3 years ago

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
Eric Oldfield, Bing O’Dowd, Sandhya Bansal, Wen Xuan Law, Guodong Rao, Sergei A. Dikanov
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, from Pichia pastoris). Here, we expressed active LOX from Drosophila 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 center—finding 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.

Publisher URL: http://dx.doi.org/10.1021/acs.biochem.7b00308

DOI: 10.1021/acs.biochem.7b00308

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