Activity And Membrane Binding Of Retinol Dehydrogenase-11 And Its Deletants

Retinol dehydrogenases (RDHs) are enzymes that catalyze the interconversion between retinol and retinal. Not much is known concerning these RDHs. Indeed, the exact physiological role of many isoforms of RDH and their membrane binding remain unknown. In this work, we have overexpressed, purified and characterized the membrane binding of one isoform, RDH-11. The cDNA of RDH-11 and of the truncated RDH-11 (N-terminal deletion, N-del RDH-11) have been cloned in the pET28a plasmid. Those proteins have been overexpressed in E. coli, purified by affinity chromatography, and then concentrated by ultrafiltration. Their activity has been measured in the presence of its substrate, all-trans retinal, and the reaction was initiated by the addition of its cofactor, NADPH. The reaction products have been analyzed by HPLC. The data showed a very high activity of RDH-11. Indeed, 1 μg of protein was enough to convert nearly 100% of all-trans retinal to all-trans retinol. Membrane binding measurements have been carried out by the injecting RDH-11, N-del RDH-11 and the synthetic N-terminal peptide (NTP) into the subphase of a phospholipid monolayer at the air-water interface. Their kinetics of monolayer binding, monitored by surface pressure measurements, increases as follows : NTP > RDH-11 > N-del RDH-11. Moreover, measurements by polarization-modulated infrared reflection absorption spectroscopy have allowed to confirm the alpha helical structure of the NTP and to determine its orientation as well as to compare the structure and orientation of RDH-11 and N-del RDH-11. For example, compared to the pure protein, N-del RDH-11 undergoes a conformational change upon monolayer binding.