ORCID Profile
0000-0002-0290-6565
Current Organisation
University of Oxford
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Publisher: Elsevier BV
Date: 08-2007
Publisher: American Chemical Society (ACS)
Date: 08-04-2005
DOI: 10.1021/BI047478Q
Abstract: Isopenicillin N synthase (IPNS), a non-heme iron(II)-dependent oxidase, catalyzes conversion of the tripeptide delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-valine (ACV) to bicyclic isopenicillin N (IPN), concomitant with the reduction of dioxygen to two molecules of water. Incubation of the "truncated"substrate analogues delta-(l-alpha-aminoadipoyl)-l-cysteinyl-glycine (ACG) and delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-alanine (ACA) with IPNS has previously been shown to afford acyclic products, in which the substrate cysteinyl residue has undergone a two-electron oxidation. We report X-ray crystal structures for the anaerobic IPNS/Fe(II)/ACG and IPNS/Fe(II)/ACA complexes, both in the absence and presence of the dioxygen analogue nitric oxide. The overall protein structures are very similar to those of the corresponding IPNS/Fe(II)/ACV complexes however, significant differences are apparent in the vicinity of the active site iron. The structure of the IPNS/Fe(II)/ACG complex reveals that the C-terminal carboxylate of this substrate is oriented toward the active site iron atom, apparently hydrogen-bonded to an additional water ligand at the metal this is a different binding mode to that observed in the IPNS/Fe(II)/ACV complex. ACA binds to the metal in a manner that is intermediate between those observed for ACV and ACG. The addition of NO to these complexes initiates conformational changes such that both the IPNS/Fe(II)/ACG/NO and IPNS/Fe(II)/ACA/NO structures closely resemble the IPNS/Fe(II)/ACV/NO complex. These results further demonstrate the feasibility of metal-centered rearrangements in catalysis by non-heme iron enzymes and provide insight into the delicate balance between hydrophilic-hydrophobic interactions and steric effects in the IPNS active site.
Publisher: Public Library of Science (PLoS)
Date: 06-07-2015
Publisher: Wiley
Date: 21-03-2016
Publisher: Elsevier BV
Date: 05-2011
Publisher: American Chemical Society (ACS)
Date: 11-04-2014
DOI: 10.1021/BI500086P
Abstract: Deacetoxycephalosporin C synthase (DAOCS) catalyzes the oxidative ring expansion of penicillin N (penN) to give deacetoxycephalosporin C (DAOC), which is the committed step in the biosynthesis of the clinically important cephalosporin antibiotics. DAOCS belongs to the family of non-heme iron(II) and 2-oxoglutarate (2OG) dependent oxygenases, which have substantially conserved active sites and are proposed to employ a consensus mechanism proceeding via formation of an enzyme·Fe(II)·2OG·substrate ternary complex. Previously reported kinetic and crystallographic studies led to the proposal of an unusual "ping-pong" mechanism for DAOCS, which was significantly different from other members of the 2OG oxygenase superfamily. Here we report pre-steady-state kinetics and binding studies employing mass spectrometry and NMR on the DAOCS-catalyzed penN ring expansion that demonstrate the viability of ternary complex formation in DAOCS catalysis, arguing for the generality of the proposed consensus mechanism for 2OG oxygenases.
Publisher: Wiley
Date: 13-01-2012
Publisher: American Chemical Society (ACS)
Date: 11-07-2012
DOI: 10.1021/JM300677J
Publisher: Wiley
Date: 30-08-2005
DOI: 10.1016/J.FEBSLET.2005.08.033
Abstract: The ferrous iron and 2-oxoglutarate (2OG) dependent oxygenases catalyse two electron oxidation reactions by coupling the oxidation of substrate to the oxidative decarboxylation of 2OG, giving succinate and carbon dioxide coproducts. The evidence available on the level of incorporation of one atom from dioxygen into succinate is inconclusive. Here, we demonstrate that five members of the 2OG oxygenase family, AlkB from Escherichia coli, anthocyanidin synthase and flavonol synthase from Arabidopsis thaliana, and prolyl hydroxylase domain enzyme 2 and factor inhibiting hypoxia-inducible factor-1 from Homo sapiens all incorporate a single oxygen atom, almost exclusively derived from dioxygen, into the succinate co-product.
Publisher: Elsevier BV
Date: 08-2012
DOI: 10.1016/J.BMCL.2012.06.024
Abstract: γ-Butyrobetaine hydroxylase (BBOX) is a 2-oxoglutarate and Fe(II)-dependent oxygenase that catalyses the final step of L-carnitine biosynthesis in animals. BBOX catalyses the oxidation of 3-(2,2,2-trimethylhydrazinium)propionate (THP), a clinically used BBOX inhibitor, to form multiple products including 3-amino-4-(methyamino)butanoic acid (AMBA), which is proposed to be formed via a Stevens type rearrangement mechanism. We report the synthesis of AMBA and confirm that it is a product of the BBOX catalysed oxidation of THP. AMBA reacts with formaldehyde, which is produced enzymatically by BBOX, to give a cyclic adduct.
