The use of active site mutants of cytochrome P450(cam) in chemical synthesis
<p>This thesis describes a study of the substrate selectivity of active site mutants of the monooxygenase cytochrome P450<sub>cam</sub>. A range of mutants was constructed which replaced the phenolic side-chain at the Tyr-96 position by various hydrophobic amino acid residues. The...
| Hlavní autoři: | , |
|---|---|
| Médium: | Diplomová práce |
| Jazyk: | English |
| Vydáno: |
1999
|
| Témata: |
| _version_ | 1826281487681454080 |
|---|---|
| author | Bell, S Bell, Stephen Graham |
| author_facet | Bell, S Bell, Stephen Graham |
| author_sort | Bell, S |
| collection | OXFORD |
| description | <p>This thesis describes a study of the substrate selectivity of active site mutants of the monooxygenase cytochrome P450<sub>cam</sub>. A range of mutants was constructed which replaced the phenolic side-chain at the Tyr-96 position by various hydrophobic amino acid residues. These 'hydrophobic mutants' were then combined with other mutations around the active site (Val-247, Phe-87, Ile-395 and Phe-193) which altered the space available at different positions in the active site. These mutants were then tested with an <em>in vitro</em> reconstituted P450<sub>cam</sub> system with a range of substrates related to diphenylmethane and phenylcylcohexane. All of these large compounds were poor substrates for the wild-type enzyme. It was found that it was necessary to increase both the space available in the active site and the active site hydrophobicity to achieve substrate turnover. The substrates were oxidised preferentially on the aliphatic cyclohexyl ring over the more constrained phenyl ring suggesting that the active site is predisposed to binding the cyclohexyl ring close to the haem. Hydroxylation using the <em>in vitro</em> reconstituted P450<sub>cam</sub> system is limited by catalyst lifetime and the need for the expensive cofactor NADH. For P450<sub>cam</sub> hydroxylation to become a viable synthetic method it is necessary to find ways to bypass the use of NADH. For this reason various self-sufficient P450<sub>cam</sub> system were constructed and expressed in <em>E. coli</em>. The best of these, despite limited protein expression, was found to turnover camphor with the wild-type P450<sub>cam</sub> enzyme and other substrates with the Y96A mutant. The <em>in vivo</em> catalytic system was then used to screen many P450<sub>cam</sub> mutants for the oxidation of natural products, monoterpenes and sesquiterpenes (e.g. limonene, pinene and valencene). Most of the target substrates are not oxidised by the wild-type enzyme but all are hydroxylated by some if not all of the P450<sub>cam</sub> mutants with different degrees of selectivity. Some of the products identified so far are important compounds in the field of flavour and fragrance chemistry (e.g. verbenol and nookatone).</p> |
| first_indexed | 2024-03-07T00:29:33Z |
| format | Thesis |
| id | oxford-uuid:7f48cf79-37b0-45cd-a40e-e971af466cff |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T00:29:33Z |
| publishDate | 1999 |
| record_format | dspace |
| spelling | oxford-uuid:7f48cf79-37b0-45cd-a40e-e971af466cff2022-03-26T21:16:07ZThe use of active site mutants of cytochrome P450(cam) in chemical synthesisThesishttp://purl.org/coar/resource_type/c_db06uuid:7f48cf79-37b0-45cd-a40e-e971af466cffCytochrome oxidaseCytochrome P-450DerivativesCytochromesEnglishPolonsky Theses Digitisation Project1999Bell, SBell, Stephen Graham<p>This thesis describes a study of the substrate selectivity of active site mutants of the monooxygenase cytochrome P450<sub>cam</sub>. A range of mutants was constructed which replaced the phenolic side-chain at the Tyr-96 position by various hydrophobic amino acid residues. These 'hydrophobic mutants' were then combined with other mutations around the active site (Val-247, Phe-87, Ile-395 and Phe-193) which altered the space available at different positions in the active site. These mutants were then tested with an <em>in vitro</em> reconstituted P450<sub>cam</sub> system with a range of substrates related to diphenylmethane and phenylcylcohexane. All of these large compounds were poor substrates for the wild-type enzyme. It was found that it was necessary to increase both the space available in the active site and the active site hydrophobicity to achieve substrate turnover. The substrates were oxidised preferentially on the aliphatic cyclohexyl ring over the more constrained phenyl ring suggesting that the active site is predisposed to binding the cyclohexyl ring close to the haem. Hydroxylation using the <em>in vitro</em> reconstituted P450<sub>cam</sub> system is limited by catalyst lifetime and the need for the expensive cofactor NADH. For P450<sub>cam</sub> hydroxylation to become a viable synthetic method it is necessary to find ways to bypass the use of NADH. For this reason various self-sufficient P450<sub>cam</sub> system were constructed and expressed in <em>E. coli</em>. The best of these, despite limited protein expression, was found to turnover camphor with the wild-type P450<sub>cam</sub> enzyme and other substrates with the Y96A mutant. The <em>in vivo</em> catalytic system was then used to screen many P450<sub>cam</sub> mutants for the oxidation of natural products, monoterpenes and sesquiterpenes (e.g. limonene, pinene and valencene). Most of the target substrates are not oxidised by the wild-type enzyme but all are hydroxylated by some if not all of the P450<sub>cam</sub> mutants with different degrees of selectivity. Some of the products identified so far are important compounds in the field of flavour and fragrance chemistry (e.g. verbenol and nookatone).</p> |
| spellingShingle | Cytochrome oxidase Cytochrome P-450 Derivatives Cytochromes Bell, S Bell, Stephen Graham The use of active site mutants of cytochrome P450(cam) in chemical synthesis |
| title | The use of active site mutants of cytochrome P450(cam) in chemical synthesis |
| title_full | The use of active site mutants of cytochrome P450(cam) in chemical synthesis |
| title_fullStr | The use of active site mutants of cytochrome P450(cam) in chemical synthesis |
| title_full_unstemmed | The use of active site mutants of cytochrome P450(cam) in chemical synthesis |
| title_short | The use of active site mutants of cytochrome P450(cam) in chemical synthesis |
| title_sort | use of active site mutants of cytochrome p450 cam in chemical synthesis |
| topic | Cytochrome oxidase Cytochrome P-450 Derivatives Cytochromes |
| work_keys_str_mv | AT bells theuseofactivesitemutantsofcytochromep450caminchemicalsynthesis AT bellstephengraham theuseofactivesitemutantsofcytochromep450caminchemicalsynthesis AT bells useofactivesitemutantsofcytochromep450caminchemicalsynthesis AT bellstephengraham useofactivesitemutantsofcytochromep450caminchemicalsynthesis |