A proposed architecture for lecithin cholesterol acyl transferase (LCAT): Identification of the catalytic triad and molecular modeling

• Published in: Protein science Vol. 7; no. 3; pp. 587 - 599
• Main Authors: Peelman, F., Vinaimont, N., Vanloo, B., Labeur, C., Rosseneu, M., Verhee, A., Verschelde, J‐L., Vanderckove, J., Tavernier, J., Seguret‐Mace, S., Duverger, N., Hutchinson, G.
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01-03-1998
• Subjects: Amino Acid Sequence; Binding Sites; Candida - enzymology; Catalysis; catalytic residues; Computer Simulation; Fungal Proteins; Histidine; Humans; lecithin cholesterol acyl transferase; lipase; Lipase - ultrastructure; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphatidylcholine-Sterol O-Acyltransferase - metabolism; Phosphatidylcholine-Sterol O-Acyltransferase - ultrastructure; protein fold; Protein Structure, Secondary; Sequence Alignment; Structure-Activity Relationship; threading
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.5560070307

The enzyme cholesterol lecithin acyl transferase (LCAT) shares the Ser/Asp‐Glu/His triad with lipases, esterases and proteases, but the low level of sequence homology between LCAT and these enzymes did not allow for the LCAT fold to be identified yet. We, therefore, relied upon structural homology calculations using threading methods based on alignment of the sequence against a library of solved three‐dimensional protein structures, for prediction of the LCAT fold. We propose that LCAT, like lipases, belongs to the α/β hydrolase fold family, and that the central domain of LCAT consists of seven conserved parallel beta‐strands connected by four α‐helices and separated by loops. We used the conserved features of this protein fold for the prediction of functional domains in LCAT, and carried out site‐directed mutagenesis for the localization of the active site residues. The wild‐type enzyme and mutants were expressed in Cos‐1 cells. LCAT mass was measured by ELISA, and enzymatic activity was measured on recombinant HDL, on LDL and on a monomelic substrate. We identified D345 and H377 as the catalytic residues of LCAT, together with F103 and L182 as the oxyanion hole residues. In analogy with lipases, we further propose that a potential “lid” domain at residues 50‐74 of LCAT might be involved in the enzyme‐substrate interaction. Molecular modeling of human LCAT was carried out using human pancreatic and Candida antarctica lipases as templates. The three‐dimensional model proposed here is compatible with the position of natural mutants for either LCAT deficiency or Fish‐eye disease. It enables moreover prediction of the LCAT domains involved in the interaction with the phospholipid and cholesterol substrates.