Browsing by Author "Mulelu, Andani E"

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    Engineering pH-tolerant mutants of a cyanide dihydratase
    (Springer Verlag, 2012) Wang, Lan; Watermeyer, Jean M; Mulelu, Andani E; Sewell, Trevor B; Benedik, Michael J
    Cyanide dihydratase is an enzyme in the nitrilase family capable of transforming cyanide to formate and ammonia. This reaction has been exploited for the bioremediation of cyanide in wastewater streams, but extending the pH operating range of the enzyme would improve its utility. In this work, we describe mutants of Bacillus pumilus C1 cyanide dihydratase (CynDpum) with improved activity at higher pH. Error-prone PCR was used to construct a library of CynDpum mutants, and a high-throughput screening system was developed to screen the library for improved activity at pH 10. Two mutant alleles were identified that allowed cells to degrade cyanide in solutions at pH 10, whereas the wild-type was inactive above pH 9. The mutant alleles each encoded three different amino acid substitutions, but for one of those, a single change, E327G, accounted for the phenotype. The purified proteins containing multiple mutations were five times more active than the wild-type enzyme at pH 9, but all purified enzymes lost activity at pH 10. The mutation Q86R resulted in the formation of significantly longer fibers at low pH, and both E327G and Q86R contributed to the persistence of active oligomeric assemblies at pH 9. In addition, the mutant enzymes proved to be more thermostable than the wild type, suggesting improved physical stability rather than any change in chemistry accounts for their increased pH tolerance.
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    Probing C-terminal interactions of the Pseudomonas stutzeri cyanide-degrading CynD protein
    (Springer Verlag, 2015) Crum, Mary Abou-Nader; Park, Jason M; Mulelu, Andani E; Sewell, Trevor B; Benedik, Michael J
    The cyanide dihydratases from Bacillus pumilus and Pseudomonas stutzeri share high amino acid sequence similarity throughout except for their highly divergent C-termini. However, deletion or exchange of the C-termini had different effects upon each enzyme. Here we extended previous studies and investigated how the C-terminus affects the activity and stability of three nitrilases, the cyanide dihydratases from B. pumilus (CynDpum) and P. stutzeri (CynDstut) and the cyanide hydratase from Neurospora crassa. Enzymes in which the C-terminal residues were deleted decreased in both activity and thermostability with increasing deletion lengths. However, CynDstut was more sensitive to such truncation than the other two enzymes. A domain of the P. stutzeri CynDstut C-terminus not found in the other enzymes, 306GERDST311, was shown to be necessary for functionality and explains the inactivity of the previously described CynDstut-pum hybrid. This suggests that the B. pumilus C-terminus, which lacks this motif, may have specific interactions elsewhere in the protein, preventing it from acting in trans on a heterologous CynD protein. We identify the dimerization interface A-surface region 195–206 (A2) from CynDpum as this interaction site. However, this A2 region did not rescue activity in C-terminally truncated CynDstutΔ302 or enhance the activity of full-length CynDstut and therefore does not act as a general stability motif.