Substrate specificities and mechanism in the enzymic processing of vitamin A into 11-cis-retinol

Abstract

The biosynthesis of 11-cis-retinol in the retinal pigment epithelium requires two consecutive enzymatic reactions. The first involves the esterification of all-trans-retinol by lecithin retinol acyltransferase (LRAT). The second reaction involves the direct conversion of an all-trans-retinyl ester into 11-cis-retinol by an isomerase-like enzyme. This latter reaction couples the free energy of hydrolysis of an ester to the thermodynamically uphill trans to cis conversion, thus providing the energy to drive the latter process. In this paper both enzymes are studied with respect to their substrate specificities to provide information on mechanism. The isomerase is shown to be highly specific with respect to the ionylidene ring system and substitution at C15, whereas sterically bulkier substituents at C9 and C11 are permitted. C5 and C13 demethyl retinoids are isomerized, removing from consideration isomerization mechanisms involving C-H abstraction at the C5 or C13 methyl groups of the retinoid. On the other hand, C9 demethyl retinoids are not isomerized. A C-H abstraction mechanism is unlikely at the C9 methyl group as well, because no kinetic deuterium isotope effect is found with all-trans-19,19,19-trideuterioretinoids and isomerization of unlabeled retinoids occurs without the incorporation of deuterium when the isomerization is performed in D2O. LRAT proved to be broadly specific for retinols but was relatively inert with other hydrophobic alcohols including cholesterol. The enzyme is also highly specific for phosphatidylcholine analogues versus other potential membranous acyl donors such as phosphatidylethanolamine and phosphatidylserine.

RPE65, Visual Cycle Retinol Isomerase, Is Not Inherently 11-cis-specific

“…Two possible mechanisms have received most attention: one an S N 2Ј mechanism of nucleophile addition to C 11 (29) and the other a carbocation-mediated mechanism (35). Although Rando et al (29,30,42) propose an S N 2Ј mechanism as consistent with their data, they, explicitly, do not rule out a carbonium (i.e. carbocation) mechanism.…”

“…Based on three considerations, 1) that RPE65 belongs to a family of monooxygenases with a catalytic iron center capable of activating oxygen, 2) that, concomitant with inversion of stereochemistry, the oxygen bound to C 15 is exchanged in the retinol product, and 3) that mutating active site residues modulate the 11-cis/13-cis-retinol ratio, we propose that RPE65 isomerization involves a cationic delocalized intermediate (carbocation or radical cation), which could employ an isomerooxygenase rather than an isomerohydrolase mechanism (29,30,42). Because a radical cation intermediate has been postulated for the ACO reaction mechanism (50), we must also consider this avenue.…”

Metabolic Basis of Visual Cycle Inhibition by Retinoid and Nonretinoid Compounds in the Vertebrate Retina

“…6C). Unfortunately, LRAT is highly specific for retinoid derivatives, so its benefits are limited to these compounds (79). Hydrophobic alcohols like geraniol and tetradeca-nol are poor LRAT substrates, and nonretinoid amines like farnesylamine are even worse than their alcohol analogs.…”

Relative contribution of α-carotene to postprandial vitamin A concentrations in healthy humans after carrot consumption

“…LRAT is thought to be the primary enzyme responsible for catalyzing retinol esterification in the intestine (33) and is likely the primary enzyme responsible for esterifying a-retinol. Previous reports demonstrate that LRAT has higher affinity for all-trans retinol compared with 13-cis-or 3-dehydroretinol (42) or b-apo-carotenols of reduced chain lengths (43). Alteration of the retinoid substrate may also modify the fatty acid preferences of this enzyme for esterification.…”