Publisher: Elsevier BV
Date: 08-2015
Publisher: American Society for Microbiology
Date: 17-09-2021
DOI: 10.1128/AAC.00936-21
Abstract: The structural ersity in metallo-β-lactamases (MBLs), especially in the vicinity of the active site, has been a major hurdle in the development of clinically effective inhibitors. Representatives from three variants of the B3 MBL subclass, containing either the canonical HHH/DHH active-site motif (present in the majority of MBLs in this subclass) or the QHH/DHH (B3-Q) or HRH/DQK (B3-RQK) variations were reported previously.
Publisher: Wiley
Date: 25-05-2012
Publisher: Public Library of Science (PLoS)
Date: 16-12-2015
Publisher: Springer Science and Business Media LLC
Date: 05-03-2014
DOI: 10.1038/NCOMMS4423
Publisher: American Chemical Society (ACS)
Date: 21-12-2009
DOI: 10.1021/JM901537Q
Abstract: This report demonstrates that solvent water relaxation measurements can be used for quantitative screening of ligand binding and for mechanistic investigations of enzymes containing paramagnetic metal centers by using conventional NMR instrumentation at high field. The method was exemplified using prolyl hydroxylase domain containing enzyme 2 (PHD2), a human enzyme involved in hypoxic sensing, with Mn(II) substituting for Fe(II) at the active site. K(D) values were determined for inhibitors that hinder access of water to the paramagnetic center. This technique is also useful for investigating the mechanism of suitable metalloenzymes, including order of ligand binding and modes of inhibition.
Publisher: Royal Society of Chemistry (RSC)
Date: 2017
DOI: 10.1039/C7SC02103H
Abstract: Four compounds in clinical trials for anaemia treatment are potent inhibitors of the hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs), but differ in potency and how they interact with HIF at the PHD active site.
Publisher: Wiley
Date: 22-06-2012
Abstract: Fluoride assays for oxygenases: The 2-oxoglutarate-dependent oxygenase BBOX catalyses the final step in carnitine biosynthesis and is a medicinal chemistry target. We report that BBOX can hydroxylate fluorinated substrates analogues with subsequent release of a fluoride ion, thereby enabling an efficient fluorescence-based assay.
Publisher: Royal Society of Chemistry (RSC)
Date: 2014
DOI: 10.1039/C3CC47581F
Abstract: A convenient method for the synthesis of fluoromethylated carnitine biosynthesis intermediates, i.e. fluorinated derivatives of γ-butyrobetaine and trimethyllysine, is described. The fluoromethylated probes were useful in both in vitro and cell based assays employing (19)F NMR and LC-MS analyses.
Publisher: Informa UK Limited
Date: 07-2020
Publisher: Elsevier BV
Date: 02-2014
Publisher: Elsevier BV
Date: 06-2002
DOI: 10.1016/S0960-894X(02)00219-6
Abstract: The hypoxic response in animals is mediated by hydroxylation of proline residues in the alpha-subunit of hypoxia inducible factor (HIF). Hydroxylation is catalysed by prolyl-4-hydroxylases (PHD isozymes in humans) which are iron(II) and 2-oxoglutarate dependent oxygenases. Mutation of the arginine proposed to bind 2-oxoglutarate and of the 2His-1-carboxylate iron(II) binding motif in PHD1 dramatically reduces its activity. The source of the oxygen of the product alcohol is (>95%) dioxygen.
Publisher: Springer Science and Business Media LLC
Date: 28-10-2019
DOI: 10.1038/S41467-019-12711-7
Abstract: AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1–3, 2–4, 5–6 disulfide bonding pattern an unexpected Cys3–4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases.
Publisher: Royal Society of Chemistry (RSC)
Date: 2014
DOI: 10.1039/C4OB01167H
Abstract: BBOX is a 2-oxoglutarate dependent oxygenase that can catalyse formation of vicinal diols and amino alcohols.
Publisher: Wiley
Date: 27-08-2014
Publisher: Elsevier BV
Date: 12-2010
DOI: 10.1016/J.CHEMBIOL.2010.09.016
Abstract: The final step in carnitine biosynthesis is catalyzed by γ-butyrobetaine (γBB) hydroxylase (BBOX), an iron/2-oxoglutarate (2OG) dependent oxygenase. BBOX is inhibited by trimethylhydrazine-propionate (THP), a clinically used compound. We report structural and mechanistic studies on BBOX and its reaction with THP. Crystallographic and sequence analyses reveal that BBOX and trimethyllysine hydroxylase form a subfamily of 2OG oxygenases that dimerize using an N-terminal domain. The crystal structure reveals the active site is enclosed and how THP competes with γBB. THP is a substrate giving formaldehyde (supporting structural links with histone demethylases), dimethylamine, malonic acid semi-aldehyde, and an unexpected product with an additional carbon-carbon bond resulting from N-demethylation coupled to oxidative rearrangement, likely via an unusual radical mechanism. The results provide a basis for development of improved BBOX inhibitors and may inspire the discovery of additional rearrangement reactions.
