Browsing by Author "Sewell, Trevor B"

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    Characterization of domain-selective inhibitor binding in angiotensin-converting enzyme using a novel derivative of lisinopril
    (Portland Press, 2010) Watermeyer, Jean M; Kröger, Wendy L; O'Neill, Hester G; Sewell, Trevor B; Sturrock, Edward D
    Human ACE (angiotensin-converting enzyme) (EC 3.4.15.1) is an important drug target because of its role in the regulation of blood pressure via the renin–angiotensin–aldosterone system. Somatic ACE comprises two homologous domains, the differing substrate preferences of which present a new avenue for domainselective inhibitor design. We have co-crystallized lisW-S, a Cdomain-selective derivative of the drug lisinopril, with human testis ACE and determined a structure using X-ray crystallography to a resolution of 2.30 Å (1 Å = 0.1 nm). In this structure, lisW-S is seen to have a similar binding mode to its parent compound lisinopril, but the P2 tryptophan moiety takes a different conformation to that seen in other inhibitors having a tryptophan residue in this position. We have examined further the domain-specific interactions of this inhibitor by mutating Cdomain-specific active-site residues to their N domain equivalents, then assessing the effect of the mutation on inhibition by lisWS using a fluorescence-based assay. Kinetics analysis shows a 258-fold domain-selectivity that is largely due to the co-operative effect of C-domain-specific residues in the S2 subsite. The high affinity and selectivity of this inhibitor make it a good lead candidate for cardiovascular drug development.
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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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    Engineering pH-tolerant mutants of a cyanide dihydratase
    (Springer Verlag, 2012) Wang, Lan; Watermeyer, Jean M; Mulelu, Andani E; Sewell, Trevor B; Benedik, Michael J
    Cyanide dihydratase is an enzyme in the nitrilase family capable of transforming cyanide to formate and ammonia. This reaction has been exploited for the bioremediation of cyanide in wastewater streams, but extending the pH operating range of the enzyme would improve its utility. In this work, we describe mutants of Bacillus pumilus C1 cyanide dihydratase (CynDpum) with improved activity at higher pH. Error-prone PCR was used to construct a library of CynDpum mutants, and a high-throughput screening system was developed to screen the library for improved activity at pH 10. Two mutant alleles were identified that allowed cells to degrade cyanide in solutions at pH 10, whereas the wild-type was inactive above pH 9. The mutant alleles each encoded three different amino acid substitutions, but for one of those, a single change, E327G, accounted for the phenotype. The purified proteins containing multiple mutations were five times more active than the wild-type enzyme at pH 9, but all purified enzymes lost activity at pH 10. The mutation Q86R resulted in the formation of significantly longer fibers at low pH, and both E327G and Q86R contributed to the persistence of active oligomeric assemblies at pH 9. In addition, the mutant enzymes proved to be more thermostable than the wild type, suggesting improved physical stability rather than any change in chemistry accounts for their increased pH tolerance.
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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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    Immobilisation and characterisation of biocatalytic co-factor recycling enzymes, glucose dehydrogenase and NADH oxidase, on aldehyde functional ReSyn™ polymer microspheres
    (Elsevier, 2012) Twala, Busisiwe V; Sewell, Trevor B; Jordaan, Justin
    The use of enzymes in industrial applications is limited by their instability, cost and difficulty in their recovery and re-use. Immobilisation is a technique which has been shown to alleviate these limitations in biocatalysis. Here we describe the immobilisation of two biocatalytically relevant co-factor recycling enzymes, glucose dehydrogenase (GDH) and NADH oxidase (NOD) on aldehyde functional ReSyn™ polymer microspheres with varying functional group densities. The successful immobilisation of the enzymes on this new high capacity microsphere technology resulted in the maintenance of activity of ∼40% for GDH and a maximum of 15.4% for NOD. The microsphere variant with highest functional group density of ∼3500 μmol g−1 displayed the highest specific activity for the immobilisation of both enzymes at 33.22 U mg−1 and 6.75 U mg−1 for GDH and NOD with respective loading capacities of 51% (0.51 mg mg−1) and 129% (1.29 mg mg−1). The immobilised GDH further displayed improved activity in the acidic pH range. Both enzymes displayed improved pH and thermal stability with the most pronounced thermal stability for GDH displayed on ReSyn™ A during temperature incubation at 65 °C with a 13.59 fold increase, and NOD with a 2.25-fold improvement at 45 °C on the same microsphere variant. An important finding is the suitability of the microspheres for stabilisation of the multimeric protein GDH.
