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. 2010 Sep 17;285(38):29217-22.
doi: 10.1074/jbc.M110.152314. Epub 2010 Jul 12.

An acyl-covalent enzyme intermediate of lecithin:retinol acyltransferase

Marcin Golczak  1 , Krzysztof Palczewski
Affiliations

An acyl-covalent enzyme intermediate of lecithin:retinol acyltransferase

Marcin Golczak et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
Purification and acyltransferase activity of GST-tLRAT. A, graphic representation of GST-tLRAT fusion protein. Molecular masses shown in the picture represent protein fragments liberated upon digestion with thrombin. B, protein purification on a glutathione affinity column. The black arrowhead indicates the position of expressed protein in a 12% polyacrylamide gel. C and D, HPLC separation of retinoids extracted from the acyltransferase activity assay performed with crude bacterial extract (left) and purified fusion protein (right) in the presence of 7:0 PC and all-trans-retinol as substrates. Formation of retinyl heptanoate indicates robust enzymatic activity of soluble GST-tLRAT fusion protein.
FIGURE 2.
FIGURE 2.
Electrospray ionization mass spectra of intact GST-tLRAT. A, purified protein spectrum shows a broad distribution of charged states (27+ to 69+). Deconvolution of GST-tLRAT spectrum reveals a protein mass of 44,672 Da (inset), which is identical to the theoretical mass of GST-tLRAT. B, incubation of the protein with 1 mm of 7:0 PC led to a 112-Da shift in experimentally obtained protein mass (inset). C, zoomed spectrum displays a group of selected multiply charged protein ions for the unmodified and acylated protein. Upon incubation with 0.3 mm of 7:0 PC, the protein became covalently modified as indicated by an additional peak at the higher m/z values marked by asterisks. D, deconvoluted mass spectrum of purified GST-tLRAT (black trace) reveals an additional peak at 44,542 Da corresponding to the N-terminal Met cleavage product (Δ = 130 ± 2 Da). The red trace represents protein mass upon incubation with the lipid substrate. The 112-Da shift in mass indicates covalent protein modification with a C7 acyl chain.
FIGURE 3.
FIGURE 3.
Determination of the acylation site by tandem MS. The spectra were recorded for the 2+ precursor ion of a peptide containing Cys161. The protein modification upon incubation with PC caused a shift in masses corresponding to a series of y and b ions shown in blue and red for 6:0 PC and 8:0 PC, respectively.
FIGURE 4.
FIGURE 4.
Correlation between enzyme acylation, its enzymatic activity and critical micelle concentration values of tested lipid substrates. A and B, formation of acyl-intermediates in GST-tLRAT (marked with asterisk in A) correlates with transfer of the acyl group to retinol observed in enzymatic assays (B). An increase in fatty acyl chain length markedly reduced the catalytic activity of soluble tLRAT. This decrease in enzymatic activity correlated with the declining concentration of monomeric lipid substrate as determined by critical micelle concentration values. The critical micelle concentration values for each of the tested PCs are cited from the Avanti Lipids Polar database available on the company website. The lipid concentration in each experimental set was fixed at 1 mm. C, enzymatic activity of the full-length, membrane-bound LRAT in the presence of lipid substrates with fatty acyl chains of different lengths. UV-treated bovine retinal pigment epithelium microsomes were incubated with 200 μm of tested PCs and 10 μm all-trans-retinol for 20 min at 37 °C, and retinoid composition was subsequently examined by HPLC. Chromatogram signals were normalized with respect to the intensity of a retinyl palmitate peak.
FIGURE 5.
FIGURE 5.
Schematic representation of LRAT enzymatic activity mechanism.
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