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11 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I24-Microfocus Macromolecular Crystallography	Acceptance and kinetic resolution of α-methyl-substituted aldehydes by norcoclaurine synthases Rebecca Roddan , Gudrun Gygli , Altin Sula , Daniel Méndez-sánchez , Jürgen Pleiss , John M. Ward , Nicholas H. Keep , Helen C. Hailes Acs Catalysis DOI: 10.1021/acscatal.9b02699 Diamond Proposal Number(s): [17201] Show Abstract ▼ Abstract: Norcoclaurine synthase (NCS) catalyzes a stereoselective Pictet−Spengler reaction to give the key intermediate, (S)-norcoclaurine in benzylisoquinoline alkaloid biosynthesis. This family of alkaloids contains many bioactive molecules including morphine and berberine. Recently, NCS has been demonstrated to accept a variety of aldehydes and some ketones as substrates, leading to a range of chiral tetrahydroisoquinoline (THIQ) products. Here, we report the unusual acceptance of α-substituted aldehydes, in particular α-methyl-substituted aldehydes, by wild-type Thalictrum flavum NCS (Δ33Tf NCS) to give THIQ products. Moreover, the kinetic resolution of several α- substituted aldehydes to give THIQs with two defined chiral centers in a single step with high conversions was achieved. Several dopamine analogues were also accepted as substrates, and reactions were amenable to scale up. Active site mutants of TfNCS were then used, which demonstrated the potential to enhance the stereoselectivities in the reaction and improve yields. The rationale for the acceptance of these substrates and improved activity with different mutants has been gained from a co-crystallized structure of Δ33TfNCS with a nonproductive mimic of a reaction intermediate bound in the active site. Finally, molecular dynamics simulations were performed to study the binding of dopamine and an α-substituted aldehyde and provided further insight into the reaction with these substrates.	Sep 2019
I02-Macromolecular Crystallography	Terminal regions confer plasticity to the tetrameric assembly of human HspB2 and HspB3 Alice Clark , Wilma Vree Egberts , Frances D. L. Kondrat , Gillian R. Hilton , Nicholas J. Ray , Ambrose R. Cole , John A. Carver , Justin L. P. Benesch , Nicholas Keep , Wilbert C. Boelens , Christine Slingsby Journal Of Molecular Biology DOI: 10.1016/j.jmb.2018.06.047 Diamond Proposal Number(s): [7197] Open Access Show Abstract ▼ Abstract: Heterogeneity in small heat shock proteins (sHsps) spans multiple spatiotemporal regimes – from fast fluctuations of part of the protein, to conformational variability of tertiary structure, plasticity of the interfaces, and polydispersity of the inter-converting, and co-assembling oligomers. This heterogeneity and dynamic nature of sHsps has significantly hindered their structural characterisation. Atomic-coordinates are particularly lacking for vertebrate sHsps, where most available structures are of extensively truncated homomers. sHsps play important roles in maintaining protein levels in the cell and therefore in organismal health and disease. HspB2 and HspB3 are vertebrate sHsps that are found co-assembled in neuromuscular cells, and variants thereof are associated with disease. Here, we present the structure of human HspB2/B3, which crystallised as a hetero-tetramer in a 3:1 ratio. In the HspB2/B3 tetramer, the four α-crystallin domains (ACDs) assemble into a flattened tetrahedron which is pierced by two non-intersecting approximate dyads. Assembly is mediated by flexible “nuts and bolts” involving IXI/V motifs from terminal regions filling ACD pockets. Parts of the N-terminal region bind in an unfolded conformation into the anti-parallel shared ACD dimer grooves. Tracts of the terminal regions are not resolved, most likely due to their disorder in the crystal lattice. This first structure of a full-length human sHsp heteromer reveals the heterogeneous interactions of the terminal regions and suggests a plasticity that is important for the cytoprotective functions of sHsps.	Jun 2018
