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31 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Structural analysis of the 2-oxoglutarate binding site of the circadian rhythm linked oxygenase JMJD5 Md. Saiful Islam , Marios Markoulides , Rasheduzzaman Chowdhury , Christopher J. Schofield Scientific Reports 12 DOI: 10.1038/s41598-022-24154-0 Diamond Proposal Number(s): [9306] Open Access Show Abstract ▼ Abstract: JmjC (Jumonji-C) domain-containing 5 (JMJD5) plays important roles in circadian regulation in plants and humans and is involved in embryonic development and cell proliferation. JMJD5 is a 2-oxoglutarate (2OG) and Fe(II) dependent oxygenase of the JmjC subfamily, which includes histone Nε-methyl lysine-demethylases (KDMs) and hydroxylases catalysing formation of stable alcohol products. JMJD5 is reported to have KDM activity, but has been shown to catalyse C-3 hydroxylation of arginine residues in sequences from human regulator of chromosome condensation domain-containing protein 1 (RCCD1) and ribosomal protein S6 (RPS6) in vitro. We report crystallographic analyses of human JMJD5 complexed with 2OG analogues, including the widely used hypoxia mimic pyridine-2,4-dicarboxylate, both D- and L-enantiomers of the oncometabolite 2-hydroxyglutarate, and a cyclic N-hydroxyimide. The results support the assignment of JMJD5 as a protein hydroxylase and reveal JMJD5 has an unusually compact 2OG binding pocket suitable for exploitation in development of selective inhibitors. They will be useful in the development of chemical probes to investigate the physiologically relevant roles of JMJD5 in circadian rhythm and development and explore its potential as a medicinal chemistry target.	Nov 2022
I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Conservation of the unusual dimeric JmjC fold of JMJD7 from Drosophila melanogaster to humans Rasheduzzaman Chowdhury , Martine I. Abboud , James Wiley , Anthony Tumber , Suzana Markolovic , Christopher J. Schofield Scientific Reports 12 DOI: 10.1038/s41598-022-10028-y Open Access Show Abstract ▼ Abstract: The JmjC family of 2-oxoglutarate dependent oxygenases catalyse a range of hydroxylation and demethylation reactions in humans and other animals. Jumonji domain-containing 7 (JMJD7) is a JmjC (3S)-lysyl-hydroxylase that catalyses the modification of Developmentally Regulated GTP Binding Proteins 1 and 2 (DRG1 and 2); JMJD7 has also been reported to have histone endopeptidase activity. Here we report biophysical and biochemical studies on JMJD7 from Drosophila melanogaster (dmJMJD7). Notably, crystallographic analyses reveal that the unusual dimerization mode of JMJD7, which involves interactions between both the N- and C-terminal regions of both dmJMJD7 monomers and disulfide formation, is conserved in human JMJD7 (hsJMJD7). The results further support the assignment of JMJD7 as a lysyl hydroxylase and will help enable the development of selective inhibitors for it and other JmjC oxygenases.	Apr 2022
I04-Macromolecular Crystallography	Structural basis of prolyl hydroxylase domain inhibition by molidustat William D. Figg , Michael A. Mcdonough , Rasheduzzaman Chowdhury , Yu Nakashima , Zhihong Zhang , James P. Holt‐martyn , Alen Krajnc , Christopher J. Schofield Chemmedchem 5 DOI: 10.1002/cmdc.202100133 Diamond Proposal Number(s): [18069] Open Access Show Abstract ▼ Abstract: Human prolyl‐hydroxylases (PHDs) are hypoxia‐sensing 2‐oxoglutarate (2OG) oxygenases, catalysis by which suppresses the transcription of hypoxia‐inducible factor target genes. PHD inhibition enables the treatment of anaemia/ischaemia‐related disease. The PHD inhibitor Molidustat is approved for the treatment of renal anaemia; it differs from other approved/late‐stage PHD inhibitors in lacking a glycinamide side chain. The first reported crystal structures of Molidustat and IOX4 (a brain‐penetrating derivative) complexed with PHD2 reveal how their contiguous triazole, pyrazolone and pyrimidine/pyridine rings bind at the active site. The inhibitors bind to the active‐site metal in a bidentate manner through their pyrazolone and pyrimidine nitrogens, with the triazole π‐π‐stacking with Tyr303 in the 2OG binding pocket. Comparison of the new structures with other PHD inhibitor complexes reveals differences in the conformations of Tyr303, Tyr310, and a mobile loop linking β2–β3, which are involved in dynamic substrate binding/product release.	Apr 2021