Publisher: American Chemical Society (ACS)
Date: 15-08-2013
DOI: 10.1021/CB400200H
Abstract: β-Lactam antibiotics react with penicillin binding proteins (PBPs) to form relatively stable acyl-enzyme complexes. We describe structures derived from the reaction of piperacillin with PBP3 (Pseudomonas aeruginosa) including not only the anticipated acyl-enzyme complex but also an unprecedented complex with (5S)-penicilloic acid, which was formed by C-5 epimerization of the nascent (5R)-penicilloic acid product. Formation of the complex was confirmed by solution studies, including NMR. Together, these results will be useful in the design of new PBP inhibitors and raise the possibility that noncovalent PBP inhibition by penicilloic acids may be of clinical relevance.
Publisher: Oxford University Press (OUP)
Date: 16-10-2014
DOI: 10.1093/JAC/DKU403
Publisher: Portland Press Ltd.
Date: 15-06-2003
DOI: 10.1042/BJ20021627
Abstract: Isopenicillin N synthase (IPNS) is a non-haem iron(II) oxidase which catalyses the biosynthesis of isopenicillin N from the tripeptide δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine (ACV). Herein we report crystallographic studies to investigate the reaction of IPNS with the truncated substrate analogue δ-(l-α-aminoadipoyl)-l-cysteinyl-d-α-aminobutyrate (ACAb). It has been reported previously that this analogue gives rise to three β-lactam products when incubated with IPNS: two methyl penams and a cepham. Crystal structures of the IPNS–Fe(II)–ACAb and IPNS–Fe(II)–ACAb–NO complexes have now been solved and are reported herein. These structures and modelling studies based on them shed light on the diminished product selectivity shown by IPNS in its reaction with ACAb and further rationalize the presence of certain key residues at the IPNS active site.
Publisher: American Chemical Society (ACS)
Date: 11-11-2021
Publisher: Springer Science and Business Media LLC
Date: 26-08-2016
DOI: 10.1038/NCOMMS12673
Abstract: The response to hypoxia in animals involves the expression of multiple genes regulated by the αβ-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1–3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel–Lindau protein (VHL)–elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors.
Publisher: Proceedings of the National Academy of Sciences
Date: 02-09-2014
Abstract: The Fe(II)- and 2-oxoglutarate (2OG)-dependent hypoxia-inducible transcription factor prolyl-hydroxylases play a central role in human oxygen sensing and are related to other prolyl-hydroxylases involved in eukaryotic collagen biosynthesis and ribosomal modification. The finding that a PHD-related prolyl-hydroxylase in Pseudomonas spp. regulates pyocyanin biosynthesis supports prokaryotic origins for the eukaryotic prolyl-hydroxylases. The identification of the switch I loop of elongation factor Tu (EF-Tu) as a Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) substrate provides evidence of roles for 2OG oxygenases in both translational and transcriptional regulation. A structure of the PPHD:EF-Tu complex, the first to the authors' knowledge of a 2OG oxygenase with its intact protein substrate, reveals that major conformational changes occur in both PPHD and EF-Tu and will be useful in the design of new prolyl-hydroxylase inhibitors.
Publisher: Public Library of Science (PLoS)
Date: 12-02-2014
Publisher: Royal Society of Chemistry (RSC)
Date: 2018
DOI: 10.1039/C8SC00286J
Abstract: Tight, non-active site binding cyclic peptides are promising affinity reagents for studying proteins and their interactions.
Publisher: Wiley
Date: 26-09-2018
Abstract: In animals, the response to chronic hypoxia is mediated by upregulation of the α,β-heterodimeric hypoxia-inducible factors (HIFs). Levels of HIFα isoforms, but not HIFβ, are regulated by their post-translational modification as catalysed by prolyl hydroxylase domain enzymes (PHDs). Different roles for the human HIF-1/2α isoforms and their two oxygen-dependent degradation domains (ODDs) are proposed. We report kinetic and NMR analyses of the ODD selectivity of the catalytic domain of wild-type PHD2 (which is conserved in nearly all animals) and clinically observed variants. Studies using Ala scanning and "hybrid" ODD peptides imply that the relatively rigid conformation of the (hydroxylated) proline plays an important role in ODD binding. They also reveal differential roles in binding for the residues on the N- and C-terminal sides of the substrate proline. The overall results indicate how the PHDs achieve selectivity for HIFα ODDs and might be of use in identifying substrate-selective PHD inhibitors.
