic network model (ENM), and QM/MM study of the oxidative mechanism at the catalytic site of AlkB. D12 remained stably bound to the catalytic site during MD runs, coordinated by hydrophobic residues I267, L263, L264, and I133, critical to substrate stabilization. MD simulations further showed that D12 could exit the catalytic site via a well-defined translocation pathway gated by I54, S49, and F46, through two intermediate states before nearly leaving the protein, and diffuse back to the active site. Several salt bridges and conserved AlkB-AlkG contacts were enhanced upon substrate binding. Substrate binding also mediated the association/dissociation events required for efficient electron transfer. It promoted tighter coupling between the diiron center in AlkB and the iron in AlkG. The allosteric effects relevant to enzymatic activity regulated by the substrate binding and channeling were further delineated by ENM analysis which confirmed a strong coupling spanning the site of entry from the membrane, the catalytic site and the AlkB-AlkG interface. Our study provides new insights into key sites that could be targeted for developing AlkB-variants with desirable alkane conversion functions.
跨膜烷单加氧酶AlkB和铁氧化还原蛋白AlkG形成一个电子转移复合物,可将末端烷烃羟基化生成醇类。最近对AlkB - AlkG复合物(FtAlkBG)的冷冻电镜研究揭示了其结构,包括在双铁活性位点中的十二烷(D12)底物。然而,FtAlkBG的分子作用机制仍然未知。在这里,我们通过多尺度计算研究了FtAlkBG的动力学和相互作用,包括分子动力学(MD)模拟、弹性网络模型(ENM)以及对AlkB催化位点氧化机制的量子力学/分子力学(QM/MM)研究。在MD运行过程中,D12通过疏水残基I267、L263、L264和I133配位,稳定地结合在催化位点,这些残基对底物稳定至关重要。MD模拟进一步表明,D12可以通过由I54、S49和F46控制的明确的易位途径离开催化位点,在几乎离开蛋白质之前经过两个中间状态,然后扩散回活性位点。底物结合后,几个盐桥和保守的AlkB - AlkG接触得到增强。底物结合还介导了有效电子转移所需的缔合/解离事件。它促进了AlkB中的双铁中心与AlkG中的铁之间更紧密的耦合。通过ENM分析进一步描绘了与底物结合和通道化调节的酶活性相关的变构效应,该分析证实了从膜进入位点、催化位点和AlkB - AlkG界面之间存在强耦合。我们的研究为开发具有理想烷烃转化功能的AlkB变体的关键靶点提供了新的见解。