Factors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1

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dc.contributor.advisorSewell, Bryan Trevoren_ZA
dc.contributor.advisorWoodward, J Den_ZA
dc.contributor.authorMulelu, Andani Errolen_ZA
dc.date.accessioned2017-06-01T10:07:45Z
dc.date.available2017-06-01T10:07:45Z
dc.date.issued2017en_ZA
dc.description.abstractThe 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.en_ZA
dc.identifier.apacitationMulelu, A. E. (2017). <i>Factors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1</i>. (Thesis). University of Cape Town ,Faculty of Health Sciences ,Division of Medical Biochemistry & Structural Biology. Retrieved from http://hdl.handle.net/11427/24446en_ZA
dc.identifier.chicagocitationMulelu, Andani Errol. <i>"Factors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1."</i> Thesis., University of Cape Town ,Faculty of Health Sciences ,Division of Medical Biochemistry & Structural Biology, 2017. http://hdl.handle.net/11427/24446en_ZA
dc.identifier.citationMulelu, A. 2017. Factors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Mulelu, Andani Errol AB - 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. DA - 2017 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2017 T1 - Factors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1 TI - Factors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1 UR - http://hdl.handle.net/11427/24446 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/24446
dc.identifier.vancouvercitationMulelu AE. Factors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1. [Thesis]. University of Cape Town ,Faculty of Health Sciences ,Division of Medical Biochemistry & Structural Biology, 2017 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/24446en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDivision of Medical Biochemistry and Structural Biology
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherMedical Biochemistryen_ZA
dc.titleFactors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1en_ZA
dc.typeDoctoral Thesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnamePhDen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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