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Conversion of the Sulfhydryl Oxidase Augmenter of Liver Regeneration into a Selenoprotein

Authors :
Sharon Rozovsky
Colin Thorpe
Stephanie Aron Schaefer
Ming Dong
Brian J. Bahnson
Source :
Biophysical Journal. 104(2)
Publication Year :
2013
Publisher :
Elsevier BV, 2013.

Abstract

Augmenter of liver regeneration (ALR) is a flavin-dependent sulfhydryl oxidase with roles in mitochondrial oxidative protein folding and cellular signaling. To study ALR's reaction mechanism we have prepared a form of the enzyme in which sulfur was replaced with selenium. The selenium-rich ALR is catalytically active, thermally stable, and its structure is almost identical to that of the native ALR. The presence of selenium in the active site leads to the formation of a charge-transfer complex during turnover, as detected by visible spectroscopy. To further demonstrate the role selenium plays in ALR's active site, we have utilized E. coli's selenium insertion machinery to introduce selenium in a site-specific manner. using this method we are able to convert ALR's redox active CxxC motif to a selenocysteine containing CxxU motif. Our results demonstrate that the selenocysteine proximal to the FAD cofactor is sufficient to cause a charge-transfer complex during turnover. In addition, 77Se NMR spectroscopy was used to probe locations typically occupied by sulfur - an insensitive nucleus that is not amendable for NMR studies of proteins. Biological 77Se NMR has so far been underutilized due to the challenges of isotopically enriching protein with 77Se. Here, we have developed a method to introduce 77Se by heterologous expression in E. coli. We report the NMR spectra of ALR bound to oxidized and reduced FAD. An unidentified resonance appears only in the presence of the reducing agent and disappears readily upon exposure to air and subsequent reoxidation of the flavin. Hence, 77Se NMR spectroscopy can be used to directly probe the chemical environment surrounding the sulfur/selenium sites as a function of their redox state.

Details

ISSN :
00063495
Volume :
104
Issue :
2
Database :
OpenAIRE
Journal :
Biophysical Journal
Accession number :
edsair.doi.dedup.....e497b1c7f26d76145605c34b255631e1
Full Text :
https://doi.org/10.1016/j.bpj.2012.11.2208