Characterization and assembly of the Pseudomonas aeruginosa aspartate transcarbamoylase-pseudo dihydroorotase complex
| Autor: | Asmita Vaishnav, Brian F.P. Edwards, Chandni Patel, David R. Evans |
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| Rok vydání: | 2020 |
| Předmět: |
Models
Molecular Amino Acid Motifs Cooperativity Regulatory site Pathology and Laboratory Medicine Physical Chemistry Biochemistry 01 natural sciences Protein Structure Secondary Protein structure Catalytic Domain Medicine and Health Sciences Cross-Linking Aspartate Carbamoyltransferase Nucleotide Enzyme Chemistry Materials Dihydroorotase Gel Electrophoresis chemistry.chemical_classification 0303 health sciences Multidisciplinary biology Circular Dichroism Chromatographic Techniques Pseudomonas Aeruginosa Bacterial Pathogens Chemistry Molecular Mass Aspartate carbamoyltransferase Medical Microbiology Physical Sciences Thermodynamics Medicine Pathogens Research Article Protein Binding Stereochemistry Science Materials Science Size-Exclusion Chromatography Research and Analysis Methods Microbiology Phosphates Enzyme Regulation Electrophoretic Techniques 03 medical and health sciences Allosteric Regulation Bacterial Proteins Pseudomonas Dimers Microbial Pathogens 030304 developmental biology Bacteria Chemical Bonding 010405 organic chemistry Organisms Chemical Compounds Biology and Life Sciences Proteins Active site Polymer Chemistry 0104 chemical sciences Dodecameric protein Chemical Properties chemistry Oligomers Enzymology Biocatalysis biology.protein Protein Multimerization |
| Zdroj: | PLoS ONE, Vol 15, Iss 3, p e0229494 (2020) PLoS ONE |
| ISSN: | 1932-6203 |
| DOI: | 10.1371/journal.pone.0229494 |
| Popis: | Pseudomonas aeruginosa is a virulent pathogen that has become more threatening with the emergence of multidrug resistance. The aspartate transcarbamoylase (ATCase) of this organism is a dodecamer comprised of six 37 kDa catalytic chains and six 45 kDa chains homologous to dihydroorotase (pDHO). The pDHO chain is inactive but is necessary for ATCase activity. A stoichiometric mixture of the subunits associates into a dodecamer with full ATCase activity. Unlike other known ATCases, the P. aeruginosa catalytic chain does not spontaneously assemble into a trimer. Chemical-crosslinking and size-exclusion chromatography showed that P. aeruginosa ATCase is monomeric which accounts for its lack of catalytic activity since the active site is a composite comprised of residues from adjacent monomers in the trimer. Circular dichroism spectroscopy indicated that the ATCase chain adopts a structure that contains secondary structure elements although neither the ATCase nor the pDHO subunits are very stable as determined by a thermal shift assay. Formation of the complex increases the melting temperature by about 30°C. The ATCase is strongly inhibited by all nucleotide di- and triphosphates and exhibits extreme cooperativity. Previous studies suggested that the regulatory site is located in an 11-residue extension of the amino end of the catalytic chain. However, deletion of the extensions did not affect catalytic activity, nucleotide inhibition or the assembly of the dodecamer. Nucleotides destabilized the dodecamer which probably accounts for the inhibition and apparent cooperativity of the substrate saturation curves. Contrary to previous interpretations, these results suggest that P. aeruginosa ATCase is not allosterically regulated by nucleotides. |
| Databáze: | OpenAIRE |
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