Modification of iron binding ligands in isopenicillin n synthase
<p>Isopenicillin N synthase (IPNS) is a non-haem iron dependent dioxygenase which catalyses the oxidative conversion of anddelta;-(<em>L</em>-andalpha;-aminoadipoyl)-<em>L</em>-cysteinyl-<em>D</em>-valine (ACV) to isopenicillin N (IPN). S...
| Váldodahkkit: | , |
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| Eará dahkkit: | |
| Materiálatiipa: | Oahppočájánas |
| Giella: | English |
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1998
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| Fáttát: |
| _version_ | 1826306317020561408 |
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| author | Sami, M Sami, Malkit |
| author2 | Schofield, C |
| author_facet | Schofield, C Sami, M Sami, Malkit |
| author_sort | Sami, M |
| collection | OXFORD |
| description | <p>Isopenicillin N synthase (IPNS) is a non-haem iron dependent dioxygenase which catalyses the oxidative conversion of anddelta;-(<em>L</em>-andalpha;-aminoadipoyl)-<em>L</em>-cysteinyl-<em>D</em>-valine (ACV) to isopenicillin N (IPN). Sequence comparisons between IPNS isozymes reveal the complete conservation of two histidine (His214, His270), one aspartate (Asp216) [also known as the '2-His-l-carboxylate' motif] and one glutamine (Gln330) residue. The crystal structure of IPNS (<em>Aspergillus nidulans</em>) active site (in the absence of ACV) revealed an octahedrally coordinated manganese atom surrounded by these four protein ligands and two water molecules.</p> <p>The role of the four conserved metal binding ligands was investigated using site directed mutagenesis. The results demonstrated that ligation of the iron with Gln330 was not essential for the catalytic activity of IPNS. In contrast, ligation of the iron with the three remaining metal ligands was indispensable for catalytic activity. Additionally, it was demonstrated that the conserved Asp216 residue may be substituted by a glutamate residue (D216E) with significant retention of catalytic activity. Crystallographic and spectroscopic evidence suggested that the D216E mutant bound both iron and ACV in a similar way to wild-type IPNS.</p> <p>The inactivation of wild-type IPNS was examined under <em>in vitro</em> assay conditions. This study showed that inactivation of IPNS results (minimally) from a slow non-oxidative pathway (in buffer alone) and a fast oxidative pathway <em>via</em> Udenfriend's chemistry (ferrous iron, ascorbate, and oxygen). The oxidative inactivation pathway was substantially reduced by the inclusion of catalase in the assay mixture, thus indicating that oxidative IPNS inactivation results (in part) from the generation of hydrogen peroxide in solution. Inactivation was also accompanied by a slow fragmentation of intact IPNS into (at least) five oligopeptides (observed by sodium dodecyl sulphate polyacrylamide gel electrophoresis). N-Terminal sequencing analyses confirmed that the fragmentation resulted from at least two cleavage sites within the active site (between Asp216-Val217 and Val272-Lys273).</p> |
| first_indexed | 2024-03-07T06:46:07Z |
| format | Thesis |
| id | oxford-uuid:fae89f76-345a-4c57-a37d-a78db8885be7 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T06:46:07Z |
| publishDate | 1998 |
| record_format | dspace |
| spelling | oxford-uuid:fae89f76-345a-4c57-a37d-a78db8885be72022-03-27T13:09:48ZModification of iron binding ligands in isopenicillin n synthaseThesishttp://purl.org/coar/resource_type/c_db06uuid:fae89f76-345a-4c57-a37d-a78db8885be7SynthesisPenicillinAspergillus nidulansLigandsEnglishPolonsky Theses Digitisation Project1998Sami, MSami, MalkitSchofield, CSchofield, C<p>Isopenicillin N synthase (IPNS) is a non-haem iron dependent dioxygenase which catalyses the oxidative conversion of anddelta;-(<em>L</em>-andalpha;-aminoadipoyl)-<em>L</em>-cysteinyl-<em>D</em>-valine (ACV) to isopenicillin N (IPN). Sequence comparisons between IPNS isozymes reveal the complete conservation of two histidine (His214, His270), one aspartate (Asp216) [also known as the '2-His-l-carboxylate' motif] and one glutamine (Gln330) residue. The crystal structure of IPNS (<em>Aspergillus nidulans</em>) active site (in the absence of ACV) revealed an octahedrally coordinated manganese atom surrounded by these four protein ligands and two water molecules.</p> <p>The role of the four conserved metal binding ligands was investigated using site directed mutagenesis. The results demonstrated that ligation of the iron with Gln330 was not essential for the catalytic activity of IPNS. In contrast, ligation of the iron with the three remaining metal ligands was indispensable for catalytic activity. Additionally, it was demonstrated that the conserved Asp216 residue may be substituted by a glutamate residue (D216E) with significant retention of catalytic activity. Crystallographic and spectroscopic evidence suggested that the D216E mutant bound both iron and ACV in a similar way to wild-type IPNS.</p> <p>The inactivation of wild-type IPNS was examined under <em>in vitro</em> assay conditions. This study showed that inactivation of IPNS results (minimally) from a slow non-oxidative pathway (in buffer alone) and a fast oxidative pathway <em>via</em> Udenfriend's chemistry (ferrous iron, ascorbate, and oxygen). The oxidative inactivation pathway was substantially reduced by the inclusion of catalase in the assay mixture, thus indicating that oxidative IPNS inactivation results (in part) from the generation of hydrogen peroxide in solution. Inactivation was also accompanied by a slow fragmentation of intact IPNS into (at least) five oligopeptides (observed by sodium dodecyl sulphate polyacrylamide gel electrophoresis). N-Terminal sequencing analyses confirmed that the fragmentation resulted from at least two cleavage sites within the active site (between Asp216-Val217 and Val272-Lys273).</p> |
| spellingShingle | Synthesis Penicillin Aspergillus nidulans Ligands Sami, M Sami, Malkit Modification of iron binding ligands in isopenicillin n synthase |
| title | Modification of iron binding ligands in isopenicillin n synthase |
| title_full | Modification of iron binding ligands in isopenicillin n synthase |
| title_fullStr | Modification of iron binding ligands in isopenicillin n synthase |
| title_full_unstemmed | Modification of iron binding ligands in isopenicillin n synthase |
| title_short | Modification of iron binding ligands in isopenicillin n synthase |
| title_sort | modification of iron binding ligands in isopenicillin n synthase |
| topic | Synthesis Penicillin Aspergillus nidulans Ligands |
| work_keys_str_mv | AT samim modificationofironbindingligandsinisopenicillinnsynthase AT samimalkit modificationofironbindingligandsinisopenicillinnsynthase |