The crystal structure of an aliphatic amidase from Geobacillus pallidus RAPc8

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dc.contributor.advisorSewell, Bryan Trevoren_ZA
dc.contributor.advisorSayed, Muhameden_ZA
dc.contributor.authorKimani, Serahen_ZA
dc.date.accessioned2014-10-29T10:05:06Z
dc.date.available2014-10-29T10:05:06Z
dc.date.issued2007en_ZA
dc.descriptionIncludes bibliographical references (leaves 123-136).en_ZA
dc.description.abstractAmidases 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.en_ZA
dc.identifier.apacitationKimani, S. (2007). <i>The crystal structure of an aliphatic amidase from Geobacillus pallidus RAPc8</i>. (Thesis). University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology. Retrieved from http://hdl.handle.net/11427/8908en_ZA
dc.identifier.chicagocitationKimani, Serah. <i>"The crystal structure of an aliphatic amidase from Geobacillus pallidus RAPc8."</i> Thesis., University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 2007. http://hdl.handle.net/11427/8908en_ZA
dc.identifier.citationKimani, S. 2007. The crystal structure of an aliphatic amidase from Geobacillus pallidus RAPc8. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Kimani, Serah AB - 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. DA - 2007 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2007 T1 - The crystal structure of an aliphatic amidase from Geobacillus pallidus RAPc8 TI - The crystal structure of an aliphatic amidase from Geobacillus pallidus RAPc8 UR - http://hdl.handle.net/11427/8908 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/8908
dc.identifier.vancouvercitationKimani S. The crystal structure of an aliphatic amidase from Geobacillus pallidus RAPc8. [Thesis]. University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 2007 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/8908en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDepartment of Molecular and Cell Biologyen_ZA
dc.publisher.facultyFaculty of Scienceen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherStructural Biologyen_ZA
dc.titleThe crystal structure of an aliphatic amidase from Geobacillus pallidus RAPc8en_ZA
dc.typeMaster Thesis
dc.type.qualificationlevelMasters
dc.type.qualificationnameMScen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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