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A Tailored Strategy to Crosslink the Aspartate Transcarbamoylase Domain of the Multienzymatic Protein CAD
CAD is a 1.5 MDa hexameric protein with four enzymatic domains responsible for initiating de novo biosynthesis of pyrimidines nucleotides: glutaminase, carbamoyl phosphate synthetase, aspartate transcarbamoylase (ATC), and dihydroorotase. Despite its central metabolic role and implication in cancer...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9863657/ https://www.ncbi.nlm.nih.gov/pubmed/36677714 http://dx.doi.org/10.3390/molecules28020660 |
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author | del Caño-Ochoa, Francisco Rubio-del-Campo, Antonio Ramón-Maiques, Santiago |
author_facet | del Caño-Ochoa, Francisco Rubio-del-Campo, Antonio Ramón-Maiques, Santiago |
author_sort | del Caño-Ochoa, Francisco |
collection | PubMed |
description | CAD is a 1.5 MDa hexameric protein with four enzymatic domains responsible for initiating de novo biosynthesis of pyrimidines nucleotides: glutaminase, carbamoyl phosphate synthetase, aspartate transcarbamoylase (ATC), and dihydroorotase. Despite its central metabolic role and implication in cancer and other diseases, our understanding of CAD is poor, and structural characterization has been frustrated by its large size and sensitivity to proteolytic cleavage. Recently, we succeeded in isolating intact CAD-like particles from the fungus Chaetomium thermophilum with high yield and purity, but their study by cryo-electron microscopy is hampered by the dissociation of the complex during sample grid preparation. Here we devised a specific crosslinking strategy to enhance the stability of this mega-enzyme. Based on the structure of the isolated C. thermophilum ATC domain, we inserted by site-directed mutagenesis two cysteines at specific locations that favored the formation of disulfide bridges and covalent oligomers. We further proved that this covalent linkage increases the stability of the ATC domain without damaging the structure or enzymatic activity. Thus, we propose that this cysteine crosslinking is a suitable strategy to strengthen the contacts between subunits in the CAD particle and facilitate its structural characterization. |
format | Online Article Text |
id | pubmed-9863657 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-98636572023-01-22 A Tailored Strategy to Crosslink the Aspartate Transcarbamoylase Domain of the Multienzymatic Protein CAD del Caño-Ochoa, Francisco Rubio-del-Campo, Antonio Ramón-Maiques, Santiago Molecules Article CAD is a 1.5 MDa hexameric protein with four enzymatic domains responsible for initiating de novo biosynthesis of pyrimidines nucleotides: glutaminase, carbamoyl phosphate synthetase, aspartate transcarbamoylase (ATC), and dihydroorotase. Despite its central metabolic role and implication in cancer and other diseases, our understanding of CAD is poor, and structural characterization has been frustrated by its large size and sensitivity to proteolytic cleavage. Recently, we succeeded in isolating intact CAD-like particles from the fungus Chaetomium thermophilum with high yield and purity, but their study by cryo-electron microscopy is hampered by the dissociation of the complex during sample grid preparation. Here we devised a specific crosslinking strategy to enhance the stability of this mega-enzyme. Based on the structure of the isolated C. thermophilum ATC domain, we inserted by site-directed mutagenesis two cysteines at specific locations that favored the formation of disulfide bridges and covalent oligomers. We further proved that this covalent linkage increases the stability of the ATC domain without damaging the structure or enzymatic activity. Thus, we propose that this cysteine crosslinking is a suitable strategy to strengthen the contacts between subunits in the CAD particle and facilitate its structural characterization. MDPI 2023-01-09 /pmc/articles/PMC9863657/ /pubmed/36677714 http://dx.doi.org/10.3390/molecules28020660 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article del Caño-Ochoa, Francisco Rubio-del-Campo, Antonio Ramón-Maiques, Santiago A Tailored Strategy to Crosslink the Aspartate Transcarbamoylase Domain of the Multienzymatic Protein CAD |
title | A Tailored Strategy to Crosslink the Aspartate Transcarbamoylase Domain of the Multienzymatic Protein CAD |
title_full | A Tailored Strategy to Crosslink the Aspartate Transcarbamoylase Domain of the Multienzymatic Protein CAD |
title_fullStr | A Tailored Strategy to Crosslink the Aspartate Transcarbamoylase Domain of the Multienzymatic Protein CAD |
title_full_unstemmed | A Tailored Strategy to Crosslink the Aspartate Transcarbamoylase Domain of the Multienzymatic Protein CAD |
title_short | A Tailored Strategy to Crosslink the Aspartate Transcarbamoylase Domain of the Multienzymatic Protein CAD |
title_sort | tailored strategy to crosslink the aspartate transcarbamoylase domain of the multienzymatic protein cad |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9863657/ https://www.ncbi.nlm.nih.gov/pubmed/36677714 http://dx.doi.org/10.3390/molecules28020660 |
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