ORCID Profile
0000-0002-4169-4278
Current Organisation
University of Oxford
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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: Oxford University Press (OUP)
Date: 16-10-2014
DOI: 10.1093/JAC/DKU403
Publisher: Elsevier BV
Date: 07-2018
Publisher: American Association for the Advancement of Science (AAAS)
Date: 05-07-2019
Abstract: The ability to sense and respond to changes in oxygen levels is critical for most forms of life. To date, mechanistic studies of this process in mammals have focused on the oxygen-sensitive stability of a transcription factor called hypoxia-inducible factor. Masson et al. discovered an enzymatic oxygen sensor in humans that is functionally identical to plant cysteine oxidases, enzymes that control responses to hypoxia in plants. The human and plant enzymes convert the N-terminal cysteine in substrate proteins to cysteine sulfinic acid, a modification that ultimately targets the proteins for degradation. Oxygen sensing is impaired in many human diseases, and further study of the human enzyme could help in the development of strategies for therapeutic intervention. Science , this issue p. 65
Publisher: Springer Science and Business Media LLC
Date: 05-03-2014
DOI: 10.1038/NCOMMS4423
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: 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: Elsevier BV
Date: 04-2016
Publisher: American Chemical Society (ACS)
Date: 21-09-2015
Publisher: Informa UK Limited
Date: 07-2020
Publisher: Springer Science and Business Media LLC
Date: 23-03-2017
DOI: 10.1038/NCOMMS14690
Abstract: Crop yield loss due to flooding is a threat to food security. Submergence-induced hypoxia in plants results in stabilization of group VII ETHYLENE RESPONSE FACTORs (ERF-VIIs), which aid survival under these adverse conditions. ERF-VII stability is controlled by the N-end rule pathway, which proposes that ERF-VII N-terminal cysteine oxidation in normoxia enables arginylation followed by proteasomal degradation. The PLANT CYSTEINE OXIDASEs (PCOs) have been identified as catalysts of this oxidation. ERF-VII stabilization in hypoxia presumably arises from reduced PCO activity. We directly demonstrate that PCO dioxygenase activity produces Cys-sulfinic acid at the N terminus of an ERF-VII peptide, which then undergoes efficient arginylation by an arginyl transferase (ATE1). This provides molecular evidence of N-terminal Cys-sulfinic acid formation and arginylation by N-end rule pathway components, and a substrate of ATE1 in plants. The PCOs and ATE1 may be viable intervention targets to stabilize N-end rule substrates, including ERF-VIIs, to enhance submergence tolerance in agriculture.
Publisher: Proceedings of the National Academy of Sciences
Date: 31-08-2020
Abstract: The plant cysteine oxidases (PCOs) have been identified as oxygen-sensing enzymes in plants, controlling hypoxia-dependent processes, including adaptive responses to flooding. As such, they are potential targets for engineering plants with enhanced flood tolerance. To approach this in a rational manner requires an understanding of how PCO structure relates to their ability to trigger hypoxic adaptation. We report the structures of two PCOs from Arabidopsis and show that the effects of mutagenesis of key amino acids at their active site are seen both in vitro and, importantly, in planta. This work provides a platform for further efforts to manipulate PCO structure and function to improve the ability of crops to withstand future climate extremes.
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.
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 Emily Flashman.