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Human oxygenase variants employing a single protein Fe(II) ligand are catalytically active Amelia Brasnett , Inga Pfeffer , Lennart Brewitz , Rasheduzzaman Chowdhury , Yu Nakashima , Anthony Tumber , Michael A. Mcdonough , Christopher Schofield Angewandte Chemie International Edition DOI: 10.1002/anie.202103711 Open Access Show Abstract ▼ Abstract: Aspartate/asparagine‐β‐hydroxylase (AspH) is a human 2‐oxoglutarate (2OG) and Fe(II) oxygenase that catalyzes C3 hydroxylations of aspartate/asparagine residues of epidermal growth factor‐like domains (EGFDs). Unusually, AspH employs two histidine residues to chelate Fe(II) rather than the typical triad of two histidine and one glutamate/aspartate residue. We report kinetic, inhibition, and crystallographic studies concerning human AspH variants in which either of its Fe(II) binding histidine residues are substituted for alanine. Both the H725A and, in particular, the H679A AspH variant retain substantial catalytic activity. Crystal structures clearly reveal metal‐ligation by only a single protein histidine ligand. The results have implications for the functional assignment of 2OG oxygenases and for the design of non‐protein biomimetic catalysts.	Apr 2021
I02-Macromolecular Crystallography	Use of cyclic peptides to induce crystallization: case study with prolyl hydroxylase domain 2 Rasheduzzaman Chowdhury , Martine I. Abboud , Tom E. Mcallister , Biswadip Banerji , Bhaskar Bhushan , John L. Sorensen , Akane Kawamura , Christopher J. Schofield Scientific Reports 10 DOI: 10.1038/s41598-020-76307-8 Diamond Proposal Number(s): [12346] Open Access Show Abstract ▼ Abstract: Crystallization is the bottleneck in macromolecular crystallography; even when a protein crystallises, crystal packing often influences ligand-binding and protein–protein interaction interfaces, which are the key points of interest for functional and drug discovery studies. The human hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) readily crystallises as a homotrimer, but with a sterically blocked active site. We explored strategies aimed at altering PHD2 crystal packing by protein modification and molecules that bind at its active site and elsewhere. Following the observation that, despite weak inhibition/binding in solution, succinamic acid derivatives readily enable PHD2 crystallization, we explored methods to induce crystallization without active site binding. Cyclic peptides obtained via mRNA display bind PHD2 tightly away from the active site. They efficiently enable PHD2 crystallization in different forms, both with/without substrates, apparently by promoting oligomerization involving binding to the C-terminal region. Although our work involves a specific case study, together with those of others, the results suggest that mRNA display-derived cyclic peptides may be useful in challenging protein crystallization cases.	Dec 2020
I03-Macromolecular Crystallography	Biochemical and biophysical analyses of hypoxia sensing prolyl hydroxylases from Dictyostelium discoideum and Toxoplasma gondii Tongri Liu , Martine I. Abboud , Rasheduzzaman Chowdhury , Anthony Tumber , Adam P. Hardy , Kerstin Lippl , Christopher T. Lohans , Elisabete Pires , James Wickens , Michael Mcdonough , Christopher M. West , Christopher J. Schofield Journal Of Biological Chemistry DOI: 10.1074/jbc.RA120.013998 Diamond Proposal Number(s): [12346] Open Access Show Abstract ▼ Abstract: In animals, the response to chronic hypoxia is mediated by prolyl-hydroxylases (PHDs) that regulate the levels of hypoxia inducible transcription factor a (HIFα). PHD homologues exist in other types of eukaryotes and prokaryotes where they act on non-HIF substrates. To gain insight into the factors underlying different PHD substrates and properties, we carried out biochemical and biophysical studies on PHD homologues from the slime mold, Dictyostelium discoideum, and the protozoan parasite, Toxoplasma gondii, both lacking HIF. The respective prolyl-hydroxylases (DdPhyA and TgPhyA) catalyze prolyl-hydroxylation of S-Phase Kinase Associated Protein 1 (Skp1), a reaction enabling adaptation to different dioxygen availability. Assays with full length Skp1 substrates reveal substantial differences in the kinetic properties of DdPhyA and TgPhyA, both with respect to each other and compared with human PHD2; consistent with cellular studies TgPhyA is more active at low dioxygen concentrations than DdPhyA. TgSkp1 is a DdPhyA substrate and DdSkp1 is a TgPhyA substrate. No cross-reactivity was detected between DdPhyA/TgPhyA substrates and human PHD2. The human Skp1 E147P variant is a DdPhyA and TgPhyA substrate, suggesting some retention of ancestral interactions. Crystallographic analysis of DdPhyA enables comparisons with homologues from humans, Trichoplax adhaerens, and prokaryotes, TgPhyA informing on differences in mobile elements involved in substrate binding and catalysis. In DdPhyA, two mobile loops that enclose substrates in the PHDs are conserved, but the C-terminal helix of the PHDs is strikingly absent. The combined results support the proposal that PHD homologues have evolved kinetic and structural features suited to their specific sensing roles.	Sep 2020
