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The crystal structure of an aliphatic amidase from Geobacillus pallidus RAPc8
Includes bibliographical references (leaves 123-136).
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| Main Author: | |
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| Other Authors: | |
| Format: | Thesis |
| Language: | English |
| Published: |
Department of Molecular and Cell Biology
2014
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| _version_ | 1867613295367684096 |
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| access_status_str | Open Access |
| author | Kimani, Serah |
| author2 | Sewell, Bryan Trevor |
| author_browse | Kimani, Serah Sewell, Bryan Trevor |
| author_facet | Sewell, Bryan Trevor Kimani, Serah |
| author_sort | Kimani, Serah |
| collection | Thesis |
| description | Includes bibliographical references (leaves 123-136). |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/8908 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:33:51.607Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2014 |
| publishDateRange | 2014 |
| publishDateSort | 2014 |
| publisher | Department of Molecular and Cell Biology |
| publisherStr | Department of Molecular and Cell Biology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/8908 The crystal structure of an aliphatic amidase from Geobacillus pallidus RAPc8 Kimani, Serah Sewell, Bryan Trevor Sayed, Muhamed Structural Biology Includes bibliographical references (leaves 123-136). Amidases are a group of carbon-nitrogen hydrolysing enzymes that catalyze the conversion of amides to corresponding carboxylic acids and ammonia. These enzymes are of great interest in synthetic industries where they are used for mass production of acidic products. Aliphatic amidase from Geobacillus pallidus RAPcS (RAPcS amidase), which belongs to the nitrilase superfamily of enzymes, has recently been characterised biochemically. It shows both amide hydrolysis and acyl transfer activities, and also exhibits stereo selectivity for some enantiomeric substrates. This enzyme can therefore be exploited in large-scale production of enantio-pure compounds. Structural characterization of this amidase would yield insights into the basis of this substrate selectivity and activity. This would inform future experiments that aims at modifying this enzyme to alter its substrate specificity. This work presents structural characterization of RAPcS amidase. Gel filtration chromatography and electron microscopic analyses provided useful information on the quaternary structure of RAPcS amidase. Crystals were grown, and an X-ray diffraction dataset to 1.9 Å collected using an in-house X-ray source. The space group of this data was determined to be primitive cubic P4₂32, and the structure was solved by molecular replacement using the backbone of the hypothetical protein PH0642 from Pyrococcus horikoshii (PDB ID, Ij31) that had all non-identical side chains substituted with alanines, as a search probe. The molecular replacement rotational and translational searches were performed using PHASER. The model was rebuilt with PHENIX before refinement using REFMAC5. The final model was of high quality with minimal errors. RAPcS amidase is homohexameric in solution and has a four-layer α-β-β-α structural fold that highly resembles nitrilase superfamily enzymes. It has an extended C-terminal tail that is essential for strengthening the interacting dimer interfaces by participating in domain swapping. The active site pocket has Glu, Lys, Cys catalytic triad that is conserved in the nitrilase superfamily. The substrate binding pocket is small in size, explaining the specificity of this enzyme for short aliphatic amides. These findings have made steps towards understanding the catalytic mechanism, and the basis for substrate specificity in this enzyme. It has also provided useful information on the overall structure, as well as the structure of the active site, not only for RAPcS amidase but also for related enzymes, which will form the basis for designing future structural characterization work in the nitrilase-related amidases. 2014-10-29T10:05:06Z 2014-10-29T10:05:06Z 2007 Master Thesis Masters MSc http://hdl.handle.net/11427/8908 eng application/pdf Department of Molecular and Cell Biology Faculty of Science University of Cape Town |
| spellingShingle | Structural Biology Kimani, Serah The crystal structure of an aliphatic amidase from Geobacillus pallidus RAPc8 |
| thesis_degree_str | Master's |
| title | The crystal structure of an aliphatic amidase from Geobacillus pallidus RAPc8 |
| title_full | The crystal structure of an aliphatic amidase from Geobacillus pallidus RAPc8 |
| title_fullStr | The crystal structure of an aliphatic amidase from Geobacillus pallidus RAPc8 |
| title_full_unstemmed | The crystal structure of an aliphatic amidase from Geobacillus pallidus RAPc8 |
| title_short | The crystal structure of an aliphatic amidase from Geobacillus pallidus RAPc8 |
| title_sort | crystal structure of an aliphatic amidase from geobacillus pallidus rapc8 |
| topic | Structural Biology |
| url | http://hdl.handle.net/11427/8908 |
| work_keys_str_mv | AT kimaniserah thecrystalstructureofanaliphaticamidasefromgeobacilluspallidusrapc8 AT kimaniserah crystalstructureofanaliphaticamidasefromgeobacilluspallidusrapc8 |