I02-Macromolecular Crystallography	Structural Evidence for the Dopamine-First Mechanism of Norcoclaurine Synthase Benjamin R. Lichman , Altin Sula , Thomas Pesnot , Helen C. Hailes , John M. Ward , Nicholas H. Keep Biochemistry DOI: 10.1021/acs.biochem.7b00769 Diamond Proposal Number(s): [12305] Show Abstract ▼ Abstract: Norcoclaurine synthase (NCS) is a Pictet-Spenglerase that catalyzes the first key step in plant benzylisoquinoline alkaloid metabolism, a compound family that includes bioactive natural products such as morphine. The enzyme has also shown great potential as a biocatalyst for the formation of chiral isoquinolines. Here we present new high-resolution X-ray crystallography data describing Thalictrum flavum NCS bound to a mechanism-inspired ligand. The structure supports two key features of the NCS “dopamine-first” mechanism: the binding of dopamine catechol to Lys-122 and the position of the carbonyl substrate binding site at the active site entrance. The catalytically vital residue Glu-110 occupies a previously unobserved ligand-bound conformation that may be catalytically significant. The potential roles of inhibitory binding and alternative amino acid conformations in the mechanism have also been revealed. This work significantly advances our understanding of the NCS mechanism and will aid future efforts to engineer the substrate scope and catalytic properties of this useful biocatalyst.	Sep 2017
I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Crystal Structures and Binding Dynamics of Odorant-Binding Protein 3 from two aphid species Megoura viciae and Nasonovia ribisnigri Tom Northey , Herbert Venthur , Filomena De Biasio , Francois Xavier Chauviac , Ambrose Cole , Karlos Antonio Lisboa Ribeiro , Gerarda Grossi , Patrizia Falabella , Linda M. Field , Nicholas H. Keep , Jing-jiang Zhou Scientific Reports 6 DOI: 10.1038/srep24739 Diamond Proposal Number(s): [7197] Open Access Show Abstract ▼ Abstract: Aphids use chemical cues to locate hosts and find mates. The vetch aphid Megoura viciae feeds exclusively on the Fabaceae, whereas the currant-lettuce aphid Nasonovia ribisnigri alternates hosts between the Grossulariaceae and Asteraceae. Both species use alarm pheromones to warn of dangers. For N. ribisnigri this pheromone is a single component (E)-β-farnesene but M. viciae uses a mixture of (E)-β-farnesene, (−)-α-pinene, β-pinene, and limonene. Odorant-binding proteins (OBP) are believed to capture and transport such semiochemicals to their receptors. Here, we report the first aphid OBP crystal structures and examine their molecular interactions with the alarm pheromone components. Our study reveals some unique structural features: 1) the lack of an internal ligand binding site; 2) a striking groove in the surface of the proteins as a putative binding site; 3) the N-terminus rather than the C-terminus occupies the site closing off the conventional OBP pocket. The results from fluorescent binding assays, molecular docking and dynamics demonstrate that OBP3 from M. viciae can bind to all four alarm pheromone components and the differential ligand binding between these very similar OBP3s from the two aphid species is determined mainly by the direct π-π interactions between ligands and the aromatic residues of OBP3s in the binding pocket.	Apr 2016
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Structure of the stationary phase survival protein YuiC from B.subtilis Doris Quay , Ambrose Cole , Adam Cryar , Konstantinos Thalassinos , Mark Williams , Sanjib Bhakta , Nicholas Keep BMC Structural Biology 15, 1349-1357 DOI: 10.1186/s12900-015-0039-z PMID: 26163297 Diamond Proposal Number(s): [7197] Open Access Show Abstract ▼ Abstract: Stationary phase survival proteins (Sps) were found in Firmicutes as having analogous domain compositions, and in some cases genome context, as the resuscitation promoting factors of Actinobacteria, but with a different putative peptidoglycan cleaving domain.	Jul 2015
