Studies of isopenicillin N synthase enzymatic properties using a continuous spectrophotometric assay.
Isopenicillin N synthase (IPNS) from Aspergillus nidulans is a no-heme iron(II)-dependent oxygenase which catalyses, in a single reaction, the bicyclisation of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine into isopenicillin N, the precursor of all other penicillins, cephalosporins and cephamyci...
| Main Authors: | , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
2000
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| _version_ | 1797079035949350912 |
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| author | Dubus, A Sami, M Brown, T Schofield, C Baldwin, J Frère, J |
| author_facet | Dubus, A Sami, M Brown, T Schofield, C Baldwin, J Frère, J |
| author_sort | Dubus, A |
| collection | OXFORD |
| description | Isopenicillin N synthase (IPNS) from Aspergillus nidulans is a no-heme iron(II)-dependent oxygenase which catalyses, in a single reaction, the bicyclisation of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine into isopenicillin N, the precursor of all other penicillins, cephalosporins and cephamycins. The IPNS reaction can be followed directly and continuously by a new assay which monitors the absorbance increase at 235 nm characteristic of penicillin nucleus formation. Using this assay, the effects of influential factors affecting the in vitro IPNS enzymatic reaction were investigated. Even under optimal conditions, enzyme inactivation occurred during catalysis. Iron(II) depletion and product inhibition were not the cause of this phenomenon, the addition of antioxidants or reducing agents failed to slow down inactivation or reactivate the enzyme. Therefore, this phenomenon appears to be irreversible and is attributed to oxidative damage caused to the enzyme by reactive oxygen species generated in solution during catalysis. Nevertheless, the steady-state kinetic parameters for the IPNS reaction were determined. |
| first_indexed | 2024-03-07T00:39:54Z |
| format | Journal article |
| id | oxford-uuid:82afdedc-747b-48d0-9e1d-2d911720e6da |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T00:39:54Z |
| publishDate | 2000 |
| record_format | dspace |
| spelling | oxford-uuid:82afdedc-747b-48d0-9e1d-2d911720e6da2022-03-26T21:39:05ZStudies of isopenicillin N synthase enzymatic properties using a continuous spectrophotometric assay.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:82afdedc-747b-48d0-9e1d-2d911720e6daEnglishSymplectic Elements at Oxford2000Dubus, ASami, MBrown, TSchofield, CBaldwin, JFrère, JIsopenicillin N synthase (IPNS) from Aspergillus nidulans is a no-heme iron(II)-dependent oxygenase which catalyses, in a single reaction, the bicyclisation of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine into isopenicillin N, the precursor of all other penicillins, cephalosporins and cephamycins. The IPNS reaction can be followed directly and continuously by a new assay which monitors the absorbance increase at 235 nm characteristic of penicillin nucleus formation. Using this assay, the effects of influential factors affecting the in vitro IPNS enzymatic reaction were investigated. Even under optimal conditions, enzyme inactivation occurred during catalysis. Iron(II) depletion and product inhibition were not the cause of this phenomenon, the addition of antioxidants or reducing agents failed to slow down inactivation or reactivate the enzyme. Therefore, this phenomenon appears to be irreversible and is attributed to oxidative damage caused to the enzyme by reactive oxygen species generated in solution during catalysis. Nevertheless, the steady-state kinetic parameters for the IPNS reaction were determined. |
| spellingShingle | Dubus, A Sami, M Brown, T Schofield, C Baldwin, J Frère, J Studies of isopenicillin N synthase enzymatic properties using a continuous spectrophotometric assay. |
| title | Studies of isopenicillin N synthase enzymatic properties using a continuous spectrophotometric assay. |
| title_full | Studies of isopenicillin N synthase enzymatic properties using a continuous spectrophotometric assay. |
| title_fullStr | Studies of isopenicillin N synthase enzymatic properties using a continuous spectrophotometric assay. |
| title_full_unstemmed | Studies of isopenicillin N synthase enzymatic properties using a continuous spectrophotometric assay. |
| title_short | Studies of isopenicillin N synthase enzymatic properties using a continuous spectrophotometric assay. |
| title_sort | studies of isopenicillin n synthase enzymatic properties using a continuous spectrophotometric assay |
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