Browsing by Subject "Cyanide hydratase"

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    The cyanide hydratase from Neurospora crassa forms a helix which has a dimeric repeat.
    (Springer Verlag, 2009) Dent, Kyle C; Weber, Brandon W; Benedik, Michael J; Sewell, Trevor B
    The fungal cyanide hydratases form a functionally specialized subset of the nitrilases which catalyze the hydrolysis of cyanide to formamide with high specificity. These hold great promise for the bioremediation of cyanide wastes. The low resolution (3.0 nm) three-dimensional reconstruction of negatively stained recombinant cyanide hydratase fibers from the saprophytic fungus Neurospora crassa by iterative helical real space reconstruction reveals that enzyme fibers display left-handed D1 S5.4 symmetry with a helical rise of 1.36 nm. This arrangement differs from previously characterized microbial nitrilases which demonstrate a structure built along similar principles but with a reduced helical twist. The cyanide hydratase assembly is stabilized by two dyadic interactions between dimers across the one-start helical groove. Docking of a homology-derived atomic model into the experimentally determined negative stain envelope suggests the location of charged residues which may form salt bridges and stabilize the helix
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    Genome mining of cyanide-degrading nitrilases from filamentous fungi
    (Springer, 2008) Basile, Lacy J; Wilson, Richard C; Sewell, B. Trevor; Benedik, Michael J
    A variety of fungal species are known to degrade cyanide through the action of cyanide hydratases, a specialized subset of nitrilases which hydrolyze cyanide to formamide. In this paper, we report on two previously unknown and uncharacterized cyanide hydratases from Neurospora crassa and Aspergillus nidulans. Recombinant forms of four cyanide hydratases from N. crassa, A. nidulans, Gibberella zeae, and Gloeocercospora sorghi were prepared after their genes were cloned with N-terminal hexahistidine purification tags, expressed in Escherichia coli, and purified using immobilized metal affinity chromatography. These enzymes were compared according to their relative specific activity, pH activity profiles, thermal stability, and ability to remediate cyanide contaminated waste water from silver and copper electroplating baths. Although all four were similar, the N. crassa cyanide hydratase (CHT) has the greatest thermal stability and widest pH range of >50% activity. N. crassa also demonstrated the highest rate of cyanide degradation in the presence of both heavy metals. The CHT of A. nidulans has the highest reaction rate of the four fungal nitrilases evaluated in this work. These data will help determine optimization procedures for the possible use of these enzymes in the bioremediation of cyanide-containing waste. Similar to known plant pathogenic fungi, both N. crassa and A. nidulans were induced to express CHT by growth in the presence of KCN.
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    Genome mining of cyanide-degrading nitrilases from filamentous fungi.
    (Springer Verlag, 2008) Basile, Lacy J; Willson, Richard C; Sewell, Trevor B; Benedik, Michael J
    A variety of fungal species are known to degrade cyanide through the action of cyanide hydratases, a specialized subset of nitrilases which hydrolyze cyanide to formamide. In this paper, we report on two previously unknown and uncharacterized cyanide hydratases from Neurospora crassa and Aspergillus nidulans. Recombinant forms of four cyanide hydratases from N. crassa, A. nidulans, Gibberella zeae, and Gloeocercospora sorghi were prepared after their genes were cloned with N-terminal hexahistidine purification tags, expressed in Escherichia coli, and purified using immobilized metal affinity chromatography. These enzymes were compared according to their relative specific activity, pH activity profiles, thermal stability, and ability to remediate cyanide contaminated waste water from silver and copper electroplating baths. Although all four were similar, the N. crassa cyanide hydratase (CHT) has the greatest thermal stability and widest pH range of >50% activity. N. crassa also demonstrated the highest rate of cyanide degradation in the presence of both heavy metals. The CHT of A. nidulans has the highest reaction rate of the four fungal nitrilases evaluated in this work. These data will help determine optimization procedures for the possible use of these enzymes in the bioremediation of cyanide-containing waste. Similar to known plant pathogenic fungi, both N. crassa and A. nidulans were induced to express CHT by growth in the presence of KCN.
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    Helical structure of unidirectionally shadowed metal replicas of cyanide hydratase from Gloeocercospora sorghi
    (Elsevier, 2008) Woodward, J D; Weber, B W; Scheffer, M P; Benedik, M J; Hoenger, A; Sewell, B T
    The helical filaments of the cyanide hydratase from Gloeocercospora sorghi have been reconstructed in three dimensions from freeze dried, unidirectionally shadowed specimens using iterative real-space helical reconstruction. The average power spectrum of all selected images has three clear reflections on different layer lines. The reconstruction is complicated by the fact that three possible indexing schemes are possible and reconstructions using the starting symmetries based on each of these indexing schemes converge on three-dimensional volumes which appear plausible. Because only one side is visible in shadowed specimens, it is necessary to examine the phases from a single filament by cryo-electron microscopy in order to make an unequivocal assignment of the symmetry. Because of the novel nature of the reconstruction method used here, conventional cryo-EM methods were also used to determine a second reconstruction, allowing us to make comparisons between the two. The filament is shown to have a left-handed one-start helix with D1 symmetry, 5.46 dimers per turn and a pitch of 7.15 nm. The reconstruction suggests the presence of an interaction across the groove not previously seen in nitrilase helical fibres.
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    Helical structure of unidirectionally shadowed metal replicas of cyanide hydratase from Gloeocercospora sorghi.
    (Elsevier, 2008) Woodward, J D; Weber, B W; Scheffer, M P; Benedik, M J; Hoenger A; Sewell, B T
    The helical filaments of the cyanide hydratase from Gloeocercospora sorghi have been reconstructed in three dimensions from freeze dried, unidirectionally shadowed specimens using iterative real-space helical reconstruction. The average power spectrum of all selected images has three clear reflections on different layer lines. The reconstruction is complicated by the fact that three possible indexing schemes are possible and reconstructions using the starting symmetries based on each of these indexing schemes converge on threedimensional volumes which appear plausible. Because only one side is visible in shadowed specimens, it is necessary to examine the phases from a single filament by cryo-electron microscopy in order to make an unequivocal assignment of the symmetry. Because of the novel nature of the reconstruction method used here, conventional cryo-EM methods were also used to determine a second reconstruction, allowing us to make comparisons between the two. The filament is shown to have a left-handed one-start helix with D1 symmetry, 5.46 dimers per turn and a pitch of 7.15 nm. The reconstruction suggests the presence of an interaction across the groove not previously seen in nitrilase helical fibres.