Jawanda Navneet, Ebalunode Jerry, Gribenko Alexey, Briggs James, Lee J Ching, Tu Shiao-Chun
Department of Biology and Biochemistry, University of Houston, 4800 Calhoun, Houston, TX 77204-5001, USA.
Arch Biochem Biophys. 2008 Apr 1;472(1):51-7. doi: 10.1016/j.abb.2008.02.006. Epub 2008 Feb 12.
Our earlier studies have shown that the Vibrio harveyi flavin reductase FRP undergoes a monomer-dimer equilibrium, and luciferase forms a functional complex with the FRP monomer but not significantly with the dimer. This work is aimed at further investigating the nature and regulation of FRP subunit interactions by computation and site-directed mutagenesis approaches. In silico mutations of a number of residues were performed, and energetic analyses led us to target residue E99, which interacts directly with R113 and R225 from the second subunit of the FRP homodimer, for detailed investigation. E99 was found non-essential to the binding of either the FMN cofactor or the substrates. However, in comparison with the native enzyme, the E99K variant was shown to have an enhanced subunit dissociation as evident from a 44-fold higher K(d) for the monomer-dimer equilibrium. The critical role of E99 in the formation of the FRP dimer has thus been demonstrated.
我们早期的研究表明，哈维弧菌黄素还原酶FRP存在单体 - 二聚体平衡，并且荧光素酶与FRP单体形成功能复合物，但与二聚体的结合不明显。这项工作旨在通过计算和定点诱变方法进一步研究FRP亚基相互作用的性质和调控。对多个残基进行了计算机模拟突变，能量分析使我们将目标锁定在残基E99上，该残基与FRP同型二聚体第二个亚基的R113和R225直接相互作用，以便进行详细研究。发现E99对FMN辅因子或底物的结合并非必需。然而，与天然酶相比，E99K变体显示出亚基解离增强，从单体 - 二聚体平衡的K(d)高出44倍可以明显看出。因此，已证明E99在FRP二聚体形成中的关键作用。
