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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Online Access: | http://hdl.handle.net/2078.1/45430 https://doi.org/10.1002/pro.5560070307 |
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ftunivlouvain:oai:dial.uclouvain.be:boreal:45430 2024-05-19T07:30:40+00:00 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, JL Labeur, C Seguret-Mace, S Duverger, N Hutchinson, G Vandekerckhove, J. Tavernier, J. Rosseneu, M UCL 1998 http://hdl.handle.net/2078.1/45430 https://doi.org/10.1002/pro.5560070307 eng eng Cambridge Univ Press boreal:45430 http://hdl.handle.net/2078.1/45430 doi:10.1002/pro.5560070307 urn:ISSN:0961-8368 urn:EISSN:1469-896X info:eu-repo/semantics/restrictedAccess Protein Science, Vol. 7, no. 3, p. 587-599 (1998) catalytic residues lecithin cholesterol acyl transferase lipase protein fold threading info:eu-repo/semantics/article 1998 ftunivlouvain https://doi.org/10.1002/pro.5560070307 2024-04-24T01:48:21Z 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. Article in Journal/Newspaper Antarc* Antarctica DIAL@UCLouvain (Université catholique de Louvain) Protein Science 7 3 587 599 |
institution |
Open Polar |
collection |
DIAL@UCLouvain (Université catholique de Louvain) |
op_collection_id |
ftunivlouvain |
language |
English |
topic |
catalytic residues lecithin cholesterol acyl transferase lipase protein fold threading |
spellingShingle |
catalytic residues lecithin cholesterol acyl transferase lipase protein fold threading Peelman, F Vinaimont, N Verhee, A Vanloo, B Verschelde, JL 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 |
topic_facet |
catalytic residues lecithin cholesterol acyl transferase lipase protein fold threading |
description |
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. |
author2 |
UCL |
format |
Article in Journal/Newspaper |
author |
Peelman, F Vinaimont, N Verhee, A Vanloo, B Verschelde, JL 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, JL 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 |
Cambridge Univ Press |
publishDate |
1998 |
url |
http://hdl.handle.net/2078.1/45430 https://doi.org/10.1002/pro.5560070307 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_source |
Protein Science, Vol. 7, no. 3, p. 587-599 (1998) |
op_relation |
boreal:45430 http://hdl.handle.net/2078.1/45430 doi:10.1002/pro.5560070307 urn:ISSN:0961-8368 urn:EISSN:1469-896X |
op_rights |
info:eu-repo/semantics/restrictedAccess |
op_doi |
https://doi.org/10.1002/pro.5560070307 |
container_title |
Protein Science |
container_volume |
7 |
container_issue |
3 |
container_start_page |
587 |
op_container_end_page |
599 |
_version_ |
1799488416121880576 |