Pulsed Electron Paramagnetic Resonance Insights into the Ligand Environment of Copper in Drosophila Lysyl Oxidase.
Pulsed Electron Paramagnetic Resonance Insights into the Ligand Environment of Copper in Drosophila Lysyl Oxidase.
复制标题
脉冲电子顺磁共振洞察果蝇赖氨酰氧化酶中铜的配体环境。
DOI：
10.1021/acs.biochem.7b00308
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发表时间：
2017
期刊：
影响因子：
2.9
通讯作者：
Oldfield,Eric
中科院分区：
文献类型：
--
作者：
Rao,Guodong;Bansal,Sandhya;Law,WenXuan;O'Dowd,Bing;Dikanov,SergeiA;Oldfield,Eric
Lysyl oxidase (LOX) is a copper amine oxidase that cross-links collagens and elastin in connective tissue and plays an important role in fibrosis, cancer development, and formation of the “metastatic niche”. Despite its important biological functions, the structure of human LOX remains unknown (unlike that of an unrelated LOX, fromPichia pastoris). Here, we expressed active LOX fromDrosophila melanogaster, DmLOXL1, a close homologue of human LOX, and characterized it by MS, UV–vis, activity, and inhibition assays. We then used bioinformatics, electron paramagnetic resonance, electron spin-echo envelope modulation, and hyperfine sublevel-correlation (HYSCORE) spectroscopies to probe Cu-ligand bonding finding direct evidence for pH-dependent Cu–His interactions. At pH = 9.3, the spectroscopic data indicated primarily a single His bound to Cu, but at pH = 7.5, there was evidence for a ∼ 1:1 mixture of species containing 1 and 3 His ligands. We then used HYSCORE to probe possible interactions between the LOX inhibitor BAPN (β-aminopropionitrile; 1-[13C15N]cyano-2-aminoethane) and the copper centerfinding none. Overall, the results are of interest since they provide new spectroscopic information about the nature of the catalytic site in LOX, an important anticancer drug target.