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    Immobilisation and characterisation of biocatalytic co-factor recycling enzymes, glucose dehydrogenase and NADH oxidase, on aldehyde functional ReSyn™ polymer microspheres.
    (Elsevier, 2012) Twalaa, Busisiwe V; Sewell, Trevor B; Jordaan, Justin
    The use of enzymes in industrial applications is limited by their instability, cost and difficulty in their recovery and re-use. Immobilisation is a technique which has been shown to alleviate these limitations in biocatalysis. Here we describe the immobilisation of two biocatalytically relevant co-factor recycling enzymes, glucose dehydrogenase (GDH) and NADH oxidase (NOD) on aldehyde functional ReSynTM polymer microspheres with varying functional group densities. The successful immobilisation ofthe enzymes on this new high capacity microsphere technology resulted in the maintenance of activity of ∼40% for GDH and a maximum of 15.4% for NOD. The microsphere variant with highest functional group density of ∼3500 mol g−1 displayed the highest specific activity for the immobilisation of both enzymes at 33.22 U mg−1 and 6.75 U mg−1 for GDH and NOD with respective loading capacities of 51% (0.51 mg mg−1) and 129% (1.29 mg mg−1). The immobilised GDH further displayed improved activity in the acidic pH range. Both enzymes displayed improved pH and thermal stability with the most pronounced thermal stability for GDH displayed on ReSynTM A during temperature incubation at 65 ◦C with a 13.59 fold increase, and NOD with a 2.25-fold improvement at 45 ◦C on the same microsphere variant. An important finding is the suitability of the microspheres for stabilisation of the multimeric protein GDH.
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    Open Access
    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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    Open Access
    The quaternary structure of the amidase from Geobacillus pallidus RAPc8 is revealed by its crystal packing
    (International Union of Crystallography, 2006) Agarkar, Vinod B; Kimani, Serah W; Cowan, Donald A; Sayed, Muhammed F R; Sewell, Trevor B
    The amidase from Geobacillus pallidus RAPc8, a moderate thermophile, is a member of the nitrilase enzyme superfamily. It converts amides to the corresponding acids and ammonia and has application as an industrial catalyst. RAPc8 amidase has been cloned and functionally expressed in Escherichia coli and has been purified by heat treatment and a number of chromatographic steps. The enzyme was crystallized using the hanging-drop vapour-diffusion method. Crystals produced in the presence of 1.2 M sodium citrate, 400 mM NaCl, 100 mM sodium acetate pH 5.6 were selected for X-ray diffraction studies. A data set having acceptable statistics to 1.96 A˚ resolution was collected under cryoconditions using an in-house X-ray source. The space group was determined to be primitive cubic P4232, with unit-cell parameter a = 130.49 ( 0.05) A˚ . The structure was solved by molecular replacement using the backbone of the hypothetical protein PH0642 from Pyrococcus horikoshii (PDB code 1j31) with all non-identical side chains substituted with alanine as a probe. There is one subunit per asymmetric unit. The subunits are packed as trimers of dimers with D3 point-group symmetry around the threefold axis in such a way that the dimer interface seen in the homologues is preserved.
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    Structure of testis ACE glycosylation mutants and evidence for conserved domain movement
    (American Chemical Society, 2006) Watermeyer, Jean M; Sewell, Trevor B; Schwager, Sylva L; Natesh, Ramanathan; Corradi, Hazel R; Acharya, Ravi K; Sturrock, Edward D
    Human angiotensin-converting enzyme is an important drug target for which little structural information has been available until recent years. The slow progress in obtaining a crystal structure was due to the problem of surface glycosylation, a difficulty that has thus far been overcome by the use of a glucosidase-1 inhibitor in the tissue culture medium. However, the prohibitive cost of these inhibitors and incomplete glucosidase inhibition makes alternative routes to minimizing the N-glycan heterogeneity desirable. Here, glycosylation in the testis isoform (tACE) has been reduced by Asn-Gln point mutations at N-glycosylation sites, and the crystal structures of mutants having two and four intact sites have been solved to 2.0 Å and 2.8 Å, respectively. Both mutants show close structural identity with the wild-type. A hinge mechanism is proposed for substrate entry into the active cleft, based on homology to human ACE2 at the levels of sequence and flexibility. This is supported by normal-mode analysis that reveals intrinsic flexibility about the active site of tACE. Subdomain II, containing bound chloride and zinc ions, is found to have greater stability than subdomain I in the structures of three ACE homologues. Crystallizable glycosylation mutants open up new possibilities for cocrystallization studies to aid the design of novel ACE inhibitors.
