Browsing by Author "Woodward, J D"

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    Factors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1
    (2017) Mulelu, Andani Errol; Sewell, Bryan Trevor; Woodward, J D
    The cyanide dihydratase enzyme from Bacillus pumilus C1 (CynDₚᵤₘ) is a member of the nitrilase superfamily and is known to specifically catalyse the conversion of cyanide into formic acid and ammonia. This enzyme is a good candidate for bioremediation of cyanide waste but the high alkaline pH of the cyanide waste water poses a problem in that it inactivates the wild type enzyme and therefore improvement of stability is required in order to synthesize an effective enzyme. Over the pH range of 6–8 the enzyme exists as short 18-subunit spirals which associate to form long, more stable helical fibres at pH 5.4. The reason for this pH dependent transition is not fully understood but it is hypothesized to be due to changes in the charge of histidine residues. The aim of this project is to obtain a high resolution structure of CynDₚᵤₘ, relate this to its function, and investigate the role of the histidines in oligomerisation with aid of the structure. Using Cryo-electron microscopy techniques a three dimensional reconstruction structure of purified CynDₚᵤₘ was obtained at a resolution of ~5Å. By flexibly fitting a CynDₚᵤₘ homology model into this high resolution structure we were able to identify amino acid residues involved in oligomerisation and stability as well as the role of the histidines, with aid from additional mutagenesis studies. Interactions at the C-interfacial region were shown to play the most crucial role in oligomerisation and included the His71-Asp275 and Arg67-Asp275 interactions. Mutations at His128, His184, His241 and His285 were shown to affect the oligomerisation of the enzyme by indirectly disrupting interactions at the interfacial regions. The Q86R+H305K+H308K+H323K mutations were shown to increase the stability of the CynDₚᵤₘ by introducing a stronger arginine-arginine interaction at the D interfacial region and a new strong interaction at the C-terminal region.
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    Helical reconstruction of Mycobacterium smegmatis Mycothiol S-conjugate amidase filaments
    (2017) Burgess, Jeremy Gareth; Sewell, Bryan Trevor; Weber, Brandon W; Woodward, J D
    The metabolic pathway of mycothiol (MSH) is a major cellular defence against oxidative stress, and several antibiotics for mycobacteria, including Mycobacterium tuberculosis. The central enzyme used in the clearance of electrophilic toxins is Mycothiol S-conjugate amidase (Mca). Mca is similar to a biosynthetic enzyme MshB, which has partial overlapping substrate activity and is the closest homologue to Mca with a known structure. The basis for the substrate specificity differences in Mca and MshB is not well understood. Several regions of low sequence similarity between MshB and Mca are contained within an active site pocket, and these may affect the observed substrate preferences. However, these regions cannot be modelled in Mca with confidence, which makes it essential to obtain a structure of Mca experimentally. Mca is also a potential drug target, and a structure of Mca would enhance the rational design of inhibitors against the enzyme. A search for crystalline forms of MsMca (Mycobacterium smegmatis Mca) led to the discovery of regular filaments, which showed helical order. Helical symmetry was estimated using power spectra from single filaments. The number of potential symmetry solutions was reduced using phase information from Fourier transforms of single filaments. Three possible solutions to the helical symmetry were suggested, two of which converged on the same symmetry parameters using Iterative Helical Real-Space Reconstruction. The first solution had a selection rule of l = 18m + n, and the second l = 20m + n. Reconstructions made from the predicted helical symmetries were compared in their power spectra and through rigid-body fitting with an atomic model of MsMca. The first reconstruction, with a final symmetry of Δφ = 20.05o and Δz = 10.27 Å, better matched the predicted helical symmetry than did the second reconstruction. However, rigid-body fitting did not indicate either reconstruction as being superior. Following this, the second reconstruction was improved using a number of additional techniques to those used in the initial reconstruction. These included the use of the fortuitous 3-fold cyclic symmetry, the removal of double-walled filaments, use of a cut-off filter for images with low correlation to projections of the 3D reconstruction, and use of a layer-line filter to reduce the noise in the images. These were used individually, then in a single reconstruction, to improve the and agreement between the predicted helical symmetry and that obtained from the reconstruction. Several of the improved reconstructions were used via rigid-body fitting to assess the favoured handedness of the filament through examination of the major interfaces between subunits. These suggest that the 3-start helix is right-handed. Future work would be to determine the handedness of the filament using alternative techniques, such as metal-shadowing. This work provides a springboard for high resolution cryo-electron microscopy, to determine a high-resolution structure of MsMca, which will enable rational inhibitor design and give the basis for the different substrate specificity in Mca and MshB.
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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.
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    Three-dimensional reconstruction of biological macromolecular complexes from in-lens scanning electron micrographs
    (Wiley, 2009) Woodward, J D; Wepf, R; Sewell, B T
    Two helical samples: F-actin and the bacteriophage T4 tail sheath were reconstructed in three dimensions from contrast enhanced (rotational shadowing and negatively stained) in-lens cryo-field emission scanning electron micrographs, using the iterative real-space helical reconstruction method. The F-actin--and bacteriophage T4 reconstructions compare favourably to an atomic model refined against fibre diffraction data and a cryo-electron microscopy reconstruction, respectively. These results show that single-particle methods, developed for macromolecules imaged in the transmission electron microscope can be applied to cryo-field emission scanning electron micrographs data with appropriate symmetry.
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    Tomography of asymmetric bulk specimens imaged by scanning electron microscopy
    (Elsevier, 2010) Woodward, J D; Sewell, B T
    The scanning electron microscope produces nanometer-resolution surface images of biological samples preserved in a life-like state. Extracting three-dimensional information from these two-dimensional images has been the subject of long and ongoing research. We present here a general method and theoretical basis for reconstructing the surfaces of SEM specimens imaged from multiple directions by back-projection. The resulting reconstructions are faithful representations of the original specimen geometry, even when the input images are blurred and have low signal-to-noise ratio.