Browsing by Subject "nitrilase"
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- ItemRestrictedC-terminal hybrid mutant of Bacillus pumilus cyanide dihydratase dramatically enhances thermal stability and pH tolerance by reinforcing oligomerization(Wiley, 2015) Crum, M A; Park, J M; Sewell, B T; Benedik M JAims: To investigate the impact of the highly variable C-terminal domain of cyanide dihydratase, a member of the nitrilase superfamily, on its activity and stability. Methods and Results: Generating and analysing the thermal stability and pH tolerance of chimeric cyanide dihydratase proteins has provided a platform to investigate domains within the C-terminus and their effect on quaternary structure of the protein. The protein oligomerization state was inferred from native protein size by gel exclusion chromatography. Conclusions: Our data indicates that the influence of the cyanide dihydratase C-terminus on thermal stability stems from its participation in oligomerization at the major C-surface interface. The formation of this surface is crucial for the activity and stability of CynD. Gel filtration chromatography of an N-terminal deletion mutant, CynDpum Δ303, revealed a defect in oligomerization, and another mutant R67C was suppressed by introduction of a heterologous C-terminus as a chimeric protein. This indicates that the C-terminus from Pseudomonas stutzeri stabilizes CynD by supporting oligomerization between dimers at the C-surface. The chimeric protein CynDpum-stut exhibited full activity at pH 9, a pH where the parent enzyme is nearly inactive, and retained 40% of its activity at pH 9 5 making it a unique pH tolerant mutant. Significance and Impact of the Study: The study characterized a chimeric protein with remarkable thermal stability and tolerance to alkaline conditions, features essential for practical application as industrial cyanide solutions are maintained as highly alkaline solutions to prevent formation of hydrogen cyanide gas
- ItemRestrictedMicrobial nitrilases: versatile, spiral forming, industrial enzymes(Wiley, 2009) Thuku, R N; Brady, D; Benedik, M J; Sewell, B TThe nitrilases are enzymes that convert nitriles to the corresponding acid and ammonia. They are members of a superfamily, which includes amidases and occur in both prokaryotes and eukaryotes. The superfamily is characterized by having a homodimeric building block with a abba–abba sandwich fold and an active site containing four positionally conserved residues: cys, glu, glu and lys. Their high chemical specificity and frequent enantioselectivity makes them attractive biocatalysts for the production of fine chemicals and pharmaceutical intermediates. Nitrilases are also used in the treatment of toxic industrial effluent and cyanide remediation. The superfamily enzymes have been visualized as dimers, tetramers, hexamers, octamers, tetradecamers, octadecamers and variable length helices, but all nitrilase oligomers have the same basic dimer interface. Moreover, in the case of the octamers, tetradecamers, octadecamers and the helices, common principles of subunit association apply. While the range of industrially interesting reactions catalysed by this enzyme class continues to increase, research efforts are still hampered by the lack of a high resolution microbial nitrilase structure which can provide insights into their specificity, enantioselectivity and the mechanism of catalysis. This review provides an overview of the current progress in elucidation of structure and function in this enzyme class and emphasizes insights that may lead to further biotechnological applications.
- ItemRestrictedPost-translational cleavage of recombinantly expressed nitrilase from Rhodococcus rhodochrous J1 yields a stable, active helical form(Wiley, 2007) Thuku, R Ndoria; Weber, Brandon W; Varsani, Arvind; Sewell, B. TrevorNitrilases convert nitriles to the corresponding carboxylic acids and ammonia. The nitrilase from Rhodococcus rhodochrous J1 is known to be inactive as a dimer but to become active on oligomerization. The recombinant enzyme undergoes post-translational cleavage at approximately residue 327, resulting in the formation of active, helical homo-oligomers. Determining the 3D structure of these helices using electron microscopy, followed by fitting the stain envelope with a model based on homology with other members of the nitrilase superfamily, enables the interacting surfaces to be identified. This also suggests that the reason for formation of the helices is related to the removal of steric hindrance arising from the 39 C-terminal amino acids from the wild-type protein. The helical form can be generated by expressing only residues 1-327.