1. Enzymatic activity of Lecithin:retinol acyltransferase: A thermostable and highly active enzyme with a likely mode of interfacial activation
- Author
-
Christian Salesse, Habib Horchani, Sylvain Bussières, Jean-Sebastien Laliberte-Gemme, Rock Breton, Line Cantin, and Mustapha Lhor
- Subjects
Circular dichroism ,Hot Temperature ,food.ingredient ,Spectrophotometry, Infrared ,Biophysics ,Biochemistry ,Lecithin ,Article ,Protein Structure, Secondary ,Substrate Specificity ,Analytical Chemistry ,food ,Lecithins ,Escherichia coli ,Humans ,Vitamin A ,Molecular Biology ,Protein secondary structure ,Micelles ,Thermostability ,chemistry.chemical_classification ,biology ,Protein Stability ,Chemistry ,Circular Dichroism ,Phospholipid Ethers ,Recombinant Proteins ,Kinetics ,Enzyme ,Rhodopsin ,Acyltransferase ,biology.protein ,Lecithin retinol acyltransferase ,Acyltransferases - Abstract
Lecithin retinol acyltransferase (LRAT) plays a major role in the vertebrate visual cycle. Indeed, it is responsible for the esterification of all-trans retinol into all-trans retinyl esters, which can then be stored in microsomes or further metabolized to produce the chromophore of rhodopsin. In the present study, a detailed characterization of the enzymatic properties of truncated LRAT (tLRAT) has been achieved using in vitro assay conditions. A much larger tLRAT activity has been obtained compared to previous reports and to an enzyme with a similar activity. In addition, tLRAT is able to hydrolyze phospholipids bearing different chain lengths with a preference for micellar aggregated substrates. It therefore presents an interfacial activation property, which is typical of classical phospholipases. Furthermore, given that stability is a very important quality of an enzyme, the influence of different parameters on the activity and stability of tLRAT has thus been studied in detail. For example, storage buffer has a strong effect on tLRAT activity and high enzyme stability has been observed at room temperature. The thermostability of tLRAT has also been investigated using circular dichroism and infrared spectroscopy. A decrease in the activity of tLRAT was observed beyond 70°C, accompanied by a modification of its secondary structure, i.e. a decrease of its α-helical content and the appearance of unordered structures and aggregated β-sheets. Nevertheless, residual activity could still be observed after heating tLRAT up to 100 °C. The results of this study highly improved our understanding of this enzyme.
- Published
- 2014