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C‐terminal hybrid mutant of Bacillus pumilus cyanide dihydratase dramatically enhances thermal stability and pH tolerance by reinforcing oligomerization
- Crum, M.A., Park, J.M., Sewell, B.T., Benedik, M.J.
- Journal of applied microbiology 2015 v.118 no.4 pp. 881-889
- Bacillus pumilus, Pseudomonas stutzeri, chimerism, gel chromatography, gels, hybrids, hydrogen cyanide, mutants, nitrilase, pH, protein quaternary structure, proteins, thermal stability
- AIMS: 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∆303, 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‐ₛₜᵤₜexhibited 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.