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    Open Access
    The structure of testis angiotensin-converting enzyme in complex with the C domain-specific inhibitor RXPA380.
    (American Chemical Society, 2007) Corradi, Hazel R; Chitapi, Itai; Sewell, Trevor B; Georgiadis, Dimitris; Dive, Vincent; Sturrock, Edward D; Acharya, Ravi K
    Angiotensin I-converting enzyme (ACE) is central to the regulation of the renin-angiotensin system and is a key therapeutic target for combating hypertension and related cardiovascular diseases. Currently available drugs bind both active sites of its two homologous domains, although it is now understood that these domains function differently in vivo. The recently solved crystal structures of both domains (N and C) open the door to new domain-specific inhibitor design, taking advantage of the differences between these two large active sites. Here we present the first crystal structure at a resolution of 2.25 Å of testis ACE (identical to the C domain of somatic ACE) with the highly C-domain-specific phosphinic inhibitor, RXPA380. Testis ACE retains the same conformation as seen in previously determined inhibitor complexes, but the RXPA380 central backbone conformation is more similar to that seen for the inhibitor captopril than enalaprilat. The RXPA380 molecule occupies more subsites of the testis ACE active site than the previously determined inhibitors and possesses bulky moieties that extend into the S2′ and S2 subsites. Thus the high affinity of RXPA380 for the testis ACE/somatic ACE C domain is explained by the interaction of these bulky moieties with residues unique to these domains, specifically Phe 391, Val 379, and Val 380, that are not found in the N domain. The characterization of the extended active site and the binding of a potent C-domain-selective inhibitor provide the first structural data for the design of truly domain-specific pharmacophores.
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    Symmetry-restrained flexible fitting for symmetric EM maps
    (Elsevier, 2011) Chan, Kwok-Yan; Gumbart, James; McGreevy, Ryan; Watermeyer, Jean M; Sewell, Trevor B; Schulten, Klaus
    Many large biological macromolecules have inherent structural symmetry, being composed of a few distinct subunits, repeated in a symmetric array. These complexes are often not amenable to traditional high-resolution structural determination methods, but can be imaged in functionally relevant states using cryo-electron microscopy (cryo-EM). A number of methods for fitting atomic-scale structures into cryo-EM maps have been developed, including the molecular dynamics flexible fitting (MDFF) method. However, quality and resolution of the cryo-EM map are the major determinants of a method's success. In order to incorporate knowledge of structural symmetry into the fitting procedure, we developed the symmetry-restrained MDFF method. The new method adds to the cryo-EM map-derived potential further restraints on the allowed conformations of a complex during fitting, thereby improving the quality of the resultant structure. The benefit of using symmetry-based restraints during fitting, particularly for medium to low-resolution data, is demonstrated for three different systems.
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    Three-dimensional structure of a type III glutamine synthetase by single-particle reconstruction
    (Elsevier, 2006) van Rooyen, Jason M; Abratt, Valerie R; Sewell, Trevor B
    GlnN, the type III glutamine synthetase (GSIII) from the medically important, anaerobic, opportunistic pathogen Bacteroides fragilis, has 82.8 kDa subunits that share only 9% sequence identity with the type I glutamine synthetases (GSI), the only family for which a structure is known. Active GlnN was found predominantly in a single peak that eluted from a calibrated gel-filtration chromatography column at a position equaivalent to 0.86(±0.08) MDa. Negative-stain electron microscopy enabled the identification of double-ringed particles and single hexameric rings (“pinwheels”) resulting from partial staining. A 2D average of these pinwheels showed marked similarity to the corresponding structures found in preparations of GSI, except that the arms of the subunits were 40% longer. Reconstructions from particles embedded in vitreous ice showed that GlnN has a double-ringed, dodecameric structure with a 6-fold dihedral space group (D6) symmetry and dimensions of 17.0 nm parallel with the 6-fold axis and 18.3 nm parallel with the 2-fold axes. The structures, combined with a sequence alignment based on structural principles, showed how many aspects of the structure of GSI, and most notably the α/β barrel fold active site were preserved. There was evidence for the presence of this structure in the reconstructed volume, thus, identifying the indentations between the pinwheel spokes as putative active sites and suggesting conservation of the overall molecular geometry found in GSI despite their low level of global homology. Furthermore, docking of GSI into the reconstruction left sufficient plausibly located unoccupied density to account for the additional residues in GSIII, thus validating the structure.