OpenUCT :: Browsing by Author "Sewell, Bryan Trevor"

Browsing by Author "Sewell, Bryan Trevor"

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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.
Open Access
Crystal structure of the Large Type III Glutamine Synthetase from Bacteroides Fragilis
(2010) van Rooyen, Jason M; Sewell, Bryan Trevor; Abratt, Valerie Rose
Glutamine synthetases are one of the most ancient functioning enzymes in existence and these large oligomeric complexes are found in all extant forms of life where they play a critical role in nitrogen metabolism. Over the past five decades, extensive biochemical studies together with structural investigations have helped build a picture of the mechanism of functioning and regulation in the GSI and GSII families. The most divergent GSIII family, however, is poorly characterized and has only recently been recognized. Structural studies, using both cryo-EM and X-ray crystallography, were undertaken on the type III GS, GlnN, from the opportunistic human pathogen, Bacteroides fragilis, with a view to better understanding the GSIII family in the light of the known structure functionrelationships of the other GS enzymes, and to investigate the potential for the design of selective inhibitors against the divergent family. A low-resolution (16 Ã) reconstruction of GlnN was first determined by single particle cryo-EM and image processing. This structure revealed that GlnN was a double-ringed dodecamer with D6 symmetry and the arrangement of active sites within the hexameric rings closely matched the GSI structure. Following the design of a rapid purification protocol and improvements to the stability and solubility of GlnN, conditions were discovered for the production of diffraction quality inhibitor-bound crystals. A second better diffracting crystal form was also produced following proteolytic processing. The crystal structure of GlnN was solved to near atomic resolution (3.0 Ã) following phase extension of low-resolution SAD phases, taking into account the cryo-EM structure. The higher resolution of the crystal structure revealed that, surprisingly, the orientation of the hexameric rings in GlnN is inverted in comparison to other families. These results have raised interesting questions surrounding the mechanism and driving forces responsible for the evolution of quaternary structure in the GS enzymes and have suggested that the GSI and GSII structure arose following truncation of a large GSIII-like ancestor. Despite the differences in higher order assembly, the GlnN monomer displayed a high degree of similarity with the GSI and GSII structures in the core active site region, thus, suggesting a conservation of reaction mechanism. Structure-based multiple sequence alignment showed that the residues forming the nucleotide binding pocket are the least conserved in the GS superfamily, and several residue positions, which represent altered modes of ligand binding, were suggested as potential avenues for the design of selective inhibitors against GlnN.
Open Access
The development of a predictive autofocus algorithm using a general image formation model
(1995) Nicolls, Frederick C; De Jager, Gerhard; Sewell, Bryan Trevor
This were outlines the development of a general imaging model for use in autofocus, astigmatism correction, and resolution analysis. The model is based on the modulation transfer function of the imaging system in the presence of aberrations, in particular defocus. The extension of the model to include astigmatism is also included. The signals used are related to the ratios of the Fourier transforms of images captured under different operating conditions. Methods are developed for working with these signals in a consistent manner. The model described is then applied to the problem of autofocus. A general autofocus algorithm is presented and results given which reﬂect the predictive properties of this model. The imaging system used for the generation of results was a scanning electron microscope, although the conclusions should be valid across a far wider range of instruments. It is however the speciﬁc requirements of the SEM that make the generalisation presented here particularly useful.
Open Access
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.
Open Access
Genetic characterization of Rhodococcus rhodochrous ATCC BAA-870 with emphasis on nitrile hydrolysing enzymes
(2013) Frederick, Joni; Sewell, Bryan Trevor; Brady, D
Rhodococcus rhodochrous ATCC BAA-870 (BAA-870) had previously been isolated on selective media for enrichment of nitrile hydrolysing bacteria. The organism was found to have a wide substrate range, with activity against aliphatics, aromatics, and aryl aliphatics, and enantioselectivity towards beta substituted nitriles and beta amino nitriles, compounds that have potential applications in the pharmaceutical industry. This makes R. rhodochrous ATCC BAA-870 potentially a versatile biocatalyst for the synthesis of a broad range of compounds with amide and carboxylic acid groups that can be derived from structurally related nitrile precursors. The selectivity of biocatalysts allows for high product yields and better atom economy than nonselective chemical methods of performing this reaction, such as acid or base hydrolysis. In order to apply BAA-870 as a nitrile biocatalyst and to mine the organism for biotechnological uses, the genome was sequenced using Solexa technology and an Illumina Genome Analyzer. The Solexa sequencing output data was analysed using the Solexa Data Analysis Pipeline and a total of 5,643,967 reads, 36-bp in length, were obtained providing 4,273,289 unique sequences. The genome sequence data was assembled using the software Edena, Velvet, and Staden. The best assembly data set was then annotated automatically using dCAS and BASys. Further matepaired sequencing, contracted to the company BaseClear® BV in Leiden, the Netherlands, was performed in order to improve the completeness of the data. The scaffolded Illumina and mate-paired sequences were further assembled and annotated using BASys. BAA-870 has a GC content of 65% and contains 6997 predicted protein-coding sequences (CDS). Of this, 54% encodes previously identified proteins of unknown function. The completed 5.83 Mb genome (with a sequencing coverage of 135 X) was submitted to the NCBI Genome data bank with accession number PRJNA78009. The genome sequence of R. rhodochrous ATCC BAA-870 is the seventh rhodococcal genome to be submitted to the NCBI and the first R. rhodochrous subtype to be sequenced. An analysis of the genome for nitrile
Open Access
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.
