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...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-01-01
|
| Series: | Molecules |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1420-3049/28/2/660 |
| _version_ | 1797438319433351168 |
|---|---|
| author | Francisco del Caño-Ochoa Antonio Rubio-del-Campo Santiago Ramón-Maiques |
| author_facet | Francisco del Caño-Ochoa Antonio Rubio-del-Campo Santiago Ramón-Maiques |
| author_sort | Francisco del Caño-Ochoa |
| collection | DOAJ |
| 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 <i>Chaetomium thermophilum</i> 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 <i>C. thermophilum</i> 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. |
| first_indexed | 2024-03-09T11:35:56Z |
| format | Article |
| id | doaj.art-6dfa8a389fd54530933797eccd34d691 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-09T11:35:56Z |
| publishDate | 2023-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-6dfa8a389fd54530933797eccd34d6912023-11-30T23:42:59ZengMDPI AGMolecules1420-30492023-01-0128266010.3390/molecules28020660A Tailored Strategy to Crosslink the Aspartate Transcarbamoylase Domain of the Multienzymatic Protein CADFrancisco del Caño-Ochoa0Antonio Rubio-del-Campo1Santiago Ramón-Maiques2Instituto de Biomedicina de Valencia (IBV), CSIC, Jaime Roig 11, 46010 Valencia, SpainInstituto de Biomedicina de Valencia (IBV), CSIC, Jaime Roig 11, 46010 Valencia, SpainInstituto de Biomedicina de Valencia (IBV), CSIC, Jaime Roig 11, 46010 Valencia, SpainCAD 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 <i>Chaetomium thermophilum</i> 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 <i>C. thermophilum</i> 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.https://www.mdpi.com/1420-3049/28/2/660nucleotide metabolismde novo pyrimidine biosynthesiscarbamoyl-phosphate synthetasedihydroorotasecysteinedisulfide bridge |
| spellingShingle | Francisco del Caño-Ochoa Antonio Rubio-del-Campo Santiago Ramón-Maiques A Tailored Strategy to Crosslink the Aspartate Transcarbamoylase Domain of the Multienzymatic Protein CAD Molecules nucleotide metabolism de novo pyrimidine biosynthesis carbamoyl-phosphate synthetase dihydroorotase cysteine disulfide bridge |
| 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 | nucleotide metabolism de novo pyrimidine biosynthesis carbamoyl-phosphate synthetase dihydroorotase cysteine disulfide bridge |
| url | https://www.mdpi.com/1420-3049/28/2/660 |
| work_keys_str_mv | AT franciscodelcanoochoa atailoredstrategytocrosslinktheaspartatetranscarbamoylasedomainofthemultienzymaticproteincad AT antoniorubiodelcampo atailoredstrategytocrosslinktheaspartatetranscarbamoylasedomainofthemultienzymaticproteincad AT santiagoramonmaiques atailoredstrategytocrosslinktheaspartatetranscarbamoylasedomainofthemultienzymaticproteincad AT franciscodelcanoochoa tailoredstrategytocrosslinktheaspartatetranscarbamoylasedomainofthemultienzymaticproteincad AT antoniorubiodelcampo tailoredstrategytocrosslinktheaspartatetranscarbamoylasedomainofthemultienzymaticproteincad AT santiagoramonmaiques tailoredstrategytocrosslinktheaspartatetranscarbamoylasedomainofthemultienzymaticproteincad |