Browsing by Subject "Structural Biology"

Now showing 1 - 4 of 4
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    Open Access
    The crystal structure of an aliphatic amidase from Geobacillus pallidus RAPc8
    (2007) Kimani, Serah; Sewell, Bryan Trevor; Sayed, Muhamed
    Amidases are a group of carbon-nitrogen hydrolysing enzymes that catalyze the conversion of amides to corresponding carboxylic acids and ammonia. These enzymes are of great interest in synthetic industries where they are used for mass production of acidic products. Aliphatic amidase from Geobacillus pallidus RAPcS (RAPcS amidase), which belongs to the nitrilase superfamily of enzymes, has recently been characterised biochemically. It shows both amide hydrolysis and acyl transfer activities, and also exhibits stereo selectivity for some enantiomeric substrates. This enzyme can therefore be exploited in large-scale production of enantio-pure compounds. Structural characterization of this amidase would yield insights into the basis of this substrate selectivity and activity. This would inform future experiments that aims at modifying this enzyme to alter its substrate specificity. This work presents structural characterization of RAPcS amidase. Gel filtration chromatography and electron microscopic analyses provided useful information on the quaternary structure of RAPcS amidase. Crystals were grown, and an X-ray diffraction dataset to 1.9 Å collected using an in-house X-ray source. The space group of this data was determined to be primitive cubic P4₂32, and the structure was solved by molecular replacement using the backbone of the hypothetical protein PH0642 from Pyrococcus horikoshii (PDB ID, Ij31) that had all non-identical side chains substituted with alanines, as a search probe. The molecular replacement rotational and translational searches were performed using PHASER. The model was rebuilt with PHENIX before refinement using REFMAC5. The final model was of high quality with minimal errors. RAPcS amidase is homohexameric in solution and has a four-layer α-β-β-α structural fold that highly resembles nitrilase superfamily enzymes. It has an extended C-terminal tail that is essential for strengthening the interacting dimer interfaces by participating in domain swapping. The active site pocket has Glu, Lys, Cys catalytic triad that is conserved in the nitrilase superfamily. The substrate binding pocket is small in size, explaining the specificity of this enzyme for short aliphatic amides. These findings have made steps towards understanding the catalytic mechanism, and the basis for substrate specificity in this enzyme. It has also provided useful information on the overall structure, as well as the structure of the active site, not only for RAPcS amidase but also for related enzymes, which will form the basis for designing future structural characterization work in the nitrilase-related amidases.
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    Open Access
    Helical structures of the cyanide degrading enzymes from Gloeocercospora sorghi and Bacillus pumilus providing insights into nitrilase quaternary interactions
    (2006) Scheffer, Margot Petra; Sewell, Bryan Trevor
    Includes bibliographical references.
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    Open Access
    The structure of testis angiotensin-converting enzyme (tACE-g13) in complex with the inhibitor RXPA380
    (2006) Chitapi, Itai; Sewell, Bryan Trevor; Sturrock, E D
    Angiotensin-converting enzyme (ACE), a zinc metalloprotease, is a key regulator of the mammalian renin-angiotensin system (RAS) Primarily, ACF is a dipeptidl peptidase which cleaves angiotensin I to produce angiotensin II, a potent vasoconstrictor. By the same enzymatic mechanism, ACE also inactivates the vasodilator bradykinin. The main overall effect of these actions is an increase in blood pressure. Several ACF inhibitors have been developed as drugs for the treatment of myocardial infarction, hypertension, kidney failure and heart failure.
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    Three-dimensional reconstruction of Heterocapsa circularisquama RNA virus by cryo-electron microscopy
    (2007) Miller, Jennifer Louise; Sewell, Bryan Trevor
    Heterocapsa circularisquama RNA virus is a non-enveloped icosahedral ssRNA virus infectious to the harmful bloom-forming dinoflagellate, H. circularisquama, and which is assumed to be the major natural agent controlling the host population. The viral capsid is constructed from a single gene product. Electron cryo-microscopy revealed that the virus has a diameter of 34 nm and T53 symmetry. The 180 quasi-equivalent monomers have an unusual arrangement in that each monomer contributes to a ‘bump’ on the surface of the protein. Though the capsid protein probably has the classic ‘jelly roll’ b-sandwich fold, this is a new packing arrangement and is distantly related to the other positive-sense ssRNA virus capsid proteins. The handedness of the structure has been determined by a novel method involving high resolution scanning electron microscopy of the negatively stained viruses and secondary electron detection.