Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities.
Deacetoxycephalosporin C synthetase (expandase) from Cephalosporium acremonium (Acremonium chrysogenum) was purified to near homogeneity as judged by SDS/polyacrylamide-gel electrophoresis. The enzyme (Mr about 40,000) exhibited a pH optimum around 7.5. It required 2-oxoglutarate (Km 0.04 mM), Fe2+...
| Main Authors: | , , , , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
1987
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| _version_ | 1797095079201996800 |
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| author | Baldwin, J Adlington, R Coates, J Crabbe, M Crouch, N Keeping, J Knight, G Schofield, C Ting, H Vallejo, C |
| author_facet | Baldwin, J Adlington, R Coates, J Crabbe, M Crouch, N Keeping, J Knight, G Schofield, C Ting, H Vallejo, C |
| author_sort | Baldwin, J |
| collection | OXFORD |
| description | Deacetoxycephalosporin C synthetase (expandase) from Cephalosporium acremonium (Acremonium chrysogenum) was purified to near homogeneity as judged by SDS/polyacrylamide-gel electrophoresis. The enzyme (Mr about 40,000) exhibited a pH optimum around 7.5. It required 2-oxoglutarate (Km 0.04 mM), Fe2+ and O2 as cofactors, and ascorbate and dithiothreitol were necessary for maximum activity. It was stable for over 4 weeks at -70 degrees C in the presence of 1 mM-dithiothreitol. Activity was inhibited by the thiol-quenching reagent N-ethylmaleimide, the metal-ion-chelating reagent bathophenanthroline, and NH4HCO3. The highly purified enzyme also showed deacetoxycephalosporin C hydroxylase (deacetylcephalosporin C synthetase) activity, indicating that both expandase and hydroxylase activities are properties of a single protein. These activities could not be separated by ion-exchange, dye-ligand, gel-filtration or hydrophobic chromatography. A beta-sulphoxide and a 3 beta-methylene hydroxy analogue of penicillin N were synthesized to test as potential intermediates in the ring-expansion reaction, Neither compound was a substrate for the enzyme. A synthetic analogue in which the 3 beta-methyl group and the 2-hydrogen atom of penicillin N were replaced by a cyclopropane ring was not a substrate but was a reversible inhibitor of the enzyme. |
| first_indexed | 2024-03-07T04:22:55Z |
| format | Journal article |
| id | oxford-uuid:cba1eb97-e137-45e1-b1f8-58965e4be068 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T04:22:55Z |
| publishDate | 1987 |
| record_format | dspace |
| spelling | oxford-uuid:cba1eb97-e137-45e1-b1f8-58965e4be0682022-03-27T07:16:13ZPurification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:cba1eb97-e137-45e1-b1f8-58965e4be068EnglishSymplectic Elements at Oxford1987Baldwin, JAdlington, RCoates, JCrabbe, MCrouch, NKeeping, JKnight, GSchofield, CTing, HVallejo, CDeacetoxycephalosporin C synthetase (expandase) from Cephalosporium acremonium (Acremonium chrysogenum) was purified to near homogeneity as judged by SDS/polyacrylamide-gel electrophoresis. The enzyme (Mr about 40,000) exhibited a pH optimum around 7.5. It required 2-oxoglutarate (Km 0.04 mM), Fe2+ and O2 as cofactors, and ascorbate and dithiothreitol were necessary for maximum activity. It was stable for over 4 weeks at -70 degrees C in the presence of 1 mM-dithiothreitol. Activity was inhibited by the thiol-quenching reagent N-ethylmaleimide, the metal-ion-chelating reagent bathophenanthroline, and NH4HCO3. The highly purified enzyme also showed deacetoxycephalosporin C hydroxylase (deacetylcephalosporin C synthetase) activity, indicating that both expandase and hydroxylase activities are properties of a single protein. These activities could not be separated by ion-exchange, dye-ligand, gel-filtration or hydrophobic chromatography. A beta-sulphoxide and a 3 beta-methylene hydroxy analogue of penicillin N were synthesized to test as potential intermediates in the ring-expansion reaction, Neither compound was a substrate for the enzyme. A synthetic analogue in which the 3 beta-methyl group and the 2-hydrogen atom of penicillin N were replaced by a cyclopropane ring was not a substrate but was a reversible inhibitor of the enzyme. |
| spellingShingle | Baldwin, J Adlington, R Coates, J Crabbe, M Crouch, N Keeping, J Knight, G Schofield, C Ting, H Vallejo, C Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities. |
| title | Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities. |
| title_full | Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities. |
| title_fullStr | Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities. |
| title_full_unstemmed | Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities. |
| title_short | Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities. |
| title_sort | purification and initial characterization of an enzyme with deacetoxycephalosporin c synthetase and hydroxylase activities |
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