Enzymatic Activity and Monolayer Binding of a Truncated Form of Lecithin Retinol Acyltransferase

Abstract

Lecithin retinol acyltransferase (LRAT) is a 230 amino acids membrane-associated protein. It has two enzymatic activities: first, it catalyzes hydrolysis of the sn-1 acyl chain of phospholipids and then transfers this acyl group to all-trans retinol to generate all-trans retinyl esters. This reaction is essential in the vertebrate visual cycle. The present study was performed to study the enzymatic activity of a truncated form of LRAT (tLRAT), where transmembrane domains have been removed. tLRAT extends from residues 31 to 196. It has been previously determined that the deleted domains of tLRAT do not contain residues known to be required for catalysis. tLRAT has been produced in E. coli and purified using affinity chromatography. Its enzymatic activity was studied using the short-chain diheptanoyl phosphatidylcholine (DHPC), which behaves like a mild detergent. The low critical micellar concentrations of DHPC allows to solubilize tLRAT and retinol. The maximal enzymatic activity of tLRAT is approximately 900 mol of ester/min • mol of protein. This value is more than 20 000 times higher than the largest enzyme activity reported in the literature. This huge difference can be explained by the use of a solution where DHPC serves both as a substrate as well as to solubililize the second substrate which highly favors the hydrolytic activity of tLRAT. Moreover, the injection tLRAT into the subphase of a phospholipid monolayer at different initial surface pressures allowed to determine the maximum insertion pressure (MIP) of tLRAT. A similar MIP of 38 mN/m has been obtained for dioleoyl phosphatidylcholine, ethanolamine and serine which is much higher than the lateral pressure of membranes. It can thus be postulated that tLRAT strongly binds membranes in the absence of its putative N- and C-terminal transmembrane domains.