Abstract
Synthesis of fatty acid retinyl esters determines systemic vitamin A levels and provides substrate for production of visual chromophore (11-cis-retinal) in vertebrates. Lecithin:retinol acyltransferase (LRAT), the main enzyme responsible for retinyl ester formation, catalyzes the transfer of an acyl group from the sn-1 position of phosphatidylcholine to retinol. To delineate the catalytic mechanism of this reaction, we expressed and purified a fully active, soluble form of this enzyme and used it to examine the possible formation of a transient acyl-enzyme intermediate. Detailed mass spectrometry analyses revealed that LRAT undergoes spontaneous, covalent modification upon incubation with a variety of phosphatidylcholine substrates. The addition of an acyl chain occurs at the Cys(161) residue, indicating formation of a thioester intermediate. This observation provides the first direct experimental evidence of thioester intermediate formation that constitutes the initial step in the proposed LRAT catalytic reaction. Additionally, we examined the effect of increasing fatty acyl side chain length in phosphatidylcholine on substrate accessibility in this reaction, which provided insights into the function of the single membrane-spanning domain of LRAT. These observations are critical to understanding the catalytic mechanism of LRAT protein family members as well as other lecithin:acyltransferases wherein Cys residues are required for catalysis.
Highlights
Fatty acyl group attached by a thioester bond to coenzyme A as a donor [12]
A number of biochemical studies involving site-directed mutagenesis and protein labeling suggest that the Lecithin:retinol acyltransferase (LRAT) reaction mechanism may involve a Cys side chain that serves as an active site nucleophile [17]
Effect of Lipid Phase on tLRAT Enzymatic Activity—By studying the relationship between LRAT activity and the length of the sn-1 fatty acyl moiety of its phosphatidylcholine substrates, we found that the soluble form of tLRAT catalyzed the most efficient retinyl ester formation in the presence of PCs with short chain fatty acids, whereas increasing the acyl chain length resulted in gradual loss of enzymatic activity accompanied by inefficient formation of the thioester intermediate (Fig. 4, A and B)
Summary
Protein Expression and Purification—Mouse LRAT cDNA encoding amino acids 30GGG...TVK186 was subcloned into a pGEX_2T vector (GE Healthcare) using BamHI and EcoRI restriction sites to generate the glutathione S-transferase fusion protein GST-tLRAT. To ensure separation putative enzyme modification, electrospray ionization-MS of retinyl esters containing acyl chains of different length, a step analyses were carried out first with intact protein where the gradient of ethyl acetate in hexane was used Self- spectrum recorded under acidic conditions revealed a broad acylation of tLRAT was detected after incubation of the protein distribution of charged states, which correspond to a protein sample with various PC substrates in 5 mM Tris/HCl, pH 8.0, with a calculated mass of 44,672 Da, identical to the theoretand 1 mM DTT. Mass Spectrometry Analysis—All of the experiments were Incubation of GST-tLRAT with PC led to rapid modification performed with a LXQ linear ion trap mass spectrometer of the protein indicated by the appearance of an additional equipped with an electrospray ionization source
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