Publisher: Wiley
Date: 21-08-2017
Publisher: Elsevier BV
Date: 09-2009
Publisher: American Chemical Society (ACS)
Date: 21-09-2015
Publisher: Portland Press Ltd.
Date: 26-11-2008
DOI: 10.1042/BJ20081238
Abstract: The transcription factor HIF (hypoxia-inducible factor) mediates a highly pleiotrophic response to hypoxia. Many recent studies have focused on defining the extent of this transcriptional response. In the present study we have analysed regulation by hypoxia among transcripts encoding human Fe(II)- and 2-oxoglutarate-dependent oxygenases. Our results show that many of these genes are regulated by hypoxia and define two groups of histone demethylases as new classes of hypoxia-regulated genes. Patterns of induction were consistent across a range of cell lines with JMJD1A (where JMJD is Jumonji-domain containing) and JMJD2B demonstrating robust, and JMJD2C more modest, up-regulation by hypoxia. Functional genetic and chromatin immunoprecipitation studies demonstrated the importance of HIF-1α in mediating these responses. Given the importance of histone methylation status in defining patterns of gene expression under different physiological and pathophysiological conditions, these findings predict a role for the HIF system in epigenetic regulation.
Publisher: Elsevier BV
Date: 04-2015
Publisher: Elsevier BV
Date: 07-2002
Publisher: American Chemical Society (ACS)
Date: 04-01-2013
DOI: 10.1021/JM301583M
Publisher: American Chemical Society (ACS)
Date: 09-07-2019
DOI: 10.1021/ACSCHEMBIO.9B00289
Abstract: Fe(II)- and 2-oxoglutarate (2OG)-dependent JumonjiC domain-containing histone demethylases (JmjC KDMs) are "epigenetic eraser" enzymes involved in the regulation of gene expression and are emerging drug targets in oncology. We screened a set of clinically used iron chelators and report that they potently inhibit JMJD2A (KDM4A)
Publisher: Elsevier BV
Date: 10-2001
DOI: 10.1016/S0092-8674(01)00507-4
Abstract: HIF is a transcriptional complex that plays a central role in mammalian oxygen homeostasis. Recent studies have defined posttranslational modification by prolyl hydroxylation as a key regulatory event that targets HIF-alpha subunits for proteasomal destruction via the von Hippel-Lindau ubiquitylation complex. Here, we define a conserved HIF-VHL-prolyl hydroxylase pathway in C. elegans, and use a genetic approach to identify EGL-9 as a dioxygenase that regulates HIF by prolyl hydroxylation. In mammalian cells, we show that the HIF-prolyl hydroxylases are represented by a series of isoforms bearing a conserved 2-histidine-1-carboxylate iron coordination motif at the catalytic site. Direct modulation of recombinant enzyme activity by graded hypoxia, iron chelation, and cobaltous ions mirrors the characteristics of HIF induction in vivo, fulfilling requirements for these enzymes being oxygen sensors that regulate HIF.
Publisher: Portland Press Ltd.
Date: 10-10-2014
DOI: 10.1042/BJ20140779
Abstract: The prolyl hydroxylase domain proteins (PHDs) catalyse the post-translational hydroxylation of the hypoxia-inducible factor (HIF), a modification that regulates the hypoxic response in humans. The PHDs are Fe(II)/2-oxoglutarate (2OG) oxygenases their catalysis is proposed to provide a link between cellular HIF levels and changes in O2 availability. Transient kinetic studies have shown that purified PHD2 reacts slowly with O2 compared with some other studied 2OG oxygenases, a property which may be related to its hypoxia-sensing role. PHD2 forms a stable complex with Fe(II) and 2OG crystallographic and kinetic analyses indicate that an Fe(II)-co-ordinated water molecule, which must be displaced before O2 binding, is relatively stable in the active site of PHD2. We used active site substitutions to investigate whether these properties are related to the slow reaction of PHD2 with O2. While disruption of 2OG binding in a R383K variant did not accelerate O2 activation, we found that substitution of the Fe(II)-binding aspartate for a glutamate residue (D315E) manifested significantly reduced Fe(II) binding, yet maintained catalytic activity with a 5-fold faster reaction with O2. The results inform on how the precise active site environment of oxygenases can affect rates of O2 activation and provide insights into limiting steps in PHD catalysis.
Publisher: Royal Society of Chemistry (RSC)
Date: 2018
DOI: 10.1039/C8CC00387D
Abstract: The binding of prolyl-hydroxylated HIF-α to PHD2 is hindered by prior 2OG binding likely, leading to the inhibition of HIF-α degradation under limiting 2OG conditions.
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Christopher Schofield.