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Factors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1
The cyanide dihydratase enzyme from Bacillus pumilus C1 (CynDₚᵤₘ) is a member of the nitrilase superfamily and is known to specifically catalyse the conversion of cyanide into formic acid and ammonia. This enzyme is a good candidate for bioremediation of cyanide waste but the high alkaline pH of the...
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| Format: | Thesis |
| Language: | English |
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Division of Medical Biochemistry and Structural Biology
2017
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| _version_ | 1867613173803122688 |
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| access_status_str | Open Access |
| author | Mulelu, Andani Errol |
| author2 | Sewell, Bryan Trevor |
| author_browse | Mulelu, Andani Errol Sewell, Bryan Trevor |
| author_facet | Sewell, Bryan Trevor Mulelu, Andani Errol |
| author_sort | Mulelu, Andani Errol |
| collection | Thesis |
| description | The cyanide dihydratase enzyme from Bacillus pumilus C1 (CynDₚᵤₘ) is a member of the nitrilase superfamily and is known to specifically catalyse the conversion of cyanide into formic acid and ammonia. This enzyme is a good candidate for bioremediation of cyanide waste but the high alkaline pH of the cyanide waste water poses a problem in that it inactivates the wild type enzyme and therefore improvement of stability is required in order to synthesize an effective enzyme. Over the pH range of 6–8 the enzyme exists as short 18-subunit spirals which associate to form long, more stable helical fibres at pH 5.4. The reason for this pH dependent transition is not fully understood but it is hypothesized to be due to changes in the charge of histidine residues. The aim of this project is to obtain a high resolution structure of CynDₚᵤₘ, relate this to its function, and investigate the role of the histidines in oligomerisation with aid of the structure. Using Cryo-electron microscopy techniques a three dimensional reconstruction structure of purified CynDₚᵤₘ was obtained at a resolution of ~5Å. By flexibly fitting a CynDₚᵤₘ homology model into this high resolution structure we were able to identify amino acid residues involved in oligomerisation and stability as well as the role of the histidines, with aid from additional mutagenesis studies. Interactions at the C-interfacial region were shown to play the most crucial role in oligomerisation and included the His71-Asp275 and Arg67-Asp275 interactions. Mutations at His128, His184, His241 and His285 were shown to affect the oligomerisation of the enzyme by indirectly disrupting interactions at the interfacial regions. The Q86R+H305K+H308K+H323K mutations were shown to increase the stability of the CynDₚᵤₘ by introducing a stronger arginine-arginine interaction at the D interfacial region and a new strong interaction at the C-terminal region. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/24446 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:31:56.645Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2017 |
| publishDateRange | 2017 |
| publishDateSort | 2017 |
| publisher | Division of Medical Biochemistry and Structural Biology |
| publisherStr | Division of Medical Biochemistry and Structural Biology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/24446 Factors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1 Mulelu, Andani Errol Sewell, Bryan Trevor Woodward, J D Medical Biochemistry The cyanide dihydratase enzyme from Bacillus pumilus C1 (CynDₚᵤₘ) is a member of the nitrilase superfamily and is known to specifically catalyse the conversion of cyanide into formic acid and ammonia. This enzyme is a good candidate for bioremediation of cyanide waste but the high alkaline pH of the cyanide waste water poses a problem in that it inactivates the wild type enzyme and therefore improvement of stability is required in order to synthesize an effective enzyme. Over the pH range of 6–8 the enzyme exists as short 18-subunit spirals which associate to form long, more stable helical fibres at pH 5.4. The reason for this pH dependent transition is not fully understood but it is hypothesized to be due to changes in the charge of histidine residues. The aim of this project is to obtain a high resolution structure of CynDₚᵤₘ, relate this to its function, and investigate the role of the histidines in oligomerisation with aid of the structure. Using Cryo-electron microscopy techniques a three dimensional reconstruction structure of purified CynDₚᵤₘ was obtained at a resolution of ~5Å. By flexibly fitting a CynDₚᵤₘ homology model into this high resolution structure we were able to identify amino acid residues involved in oligomerisation and stability as well as the role of the histidines, with aid from additional mutagenesis studies. Interactions at the C-interfacial region were shown to play the most crucial role in oligomerisation and included the His71-Asp275 and Arg67-Asp275 interactions. Mutations at His128, His184, His241 and His285 were shown to affect the oligomerisation of the enzyme by indirectly disrupting interactions at the interfacial regions. The Q86R+H305K+H308K+H323K mutations were shown to increase the stability of the CynDₚᵤₘ by introducing a stronger arginine-arginine interaction at the D interfacial region and a new strong interaction at the C-terminal region. 2017-06-01T10:07:45Z 2017-06-01T10:07:45Z 2017 Doctoral Thesis Doctoral PhD http://hdl.handle.net/11427/24446 eng application/pdf Division of Medical Biochemistry and Structural Biology Faculty of Health Sciences University of Cape Town |
| spellingShingle | Medical Biochemistry Mulelu, Andani Errol Factors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1 |
| thesis_degree_str | Doctoral |
| title | Factors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1 |
| title_full | Factors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1 |
| title_fullStr | Factors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1 |
| title_full_unstemmed | Factors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1 |
| title_short | Factors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1 |
| title_sort | factors involved in the oligomerisation of the cyanide dihydratase from bacillus pumilus c1 |
| topic | Medical Biochemistry |
| url | http://hdl.handle.net/11427/24446 |
| work_keys_str_mv | AT muleluandanierrol factorsinvolvedintheoligomerisationofthecyanidedihydratasefrombacilluspumilusc1 |