Abstract
Purpose :
Retinol dehydrogenase 11 (RDH11) is a member of the short-chain dehyrogenase/reductase family. It is expressed in the retinal pigment epithelium and likely involved in the visual cycle. Indeed, the final process of the cycle involving the reduction of 11-cis-retinol to 11-cis-retinal is conducted by several retinol dehydrogenases including RDH11. Little attention was paid on the biophysical properties of RDH11 and its membrane binding. The present study was thus aimed to characterize the structure and membrane binding of RDH11.
Methods :
RDH11 and truncated RDH11 (tRDH11, without its N-terminal alpha helical segment) have been expressed using E. coli ArcticExpress (DE3) and purified by means of its polyhistidine tag located at the C-terminus. Their catalytic activity has been assayed using all-trans retinal and its NADPH cofactor. Their membrane binding has been assessed by measuring their maximum insertion pressure (MIP) in the presence of various phospholipids. Their secondary structure upon membrane binding has been determined by infrared spectroscopy.
Results :
RDH11 was found to be highly active, readily converting all-trans retinol into all-trans retinal. The MIP measurements have shown that the N-terminal alpha helical segment of RDH11 greatly contributes to accelerate its kinetics of membrane binding. Moreover, RDH11 showed a preference for binding unsaturated phosphoethanolamine. Infrared spectroscopy revealed that RDH11 had a high content of alpha helices and that its orientation was modified upon membrane binding as well as when binding to its all-trans retinal substrate. Infrared measurements also demonstrated that RDH11 progressively binds inorganic phosphate.
Conclusions :
The N-terminal segment of RDH11 highly favors its membrane binding to unsaturated phospholipids. Membrane binding strongly influences the orientation of RDH11. The observation of a strong phosphate binding by RDH11 raises issues on its physiological role.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.