A proposed architecture for lecithin cholesterol acyl transferase (LCAT): identification of the catalytic triad and molecular modeling.
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 c...
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1998
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ftpubmed:oai:pubmedcentral.nih.gov:2143955 2023-05-15T13:38:05+02:00 A proposed architecture for lecithin cholesterol acyl transferase (LCAT): identification of the catalytic triad and molecular modeling. Peelman, F. Vinaimont, N. Verhee, A. Vanloo, B. Verschelde, J. L. Labeur, C. Seguret-Mace, S. Duverger, N. Hutchinson, G. Vandekerckhove, J. Tavernier, J. Rosseneu, M. 1998-03 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2143955 http://www.ncbi.nlm.nih.gov/pubmed/9541390 en eng Cold Spring Harbor Laboratory Press http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2143955 http://www.ncbi.nlm.nih.gov/pubmed/9541390 Research Article Text 1998 ftpubmed 2013-09-01T10:30:02Z 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 alpha/beta hydrolase fold family, and that the central domain of LCAT consists of seven conserved parallel beta-strands connected by four alpha-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 monomeric 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. Text Antarc* Antarctica PubMed Central (PMC) |
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Research Article Peelman, F. Vinaimont, N. Verhee, A. Vanloo, B. Verschelde, J. L. Labeur, C. Seguret-Mace, S. Duverger, N. Hutchinson, G. Vandekerckhove, J. Tavernier, J. Rosseneu, M. A proposed architecture for lecithin cholesterol acyl transferase (LCAT): identification of the catalytic triad and molecular modeling. |
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Research Article |
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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 alpha/beta hydrolase fold family, and that the central domain of LCAT consists of seven conserved parallel beta-strands connected by four alpha-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 monomeric 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. |
format |
Text |
author |
Peelman, F. Vinaimont, N. Verhee, A. Vanloo, B. Verschelde, J. L. Labeur, C. Seguret-Mace, S. Duverger, N. Hutchinson, G. Vandekerckhove, J. Tavernier, J. Rosseneu, M. |
author_facet |
Peelman, F. Vinaimont, N. Verhee, A. Vanloo, B. Verschelde, J. L. Labeur, C. Seguret-Mace, S. Duverger, N. Hutchinson, G. Vandekerckhove, J. Tavernier, J. Rosseneu, M. |
author_sort |
Peelman, F. |
title |
A proposed architecture for lecithin cholesterol acyl transferase (LCAT): identification of the catalytic triad and molecular modeling. |
title_short |
A proposed architecture for lecithin cholesterol acyl transferase (LCAT): identification of the catalytic triad and molecular modeling. |
title_full |
A proposed architecture for lecithin cholesterol acyl transferase (LCAT): identification of the catalytic triad and molecular modeling. |
title_fullStr |
A proposed architecture for lecithin cholesterol acyl transferase (LCAT): identification of the catalytic triad and molecular modeling. |
title_full_unstemmed |
A proposed architecture for lecithin cholesterol acyl transferase (LCAT): identification of the catalytic triad and molecular modeling. |
title_sort |
proposed architecture for lecithin cholesterol acyl transferase (lcat): identification of the catalytic triad and molecular modeling. |
publisher |
Cold Spring Harbor Laboratory Press |
publishDate |
1998 |
url |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2143955 http://www.ncbi.nlm.nih.gov/pubmed/9541390 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_relation |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2143955 http://www.ncbi.nlm.nih.gov/pubmed/9541390 |
_version_ |
1766101434094321664 |