I03-Macromolecular Crystallography	Characterisation of an oxidoreductase from the Arylamine N-acetyltransferase operon in Mycobacterium smegmatis Jim Evangelopoulos , Nora Cronin , Tina Daviter , Edith Sim , Nicholas Keep , Sanjib Bhakta Febs Journal DOI: 10.1111/j.1742-4658.2011.08382.x PMID: 21972977 Show Abstract ▼ Abstract: Mycobacterium tuberculosis, the most successful bacterial pathogen, causes tuberculosis, a disease that still causes more than 2 million deaths per year. Arylamine N-acetyltransferase is an enzyme that is conserved in most Mycobacterium spp. The nat gene belongs to an operon that is important for the intracellular survival of M. tuberculosis within macrophages. The nat operon in Mycobacterium smegmatis and other fast-growing mycobacterial species has a unique organization containing genes with uncharacterized function. Here, we describe the biochemical, biophysical and structural characterization of the MSMEG_0308 gene product (MS0308) of the M. smegmatis nat operon. While characterizing the function of MS0308, we validated the oxidoreductase property; however, we found that the enzyme was not utilizing dihydrofolate as its substrate, hence we first report that MS0308 is not a dihydrofolate reductase, as annotated in the genome. The structure of this oxidoreductase was solved at 2.0 Å in complex with the cofactor NADPH and has revealed the hydrophobic pocket where the endogenous substrate binds.	Oct 2011
I02-Macromolecular Crystallography I03-Macromolecular Crystallography	Crystal Structure of R120G Disease Mutant of Human αB-Crystallin Domain Dimer Shows Closure of a Groove A. R. Clark , C. E. Naylor , C. Bagneris , N. H. Keep , C. Slingsby Journal Of Molecular Biology 408 (1), 118-134 DOI: 10.1016/j.jmb.2011.02.020 PMID: 21329698 Open Access Show Abstract ▼ Abstract: Small heat shock proteins form large cytosolic assemblies from an α-crystallin domain (ACD) flanked by sequence extensions. Mutation of a conserved arginine in the ACD of several human small heat shock protein family members causes many common inherited diseases of the lens and neuromuscular system. The mutation R120G in αB-crystallin causes myopathy, cardiomyopathy and cataract. We have solved the X-ray structure of the excised ACD dimer of human αB R120G close to physiological pH and compared it with several recently determined wild-type vertebrate ACD dimer structures. Wild-type excised ACD dimers have a deep groove at the interface floored by a flat extended bottom sheet. Solid-state NMR studies of large assemblies of full-length αB-crystallin have shown that the groove is blocked in the ACD dimer by curvature of the bottom sheet. The crystal structure of R120G ACD dimer also reveals a closed groove, but here the bottom sheet is flat. Loss of Arg120 results in rearrangement of an extensive array of charged interactions across this interface. His83 and Asp80 on movable arches on either side of the interface close the groove by forming two new salt bridges. The residues involved in this extended set of ionic interactions are conserved in Hsp27, Hsp20, αA- and αB-crystallin sequences. They are not conserved in Hsp22, where mutation of the equivalent of Arg120 causes neuropathy. We speculate that the αB R120G mutation disturbs oligomer dynamics, causing the growth of large soluble oligomers that are toxic to cells by blocking essential processes.	Feb 2011
I04-Macromolecular Crystallography	Essential residues for the enzyme activity of ATP-dependent MurE ligase from Mycobacterium tuberculosis Chandrakala Basavannacharya , Paul Moody , Tulika Munshi , Nora Cronin , Nicholas Keep , Sanjib Bhakta Protein And Cell 1 (11), 1011 - 1022 DOI: 10.1007/s13238-010-0132-9 Show Abstract ▼ Abstract: The emergence of total drug-resistant tuberculosis (TDRTB) has made the discovery of new therapies for tuberculosis urgent. The cytoplasmic enzymes of peptidoglycan biosynthesis have generated renewed interest as attractive targets for the development of new anti-mycobacterials. One of the cytoplasmic enzymes, uridine diphosphate (UDP)-MurNAc-tripeptide ligase (MurE), catalyses the addition of meso-diaminopimelic acid (m-DAP) into peptidoglycan in Mycobacterium tuberculosis coupled to the hydrolysis of ATP. Mutants of M. tuberculosis MurE were generated by replacing K157, E220, D392, R451 with alanine and N449 with aspartate, and truncating the first 24 amino acid residues at the N-terminus of the enzyme. Analysis of the specific activity of these proteins suggested that apart from the 24 Nterminal residues, the other mutated residues are essential for catalysis. Variations in K m values for one or more substrates were observed for all mutants, except the N-terminal truncation mutant, indicating that these residues are involved in binding substrates and form part of the active site structure. These mutant proteins were also tested for their specificity for a wide range of substrates. Interestingly, the mutations K157A, E220A and D392A showed hydrolysis of ATP uncoupled from catalysis. The ATP hydrolysis rate was enhanced by at least partial occupation of the uridine nucleotide dipeptide binding site. This study provides an insight into the residues essential for the catalytic activity and substrate binding of the ATP-dependent MurE ligase. Since ATP-dependent MurE ligase is a novel drug target, the understanding of its function may lead to development of novel inhibitors against resistant forms of M. tuberculosis.	Dec 2010
