B22-Multimode InfraRed imaging And Microspectroscopy
I04-1-Macromolecular Crystallography (fixed wavelength)
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Rhiannon M.
Evans
,
Stephen E.
Beaton
,
Patricia
Rodriguez Macia
,
Yunjie
Pang
,
Kin Long
Wong
,
Leonie
Kertess
,
William K.
Myers
,
Ragnar
Bjornsson
,
Philip A.
Ash
,
Kylie A.
Vincent
,
Stephen B.
Carr
,
Fraser A.
Armstrong
Diamond Proposal Number(s):
[12346, 21651]
Open Access
Abstract: The active site of [NiFe]-hydrogenases contains a strictly-conserved pendant arginine, the guanidine head group of which is suspended immediately above the Ni and Fe atoms. Replacement of this arginine (R479) in hydrogenase-2 from E. coli results in an enzyme that is isolated with a very tightly-bound diatomic ligand attached end-on to the Ni and stabilised by hydrogen bonding to the Nζ atom of the pendant lysine and one of the three additional water molecules located in the active site of the variant. The diatomic ligand is bound under oxidising conditions and is removed only after a prolonged period of reduction with H2 and reduced methyl viologen. Once freed of the diatomic ligand, the R479K variant catalyses both H2 oxidation and evolution but with greatly decreased rates compared to the native enzyme. Key kinetic characteristics are revealed by protein film electrochemistry: most importantly, a very low activation energy for H2 oxidation that is not linked to an increased H/D isotope effect. Native electrocatalytic reversibility is retained. The results show that the sluggish kinetics observed for the lysine variant arise most obviously because the advantage of a more favourable low-energy pathway is massively offset by an extremely unfavourable activation entropy. Extensive efforts to establish the identity of the diatomic ligand, the tight binding of which is an unexpected further consequence of replacing the pendant arginine, prove inconclusive.
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Jul 2023
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I03-Macromolecular Crystallography
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Xiao
Liu
,
Raphael
Reinbold
,
Shuang
Liu
,
Ryan A.
Herold
,
Patrick
Rabe
,
Stéphanie
Duclos
,
Rahul B.
Yadav
,
Martine I.
Abboud
,
Sandrine
Thieffine
,
Fraser A.
Armstrong
,
Lennart
Brewitz
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[23459]
Open Access
Abstract: Variants of isocitrate dehydrogenase (IDH) 1 and 2 (IDH1/2) alter metabolism in cancer cells by catalyzing the NADPH-dependent reduction of 2-oxoglutate (2OG) to (2R)-hydroxyglutarate (2HG). However, it is unclear how derivatives of 2OG can affect cancer cell metabolism. Here, we used synthetic C3 and C4 alkylated 2OG derivatives to investigate the substrate selectivities of the most common cancer-associated IDH1 variant (R132H IDH1), of two cancer-associated IDH2 variants (R172K IDH2, R140Q IDH2), and of wildtype IDH1/2. Absorbance-based, NMR and electrochemical assays were employed to monitor wildtype IDH1/2 and IDH1/2 variant-catalyzed 2OG derivative turnover in the presence and absence of 2OG. Our results reveal that 2OG derivatives can serve as substrates of the investigated IDH1/2 variants, but not of wildtype IDH1/2, and have the potential to act as 2OG-competitive inhibitors. Kinetic parameters reveal that some 2OG derivatives, including the natural product 3-methyl-2OG, are equally or even more efficient IDH1/2 variant substrates compared to 2OG. Furthermore, NMR and mass spectrometry studies confirmed IDH1/2 variant-catalyzed production of alcohols in the cases of the 3-methyl-, 3-butyl-, and 3-benzyl-substituted 2OG derivatives; a crystal structure of 3-butyl-2OG with an IDH1 variant (R132C/S280F IDH1) reveals active site binding. The combined results highlight the potential for (i) IDH1/2 variant-catalyzed reduction of 2-oxoacids other than 2OG in cells, (ii) modulation of IDH1/2 variant activity by 2-oxoacid natural products, including some present in common foods, (iii) inhibition of IDH1/2 variants via active site binding rather than the established allosteric mode of inhibition, and (iv) possible use of IDH1/2 variants as biocatalysts.
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Jan 2023
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Raphael
Reinbold
,
Ingvild C.
Hvinden
,
Patrick
Rabe
,
Ryan A.
Herold
,
Alina
Finch
,
James
Wood
,
Melissa
Morgan
,
Maximillian
Staudt
,
Ian J.
Clifton
,
Fraser A.
Armstrong
,
James S. O.
Mccullagh
,
Jo
Redmond
,
Chiara
Bardella
,
Martine I.
Abboud
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[23459]
Open Access
Abstract: Ivosidenib, an inhibitor of isocitrate dehydrogenase 1 (IDH1) R132C and R132H variants, is approved for the treatment of acute myeloid leukaemia (AML). Resistance to ivosidenib due to a second site mutation of IDH1 R132C, leading to IDH1 R132C/S280F, has emerged. We describe biochemical, crystallographic, and cellular studies on the IDH1 R132C/S280F and R132H/S280F variants that inform on the mechanism of second-site resistance, which involves both modulation of inhibitor binding at the IDH1 dimer-interface and alteration of kinetic properties, which enable more efficient 2-HG production relative to IDH1 R132C and IDH1 R132H. Importantly, the biochemical and cellular results demonstrate that it should be possible to overcome S280F mediated resistance in AML patients by using alternative inhibitors, including some presently in phase 2 clinical trials.