I04-1-Macromolecular Crystallography (fixed wavelength)	A human protein hydroxylase that accepts D-residues Hwanho Choi , Adam P. Hardy , Thomas M. Leissing , Rasheduzzaman Chowdhury , Yu Nakashima , Wei Ge , Marios Markoulides , John S. Scotti , Philip A. Gerken , Helen Thorbjornsrud , Dahye Kang , Sungwoo Hong , Joongoo Lee , Michael A. Mcdonough , Hwangseo Park , Christopher J. Schofield Communications Chemistry 3 DOI: 10.1038/s42004-020-0290-5 Diamond Proposal Number(s): [18069] Open Access Show Abstract ▼ Abstract: Factor inhibiting hypoxia-inducible factor (FIH) is a 2-oxoglutarate-dependent protein hydroxylase that catalyses C3 hydroxylations of protein residues. We report FIH can accept (D)- and (L)-residues for hydroxylation. The substrate selectivity of FIH differs for (D) and (L) epimers, e.g., (D)- but not (L)-allylglycine, and conversely (L)- but not (D)-aspartate, undergo monohydroxylation, in the tested sequence context. The (L)-Leu-containing substrate undergoes FIH-catalysed monohydroxylation, whereas (D)-Leu unexpectedly undergoes dihydroxylation. Crystallographic, mass spectrometric, and DFT studies provide insights into the selectivity of FIH towards (L)- and (D)-residues. The results of this work expand the potential range of known substrates hydroxylated by isolated FIH and imply that it will be possible to generate FIH variants with altered selectivities.	May 2020
I03-Macromolecular Crystallography	Structure‐activity relationship and crystallographic studies on 4‐hydroxypyrimidine HIF prolyl hydroxylase domain inhibitors James P. Holt‐martyn , Rasheduzzaman Chowdhury , Anthony Tumber , Tzu‐lan Yeh , Martine I. Abboud , Kerstin Lippl , Christopher T. Lohans , Gareth W. Langley , William Figg , Michael A. Mcdonough , Christopher W. Pugh , Peter J. Ratcliffe , Christopher J. Schofield Chemmedchem 13 DOI: 10.1002/cmdc.201900557 Open Access Show Abstract ▼ Abstract: The 2‐oxoglutarate‐dependent hypoxia inducible factor prolyl hydroxylases (PHDs) are targets for treatment of a variety of diseases including anaemia. One PHD inhibitor is approved for use for the treatment of renal anaemia and others are in late stage clinical trials. The number of reported templates for PHD inhibition is limited. We report structure–activity relationship and crystallographic studies on a promising class of 4‐hydroxypyrimidine‐containing PHD inhibitors.	Dec 2019
I02-Macromolecular Crystallography	Biochemical and structural investigations clarify the substrate selectivity of the 2-oxoglutarate oxygenase JMJD6 Md. Saiful Islam , Michael A. Mcdonough , Rasheduzzaman Chowdhury , Joseph Gault , Amjad Khan , Elisabete Pires , Christopher J. Schofield Journal Of Biological Chemistry DOI: 10.1074/jbc.RA119.008693 Diamond Proposal Number(s): [12346] Show Abstract ▼ Abstract: JmjC domain containing protein 6 (JMJD6) is a 2-oxoglutarate (2OG)-dependent oxygenase linked to various cellular processes including splicing regulation, histone modification, transcriptional pause release, hypoxia sensing, and cancer. JMJD6 is reported to catalyze hydroxylation of lysine residue(s) of histones, the tumor suppressor protein p53, and splicing regulatory proteins, including u2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65). JMJD6 is also reported to catalyze N-demethylation of N-methylated (both mono- and di-methylated) arginine residues of histones, and other proteins including heat shock protein 70 (HSP70), oestrogen receptor α (ERα) and RNA helicase A. Here we report MS- and NMR-based kinetic assays employing purified JMJD6 and multiple substrate fragment sequences, the results of which support the assignment of purified JMJD6 as a lysyl hydroxylase. By contrast, we did not observe N-methyl arginyl N-demethylation with purified JMJD6. Biophysical analyses including crystallographic analyses of JMJD6Δ344-403 in complex with iron and 2OG supported its assignment as a lysyl-hydroxylase rather than an N-methyl arginyl-demethylase. The screening results supported some, but not all, of the assigned JMJD6 substrates and identified other potential JMJD6 substrates. We envision these results will be useful in cellular and biological work on the substrates and functions of JMJD6 and in the development of selective inhibitors of human 2OG oxygenases.	May 2019
I03-Macromolecular Crystallography	Studies on spiro[4.5]decanone prolyl hydroxylase domain inhibitors James P. Holt-Martyn , Anthony Tumber , Mohammed Z. Rahman , Kerstin Lippl , William Figg , Michael Mcdonough , Rasheduzzaman Chowdhury , Christopher J. Schofield Medchemcomm 10, 500 - 504 DOI: 10.1039/C8MD00548F Open Access Show Abstract ▼ Abstract: The 2-oxoglutarate (2OG) dependent hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are targets for treatment of anaemia and other ischaemia related diseases. PHD inhibitors are in clinical trials; however, the number of reported templates for PHD inhibition is limited. We report structure–activity relationship and crystallographic studies on spiro[4.5]decanone containing PHD inhibitors. Together with other studies, our results reveal spiro[4.5]decanones as useful templates for generation of potent and selective 2OG oxygenase inhibitors.	Apr 2019

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