Browsing Faculty of Natural Sciences by Author "Sayed, Muhammed F."
Now showing items 1-2 of 2
-
Cowan, Donald A.; Sayed, Muhammed F.; Tsekoa, Tsepo L.; Cameron, Rory A.; Sewell, B. Trevor (Academy of Science of South Africa (ASSAf), 2004)[more][less]
Abstract: Microbial nitrile hydratases are important industrial enzymes that catalyse the conversion of nitriles to the corresponding amides. Bacillus strain RAPc8 nitrile hydratase has recently been cloned and functionally expressed in E. coli. Here, the purification, crystallization and preliminary X-ray diffraction data of this nitrile hydratase are described. The heterotetrameric enzyme was crystallized using the hanging-drop vapour-diffusion method. Crystals produced in the presence of 30% PEG 400, 0.1 M MES (pH 6.5) and 0.1 M magnesium chloride were selected for X-ray diffraction studies. A data set complete to 2.5 Å was collected under cryoconditions at the in-house X-ray source at the University of the Western Cape. The space group was determined to be primitive tetragonal (P41212) with unit cell dimensions a = 106.61 Å, b = 106.61 Å, c=83.23 Å, = = =90°; with one dimer per asymmetric unit. Solution of the three-dimensional structure via molecular replacement is in progress. URI: http://hdl.handle.net/10566/167 Files in this item: 1
TsekoaPurification2004.pdf (784.0Kb) -
Kimani, Serah W.; Agarkar, Vinod B.; Cowan, Donald A.; Sayed, Muhammed F.; Sewell, B. Trevor (International Union of Crystallography, 2007)[more][less]
Abstract: The amidase from Geobacillus pallidus RAPc8, a moderate thermophile, is a member of the nitrilase superfamily and catalyzes the conversion of amides to the corresponding carboxylic acids and ammonia. It shows both amide-hydrolysis and acyl-transfer activities and also exhibits stereoselectivity for some enantiomeric substrates, thus making it a potentially important industrial catalyst. The crystal structure of G. pallidus RAPc8 amidase at a resolution of 1.9 A ˚ was solved by molecular replacement from a crystal belonging to the primitive cubic space group P4232. G. pallidus RAPc8 amidase is homohexameric in solution and its monomers have the typical nitrilase-superfamily α-β-β-α fold. Association in the hexamer preserves the eight-layered α-β-β-α:α-β-β-α structure across an interface which is conserved in the known members of the superfamily. The extended carboxy-terminal tail contributes to this conserved interface by interlocking the monomers. Analysis of the small active site of the G. pallidus RAPc8 amidase suggests that access of a water molecule to the catalytic triad (Cys, Glu, Lys) side chains would be impeded by the formation of the acyl intermediate. It is proposed that another active-site residue, Glu142, the position of which is conserved in the homologues, acts as a general base to catalyse the hydrolysis of this intermediate. The small size of the substrate-binding pocket also explains the specificity of this enzyme for short aliphatic amides and its asymmetry explains its enantioselectivity. URI: http://hdl.handle.net/10566/141 Files in this item: 1
KimaniAliphaticAmidase2007.pdf (2.155Mb)
Now showing items 1-2 of 2