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.
Open Access
Investigation of the determinants of thermal stability of the nitrile hydratase from Geobacillus pallidus RAPc8
(2016) Kianja, John Maina; Sewell, Bryan Trevor
The mechanisms of thermal stability have been a long studied subject for many years with the aim of enhancing thermal stability of protein molecules to enhance their application in industry. The nitrile hydratases group of enzymes catalyse the hydrolysis of nitriles to amides using an exothermic catalytic mechanism. Understanding and applying specific amino acid residue mutations at specific regions in protein structures has been important for engineering of thermal stability into these often tetrameric thermolabile nitrile hydratases currently used in industry globally. At the near atomic level, the interatomic interaction(s) between specific amino acid residues governs the structure and function of nitrile hydratases. This study investigated several possible interactions responsible for conferring thermal stability to several thermostability-enhanced nitrile hydratase composite mutants generated from the wild type Geobacillus pallidus RAPc8 nitrile hydratase (NHase), namely: L103S+Y127N+F36L+D4G, M43K+T150A+S169R and D96E+D167V+M188V each labelled as 9E, 9C and 8C respectively. The composite mutants were previously developed using error-prone PCR of the wild type nitrile hydratase genes coding for the alpha and beta subunits from Geobacillus pallidus RAPc8. These composite mutants presented an opportunity to understand intramolecular thermostabilising mechanisms in this nitrile hydratase. Each individual mutation found in the composite mutants, was separately introduced into the DNA coding for the Geobacillus pallidus RAPc8 NHase by site directed mutagenesis. These individual mutants were over-expressed from E. coli and purified for further study. Using activity assays and protein melting curves, their individual thermal stability contributions were determined and represented as the difference in free energy of thermal unfolding (change in Gibbs free energy) of the single and composite mutants relative to the wild type nitrile hydratase. The measured residual activity following thermal inactivation was used together with the Arrhenius equation and a three parameter non-linear fit to determine the free energy of thermal unfolding. The change in Gibbs free energy resulting from each thermostabilising mechanism coupled to the analysis of their crystal structures was used to suggest the contributing mechanisms. This study found that intersubunit interactions through hydrogen bonds and salt bridges are especially important for contributing towards thermal stability of tetrameric nitrile hydratases. Hydrophobic interaction through the formation of a water shell around hydrophobic side-chains and packing of hydrophobic side-chains was also observed to contribute to thermal stability. These results suggest a path towards rational design and engineering of thermostabilising mutations into nitrile hydratases. Increased thermostability would improve their large scale application in industry by allowing these enzymes to be more active for longer at higher temperatures and decrease the cost of amide production.
Open Access
A new crystal form of MshB from Mycobacterium tuberculosis with glycerol and acetate in the active site suggests the catalytic mechanism.
