Browsing by Author "Weber, Brandon W"

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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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    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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    Identification of a collagen type I adhesin of Bacteroides fragilis
    (Public Library of Science, 2014) Galvão, Bruna P G V; Weber, Brandon W; Rafudeen, Mohamed S; Ferreira, Eliane O; Patrick, Sheila; Abratt, Valerie R
    Bacteroides fragilis is an opportunistic pathogen which can cause life threatening infections in humans and animals. The ability to adhere to components of the extracellular matrix, including collagen, is related to bacterial host colonisation. Collagen Far Western analysis of the B. fragilis outer membrane protein (OMP) fraction revealed the presence two collagen adhesin bands of ∼31 and ∼34 kDa. The collagen adhesins in the OMP fraction were separated and isolated by two-dimensional SDS-PAGE and also purified by collagen affinity chromatography. The collagen binding proteins isolated by both these independent methods were subjected to tandem mass spectroscopy for peptide identification and matched to a single hypothetical protein encoded by B. fragilis NCTC 9343 (BF0586), conserved in YCH46 (BF0662) and 638R (BF0633) and which is designated in this study as cbp1 (collagen binding protein). Functionality of the protein was confirmed by targeted insertional mutagenesis of the cbp1 gene in B. fragilis GSH18 which resulted in the specific loss of both the ∼31 kDa and the ∼34 kDa adhesin bands. Purified his-tagged Cbp1, expressed in a B. fragilis wild-type and a glycosylation deficient mutant, confirmed that the cbp1 gene encoded the observed collagen adhesin, and showed that the 34 kDa band represents a glycosylated version of the ∼31 kDa protein. Glycosylation did not appear to be required for binding collagen. This study is the first to report the presence of collagen type I adhesin proteins in B. fragilis and to functionally identify a gene encoding a collagen binding protein.
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    The mechanism of the amidases: mutating the glutamate adjacent to the catalytic triad inactivates the enzyme due to substrate mispositioning
    (American Society for Biochemistry and Molecular Biology, 2013) Weber, Brandon W; Kimani, Serah W; Varsani, Arvind; Cowan, Donald A; Hunter, Roger; Venter, Gerhard A; Gumbart, James C; Sewell, Trevor B
    All known nitrilase superfamily amidase and carbamoylase structures have an additional glutamate thatis hydrogen bonded to the catalytic lysine in addition to the Glu, Lys, Cys “catalytic triad.” In the amidase from Geobacillus pallidus, mutating this glutamate (Glu-142) to a leucine or aspartate renders the enzyme inactive. X-ray crystal structure determination shows that the structural integrity of the enzymeismaintained despite themutation with the catalytic cysteine (Cys-166), lysine (Lys-134), and glutamate (Glu- 59)in positions similar to those of the wild-type enzyme. In the case of the E142L mutant, a chloride ion is located in the position occupied by Glu-142 O 1 in the wild-type enzyme andinteracts with the active site lysine. In the case of the E142D mutant, this site is occupied by Asp-142 O1.In neither case is an atom located at the position of Glu-142 O 2 in the wild-type enzyme. The active site cysteine of the E142Lmutant was found to form aMichael adduct with acrylamide, which is a substrate of the wild-type enzyme, due to an interaction that places the double bond of the acrylamide rather than the amide carbonyl carbon adjacent to the active site cysteine. Our results demonstrate that in the wild-type active site a crucial role is played by the hydrogen bond between Glu-142 O 2 and the substrate amino groupin positioning the substrate with the correct stereoelectronic alignment to enable the nucleophilic attack on the carbonyl carbon by the catalytic cysteine.
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    Post-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. Trevor
    Nitrilases 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.