Includes bibliographical references (leaves 81-86). / Nitrilases are moderately ubiquitous nitrile/cyanide-degrading enzymes, found in both eukaryotes (animals, fungi, plants) and prokaryotes (archaea, bacteria) which catalyse the condensation and hydrolysis of a wide range of non-peptide nitrile substrates and are involved in nitrile-posttranslational modification. As Cyanide and related compounds are used extensively by humans in various industrial processes which, due to carelessness and inadequate waste-management systems, contribute significantly to the levels of toxic cyanide contamination in the environment nitrilases have been speculated to be useful for bioremediation amongst other things.Nitrile/cyanide hydrolysing enzymes have a broad range of substrates and they function via four known pathways. Nitrilase and cyanide dihydratase completely hydrolyse nitriles and HCN respectively to yield the corresponding acid and ammonia without going via an amide intermediate. Nitrile hydratase and cyanide hydratase perform a single hydrolysis producing the corresponding amide and formamide, respectively. The nitrilases are known to form extensive quaternary structures including dimers, spirals and rods/helices. Generally microbial nitrilases exist as homo-oligomers having a large molecular weight (>300 kDa). These enzymes are known to oligomerise under conditions of substrate activation (Rhodococcus rhodocrous) and pH change as is the case for the Cyanide dihydratase from Bacilluspumilus Cl (CynDpum) which exists as a terminating spiral of -16 subunits above pH 6 but forms a long helical fibre below -pH 6. In this project the Cyanide dihydratase from strain 8A3 of B. pumilus was analysed using electron microscopy at pH of 5.4,6 and 8. These data were reconstructed at pH 6 and pH 8 using the single particle reconstruction technique to resolutions of 29A and 31A respectively. It is shown that at pH 6 the enzyme consists of 20 subunits (10 dimers) and at pH 8 22 subunits (11 dimers). These models show that CynDpum exists as an oligomeric spiral that terminates by decreasing the helical radius and tilting the terminal subunits toward the helical axis. Below pH 5.4 CynDpum from strain 8A3 does not extend into a fibre as in Cl, this is explained to be due to the lack of 3 key histidine residues found on the C-terminal tail of CynDpum which point into the inner cavity of the spiral and become charged below pH 6 producing a repulsion preventing the termination of the spiral by narrowing of the helical radius and thus encouraging extension into the helical form.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uct/oai:localhost:11427/4257 |
| Date | January 2007 |
| Creators | Eicher, Johann J |
| Publisher | University of Cape Town, Faculty of Science, Department of Molecular and Cell Biology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Master Thesis, Masters, MSc |
| Format | application/pdf |
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