I03-Macromolecular Crystallography	ATP-dependent MurE ligase in Mycobacterium tuberculosis: Biochemical and structural characterisation Chandrakala Basavannacharya , Giles Robertson , Tulika Munshi , Sanjib Bhakta , Nicholas Keep Tuberculosis 90 (1), 16 - 24 DOI: 10.1016/j.tube.2009.10.007 PMID: 19945347 Show Abstract ▼ Abstract: New therapies are required against Mycobacterium tuberculosis and its cell wall peptidoglycan biosynthesis is a potential therapeutic target. UDP-MurNAc-tripeptide ligase (MurE) is a member of the ATP-dependent ligase family, which incorporate amino acids including meso-diaminopimelic acid (m-DAP) into peptidoglycan during synthesis in a species-specific manner. In the present study, we have cloned, over-expressed, and characterised MurE from M. tuberculosis (Mtb-MurE). The crystal structure has been determined at 3.0 Å resolution in the presence of the substrate UDP-MurNAc-l-Ala-d-Glu (UAG). The activity of the enzyme was measured through estimating inorganic phosphate released in a non-radioactive high-throughput colourimetric assay. UDP-MurNAc-l-Ala-d-Glu-m-DAP (UMT) formation coupled to inorganic phosphate release was confirmed by HPLC and mass spectrometric analyses. Kinetic constants were determined for a range of natural substrates using optimised conditions. From our findings, it is evident that Mtb-MurE is highly specific in adding m-DAP to UDP-MurNAc-dipeptide and ATP-hydrolysis is an absolute requirement for its activity.	Jan 2010
I04-Macromolecular Crystallography	Crystal Structures of α-Crystallin Domain Dimers of αB-Crystallin and Hsp20 C. Bagnéris , O. A. Bateman , C. E. Naylor , N. Cronin , W. C. Boelens , N. H. Keep , C. Slingsby Journal Of Molecular Biology 392 (5), 1242 - 1252 DOI: 10.1016/j.jmb.2009.07.069 PMID: 19646995 Open Access Show Abstract ▼ Abstract: Small heat shock proteins (sHsps) are a family of large and dynamic oligomers highly expressed in long-lived cells of muscle, lens and brain. Several family members are upregulated during stress, and some are strongly cytoprotective. Their polydispersity has hindered high-resolution structure analyses, particularly for vertebrate sHsps. Here, crystal structures of excised α-crystallin domain from rat Hsp20 and that from human αB-crystallin show that they form homodimers with a shared groove at the interface by extending a β sheet. However, the two dimers differ in the register of their interfaces. The dimers have empty pockets that in large assemblies will likely be filled by hydrophobic sequence motifs from partner chains. In the Hsp20 dimer, the shared groove is partially filled by peptide in polyproline II conformation. Structural homology with other sHsp crystal structures indicates that in full-length chains the groove is likely filled by an N-terminal extension. Inside the groove is a symmetry-related functionally important arginine that is mutated, or its equivalent, in family members in a range of neuromuscular diseases and cataract. Analyses of residues within the groove of the αB-crystallin interface show that it has a high density of positive charges. The disease mutant R120G α-crystallin domain dimer was found to be more stable at acidic pH, suggesting that the mutation affects the normal dynamics of sHsp assembly. The structures provide a starting point for modelling higher assembly by defining the spatial locations of grooves and pockets in a basic dimeric assembly unit. The structures provide a high-resolution view of a candidate functional state of an sHsp that could bind non-native client proteins or specific components from cytoprotective pathways. The empty pockets and groove provide a starting model for designing drugs to inhibit those sHsps that have a negative effect on cancer treatment.	Oct 2009

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