期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 109, 期 41, 页码 E2747-E2756出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1212025109
关键词
metalloprotein; monotopic membrane protein; extended X-ray absorption fine structure; isomerohydrolase
资金
- National Institutes of Health (NIH) [EY009339, EY020551, P30-EY011373]
- NIH National Institute of Biomedical Imaging and Bioengineering Grant [P30-EB-009998]
- [5P41RR015301-10]
- [8P41GM103403-10]
RPE65 is a key metalloenzyme responsible for maintaining visual function in vertebrates. Despite extensive research on this membrane-bound retinoid isomerase, fundamental questions regarding its enzymology remain unanswered. Here, we report the crystal structure of RPE65 in a membrane-like environment. These crystals, obtained from enzymatically active, nondelipidated protein, displayed an unusual packing arrangement wherein RPE65 is embedded in a lipid-detergent sheet. Structural differences between delipidated and nondelipidated RPE65 uncovered key residues involved in substrate uptake and processing. Complementary iron K-edge X-ray absorption spectroscopy data established that RPE65 as isolated contained a divalent iron center and demonstrated the presence of a tightly bound ligand consistent with a coordinated carboxylate group. These results support the hypothesis that the Lewis acidity of iron could be used to promote ester dissociation and generation of a carbocation intermediate required for retinoid isomerization.
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