Protein-protein recognition in biological systems exhibiting highly-conserved tertiary structure: cytochrome P450
<p>Protein tertiary structure is more conserved than amino acid sequence, leading to a diverse range of functions observed in the same fold. Despite < 20 % overall sequence identity, cytochromes P450 all have the same fold. Bacterial Class I P450s receive electrons from a highly specifi...
| Glavni autori: | , |
|---|---|
| Daljnji autori: | |
| Format: | Disertacija |
| Jezik: | English |
| Izdano: |
2013
|
| Teme: |
| _version_ | 1826298053090344960 |
|---|---|
| author | Johnson, E Eachan Oliver Daniel Johnson |
| author2 | Wong, L |
| author_facet | Wong, L Johnson, E Eachan Oliver Daniel Johnson |
| author_sort | Johnson, E |
| collection | OXFORD |
| description | <p>Protein tertiary structure is more conserved than amino acid sequence, leading to a diverse range of functions observed in the same fold. Despite < 20 % overall sequence identity, cytochromes P450 all have the same fold. Bacterial Class I P450s receive electrons from a highly specific, often unidentified, ferredoxin, in which case the hemoprotein is termed “orphaned”.</p> <p>CYP199A2, a Class I P450, accepts electrons from ferredoxins Pux and HaPux. Five orientation-dependent and one orientation-independent DEER measurements on paramagnetic HaPux and spin-labelled CYP199A2 yielded vector restraints, which were applied to building a model of the CYP199A2:HaPux complex <em>in silico</em>. A different binding mode was observed compared to P450cam:Pdx and P450scc:Adx, both recently elucidated by X-ray crystallography. This protocol was also applied to the CYP101D1:Arx complex. The first three measurements indicate that this heterodimer does not have a similar orientation to CYP199A2:HaPux, P450cam:Pdx, or P450scc:Adx.</p> <p>P450cam was fused to putidatredoxin reductase (PdR) to explore the kinetic effects with a view to improving electron transfer to orphan P450s. Heme incorporation of this enzyme depends on linker length. In whole cells, the fusion was more active after longer incubations. <em>In vitro</em> kinetics of the fusion exhibited some co-operativity and enhanced kinetics over the unfused system under steady-state conditions.</p> <p>The putative iron-sulfur biosynthesis ferredoxin PuxB had been engineered by rational mutagenesis to support catalysis by CYP199A2. It was confirmed this arose from improved protein-protein recognition. Engineering of <em>E. coli</em> ferredoxin based on these findings was carried out, resulting in electron-transfer to CYP199A4 from a novel engineered alien ferredoxin.</p> |
| first_indexed | 2024-03-07T04:40:57Z |
| format | Thesis |
| id | oxford-uuid:d19f5f52-d1ce-4ec2-be83-fd52f01124f8 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T04:40:57Z |
| publishDate | 2013 |
| record_format | dspace |
| spelling | oxford-uuid:d19f5f52-d1ce-4ec2-be83-fd52f01124f82022-03-27T07:58:12ZProtein-protein recognition in biological systems exhibiting highly-conserved tertiary structure: cytochrome P450Thesishttp://purl.org/coar/resource_type/c_db06uuid:d19f5f52-d1ce-4ec2-be83-fd52f01124f8Protein chemistryPhysical & theoretical chemistryCatalysisChemical kineticsMolecular biophysics (biochemistry)Biophysical chemistryBiophysicsChemical biologySpectroscopy and molecular structureEnzymesEnglishOxford University Research Archive - Valet2013Johnson, EEachan Oliver Daniel JohnsonWong, L<p>Protein tertiary structure is more conserved than amino acid sequence, leading to a diverse range of functions observed in the same fold. Despite < 20 % overall sequence identity, cytochromes P450 all have the same fold. Bacterial Class I P450s receive electrons from a highly specific, often unidentified, ferredoxin, in which case the hemoprotein is termed “orphaned”.</p> <p>CYP199A2, a Class I P450, accepts electrons from ferredoxins Pux and HaPux. Five orientation-dependent and one orientation-independent DEER measurements on paramagnetic HaPux and spin-labelled CYP199A2 yielded vector restraints, which were applied to building a model of the CYP199A2:HaPux complex <em>in silico</em>. A different binding mode was observed compared to P450cam:Pdx and P450scc:Adx, both recently elucidated by X-ray crystallography. This protocol was also applied to the CYP101D1:Arx complex. The first three measurements indicate that this heterodimer does not have a similar orientation to CYP199A2:HaPux, P450cam:Pdx, or P450scc:Adx.</p> <p>P450cam was fused to putidatredoxin reductase (PdR) to explore the kinetic effects with a view to improving electron transfer to orphan P450s. Heme incorporation of this enzyme depends on linker length. In whole cells, the fusion was more active after longer incubations. <em>In vitro</em> kinetics of the fusion exhibited some co-operativity and enhanced kinetics over the unfused system under steady-state conditions.</p> <p>The putative iron-sulfur biosynthesis ferredoxin PuxB had been engineered by rational mutagenesis to support catalysis by CYP199A2. It was confirmed this arose from improved protein-protein recognition. Engineering of <em>E. coli</em> ferredoxin based on these findings was carried out, resulting in electron-transfer to CYP199A4 from a novel engineered alien ferredoxin.</p> |
| spellingShingle | Protein chemistry Physical & theoretical chemistry Catalysis Chemical kinetics Molecular biophysics (biochemistry) Biophysical chemistry Biophysics Chemical biology Spectroscopy and molecular structure Enzymes Johnson, E Eachan Oliver Daniel Johnson Protein-protein recognition in biological systems exhibiting highly-conserved tertiary structure: cytochrome P450 |
| title | Protein-protein recognition in biological systems exhibiting highly-conserved tertiary structure: cytochrome P450 |
| title_full | Protein-protein recognition in biological systems exhibiting highly-conserved tertiary structure: cytochrome P450 |
| title_fullStr | Protein-protein recognition in biological systems exhibiting highly-conserved tertiary structure: cytochrome P450 |
| title_full_unstemmed | Protein-protein recognition in biological systems exhibiting highly-conserved tertiary structure: cytochrome P450 |
| title_short | Protein-protein recognition in biological systems exhibiting highly-conserved tertiary structure: cytochrome P450 |
| title_sort | protein protein recognition in biological systems exhibiting highly conserved tertiary structure cytochrome p450 |
| topic | Protein chemistry Physical & theoretical chemistry Catalysis Chemical kinetics Molecular biophysics (biochemistry) Biophysical chemistry Biophysics Chemical biology Spectroscopy and molecular structure Enzymes |
| work_keys_str_mv | AT johnsone proteinproteinrecognitioninbiologicalsystemsexhibitinghighlyconservedtertiarystructurecytochromep450 AT eachanoliverdanieljohnson proteinproteinrecognitioninbiologicalsystemsexhibitinghighlyconservedtertiarystructurecytochromep450 |