(International Union of Crystallography, 2012) Broadley, Simon Gareth; Gumbart, James Conrad; Weber, Brandon William; Marakalala, Mohlopheni Jackson; Steenkamp, Daniel Jacobus; Sewell, Bryan Trevor
MshB, a zinc-based deacetylase, catalyses a step in the mycothiol biosynthetic pathway that involves the deacetylation of 1-O-(2-acetamido-2-deoxy--d-glucopyranosyl)-dmyo-inositol (GlcNAc-Ins), via cleavage of an amide bond, to 1-O-(2-amino-2-deoxy--d-glucopyranosyl)-d-myo-inositol (GlcN-Ins) and acetate. In this study, MshB was expressed, purified and crystallized. A new crystal form was encountered in 0.1 M sodium acetate, 0.2 M ammonium sulfate, 25% PEG 4000 pH 4.6. The crystals diffracted to 1.95 A˚ resolution and the resulting electron-density map revealed glycerol and the reaction product, acetate, in the active site. These ligands enabled the natural substrate GlcNAc-Ins to be modelled in the active site with some certainty. One acetate O atom is hydrogen bonded to Tyr142 and is located 2.5 A˚ from the catalytic zinc. The other acetate O atom is located 2.7 A˚ from a carboxylate O atom of Asp15. This configuration strongly suggests that Asp15 acts both as a general base catalyst in the nucleophilic attack of water on the amide carbonyl C atom and in its protonated form acts as a general acid to protonate the amide N atom. The configuration of Tyr142 differs from that observed previously in crystal structures of MshB (PDB entries 1q74 and 1q7t) and its location provides direct structural support for recently published biochemical and mutational studies suggesting that this residue is involved in a conformational change on substrate binding and contributes to the oxyanion hole that stabilizes the tetrahedral intermediate.
Open Access
Physical mapping of an early sea urchin gene battery from Parenchinus angulosus
(1988) Lawson, T N; Sewell, Bryan Trevor; Von Holt, Claus
The aim of this project was to characterise an early histone gene battery isolated from Parenchinus angulosus. An early histone gene battery (named H27) which was believed to have been isolated from Parenchinus angulosus, appeared by restriction enzyme mapping and partial sequencing to be identical to H22, an early histone gene battery isolated from Psammechinus miliaris. (This latter gene was obtained from M. Birnstiel.) This was further confirmed by electron microscopy, and proved to be a convenient testing ground for the electron microscopic techniques of denaturation mapping and heteroduplex anlysis. Another gene battery (named SU1) isolated fromParenchinus angulosus, was then characterised using the techniques developed whilst studying H27. The restriction enzyme map of this clone is different to that of H22, indicating that differences do indeed exist between these two early histone gene batteries. SU1 also showed the expected order of the five histone genes, as determined by hybridization against the coding regions of H22. The denaturation map of SU1 showed AT rich spacer regions and GC rich coding regions. Heteroduplex analysis indicated that the spacer regions between the Hl and H2A, the H2A and H3, and the H3 and the H2B gene coding areas are essentially nonhomologous. The H4 structural gene and corresponding spacer regions were not included in this analysis. Because it is known that all the five histones are coded for on the same strand of DNA in H22, and that each of the genes is transcribed in the same direction, it follows that, the same holds for, at least, the Hl, H2A, H3 and H2B genes of SU1.
Open Access
Preparation of a self-contained NADH co-factor recycling particle system
(2010) Twala, Busisiwe V; Sewell, Bryan Trevor; Jordaan, Justin
Oxidoreductases are interesting enzymes with potential applications in a number of different industries such as the textile, food and feed, chemical and biomedical industries. Oxidoreductases require the use of co-factors. These small molecules are relatively expensive and are required in stoichiometric amounts for their enzymatic reaction; this negatively impacts the economic viability of their potential applications. Several methods have been developed to counteract this problem, the most preferred of which is the enzymatic co-factor recycling method. A few methods for the co-immobilisation of enzymes and co-factors have been developed. These systems are of interest as they offer the advantages of recycling the enzymes together with the co-factor, thereby enabling re-use. The immobilisation of enzymes also provides a platform for improving their stability, activity, specificity and selectivity. Since glucose dehydrogenase (GDH) and NADH oxidase, are industrially relevant co-factor recycling enzymes for NAD(P)H and NAD+ respectively, characterisation of their immobilisation is of interest. The current work describes the use of the proprietary particle technology, termed ReSyn™, for the construction of a self-contained co-factor recycling system. The research included the optimisation of immobilisation for the individual enzymes, followed by the co-immobilisation with subsequent co-factor entrapment. The immobilised enzymes displayed improved thermal and pH stability compared to the non-immobilised enzymes. Immobilised GDH also displayed increased activity over the acidic range when compared to free GDH. The system was shown to be capable of recycling NADH/NAD+ up to at least 142 times with a specific activity of 10.18 U.mg¯1. The system was recovered and recycled with a 77% activity efficiency indicating recovery of the system and reusability. Preparation of a functional self-contained co-factor recycling system was demonstrated consisting of the biological components NADH oxidase and glucose dehydrogenase, immobilised on a polyethylenimine support with entrapped cofactor. This serves as proof-of-principle for the construction of derivative systems that could be used for the development of applications such as efficient biosynthesis, novel biosensors, diagnostic and therapeutic systems.
Open Access
Quaternary structures of the cyanide dihydratases of Bacillus pumilus C1 and Pseudomonas stutzeri AK61
(2003) Berman, Mark Nicholas; Sewell, Bryan Trevor; Meyers, Paul
Nitrilases catalyse the conversion of a nitrile to its corresponding acid and ammonia by the addition of two water molecules. Cyanide dihydratases, a subgroup of nitrilases, specifically hydrolyse cyanide to formic acid and ammonia. Nitrilases are found in a diverse collection organisms that includes plants, bacteria and fungi. They form one branch a superfamily of structurally related enzymes that are believed to have in common a unique cys-glu-Iys catalytic triad. Many nitrilases exiat as a large molecular weight oligomers of more than 300kDa. In the current study the structures of two cyanide dihydratases, from Pseudomonas stutzeri AK61 and Bacillus pumilus Cl, have solved at a resolution 2.9nm and 32nm respectively by single particle reconstruction from electron micrographs of enzyme particles stained in uranyl acetate. Each enzyme consists of a spiral structure of well-defined length. It is proposed that this arrangement of subunits occurs in many other nitrilases and that a number of unexplained observations in the literature can reconciled by this model.
Open Access
The reconstitution of the histone octamer
(1987) Greyling, H J; Von Holt, Claus; Sewell, Bryan Trevor
This thesis describes methodology for the reconstitution of the chicken erythrocyte octamer from acid-denatured histones or the natural H3-H4 tetramer and H2A-H2B dimers. Oligomeric properties of reconstituted octamers were elucidated during column chromatographic and chemical cross-linking studies. The conformational identity of the natural and reconstituted octamers was demonstrated by the ability of all preparations to crystallise as helical octamer tubes. The application of the reconstitution methodology in addressing fundamental problems of chromatin research, was demonstrated during subsequent studies, namely (i) The reconstitution of hybrid histone octamers containing a structural variant of a specific histone. These studies were undertaken to study the effect on histone-histone interactions in hybrid octamers of which erythrocyte H2B was substituted for by sea urchin sperm H2B(l) or erythrocyte H3 and H4 were substituted for by dethiolated H3 and sea urchin sperm H4 respectively. (ii) The reconstitution of an octamer suitable for the sitespecific derivatisation of a specific histone, or covalently labelled with aurothiomalate in a specific histone complex. These studies were concluded to represent general labelling strategies which may be of use in crystallographic or physico-chemical studies of nucleosome structure.
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.
Open Access
Substrate binding to MshB, a zinc-dependent deacetylase in the mycothiol biosynthetic pathway of Mycobacterium tuberculosis
(2012) Broadley, Simon Gareth; Sewell, Bryan Trevor
New drugs are needed to combat the ever-increasing incidence of drug resistant tuberculosis. Eukaryotes and Gram negative bacteria make use of a low molecular weight thiol-containing peptide, glutathione (GSH), in their defense against toxins and oxidative stress. In mycobacteria a carbohydrate-based thiol, mycothiol (MSH), is used instead. MshB catalyses the third step in the mycothiol biosynthetic pathway which involves the deacetylation of 1-O-(2-acetamido-2-deoxy-α-D-glucopyranosyl)-D-myo-inositol (GlcNAcIns), via cleavage of an amide bond, to 1-O-(2-amino-2-deoxy-α-D-glucopyranosyl)-D-myoinositol (GlcN-Ins) and acetate. MshB is a homolog of another zinc-dependent amidase, mycothiol S-conjugate amidase (Mca), which recycles GlcNAc-Ins by cleaving the similarly located amide bond after mycothiol has reacted with an electrophile. The enzymes have overlapping enzyme activity and their products feed into the same step of the mycothiol synthetic pathway. Structure-based drug design could potentially be used to develop leads that could inhibit both enzymes simultaneously. This study was motivated by the need to visualise a putative inhibitor bound to MshB. An attempt was made to co-crystallise VU5 and MshB. VU5, which comprises plumbagin tethered to a thiophenylglycoside by five methylene carbons, was found by Gammon et al. (2010) to be an inhibitor of MshB and Mca. Diffraction quality crystals were grown and data were collected at the ESRF synchrotron facility. The crystal structure was solved by molecular replacement and most notably, glycerol and acetate (a product of the natural reaction) was found bound in the active site. This allowed a greater understanding regarding important residues involved in substrate binding and the catalytic mechanism. The findings presented here provide some insight into the substrate binding mechanism and support a recent publication that infers that Tyr-142 plays an important role in the catalytic mechanism, as well as suggesting that VU5 may behave as a substrate and that DTT may act as an inhibitor.
Open Access
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.

Browsing by Author "Sewell, Bryan Trevor"

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