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Aug 2022
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B22-Multimode InfraRed imaging And Microspectroscopy
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Philip A.
Ash
,
Sophie E. T.
Kendall-Price
,
Rhiannon M.
Evans
,
Stephen
Carr
,
Amelia
Brasnett
,
Simone
Morra
,
Ricardo
Hidalgo
,
Adam J.
Healy
,
Gianfelice
Cinque
,
Mark D.
Frogley
,
Fraser A
Armstrong
,
Kylie A.
Vincent
Diamond Proposal Number(s):
[17753, 19269, 21651]
Open Access
Abstract: Controlled formation of catalytically-relevant states within crystals of complex metalloenzymes represents a significant challenge to structure-function studies. Here we show how electrochemical control over single crystals of [NiFe] hydrogenase 1 (Hyd1) from Escherichia coli makes it possible to navigate through the full array of active site states previously observed in solution. Electrochemical control is combined with synchrotron infrared microspectroscopy, which enables us to measure high signal-to-noise IR spectra in situ from a small area of crystal. The output reports on active site speciation via the vibrational stretching band positions of the endogenous CO and CN- ligands at the hydrogenase active site. Variation of pH further demonstrates how equilibria between catalytically-relevant protonation states can be deliberately perturbed in the crystals, generating a map of electrochemical potential and pH conditions which lead to enrichment of specific states. Comparison of in crystallo redox titrations with measurements in solution or of electrode-immobilised Hyd1 confirms the integrity of the proton transfer and redox environment around the active site of the enzyme in crystals. Slowed proton-transfer equilibria in the hydrogenase in crystallo reveals transitions which are only usually observable by ultrafast methods in solution. This study therefore demonstrates the possibilities of electrochemical control over single metalloenzyme crystals in stabilising specific states for further study, and extends mechanistic understanding of proton transfer during the [NiFe] hydrogenase catalytic cycle.
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Jun 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[18069]
Abstract: Hydrogenase-1 (Hyd-1) from E. coli poses a conundrum regarding the properties of electrocatalytic reversibility and associated bidirectionality now established for many redox enzymes. Its excellent H2-oxidizing activity begins only once a substantial overpo-tential is applied, and it cannot produce H2. A major reason for its unidirectional behavior is that the reduction potentials of its electron-relaying FeS clusters are too positive relative to the 2H+/H2 couple at neutral pH; consequently, electrons held within the enzyme lack the energy to drive H2 production. However, Hyd-1 is O2-tolerant and even functions in air. Changing a tyrosine (Y) or threonine (T), located on the protein surface within 10 Å of the distal [4Fe-4S] and medial [3Fe-4S] clusters, to cysteine (C), allows site-selective attachment of a silver nanocluster (AgNC), the reduced or photoexcited state of which is a powerful reduct-ant. The AgNC provides a new additional redox site, capturing externally-supplied electrons with sufficiently high energy to drive H2 production. Assemblies of Y’227C (or T’225C) with AgNCs/PMAA (PMAA = polymethylacrylate templating several AgNC) are also electroactive for H2 production at a TiO2 electrode. A colloidal system for visible-light photo-H2 generation is made by building the hybrid enzyme into a heterostructure with TiO2 and graphitic carbon nitride (g-C3N4), the resulting scaffold promoting uptake of electrons excited at the AgNC. Each hydrogenase produces 40 molecules of H2 per second and sustains 20% activity in air.
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Jun 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: Genetically engineering a cysteine (thiolate) close to the distal [4Fe–4S] cluster of a [NiFe]-hydrogenase creates a highly specific target for attachment of Ag nanoclusters templated in polymethyl acrylate, the resulting ‘hard-wired’ enzyme catalysing rapid hydrogen evolution by visible light. The rate is further enhanced by binding to metal oxide nanoparticles – results of investigations focusing on P-25 TiO2 and including anatase TiO2, rutile TiO2, ZnO, SrTiO3 and ZrO2 leading to the proposal that these act as active or structural scaffolds to promote intra-assembly electron transfer.
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Oct 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[12346]
Open Access
Abstract: Catalytic long-range proton transfer in [NiFe]-hydrogenases has long been associated with a highly conserved glutamate (E) situated within 4 Å of the active site. Substituting for glutamine (Q) in the O2-tolerant [NiFe]-hydrogenase-1 from Escherichia coli produces a variant (E28Q) with unique properties that have been investigated using protein film electrochemistry, protein film infrared electrochemistry, and X-ray crystallography. At pH 7 and moderate potential, E28Q displays approximately 1% of the activity of the native enzyme, high enough to allow detailed infrared measurements under steady-state conditions. Atomic-level crystal structures reveal partial displacement of the amide side chain by a hydroxide ion, the occupancy of which increases with pH or under oxidizing conditions supporting formation of the superoxidized state of the unusual proximal [4Fe–3S] cluster located nearby. Under these special conditions, the essential exit pathway for at least one of the H+ ions produced by H2 oxidation, and assumed to be blocked in the E28Q variant, is partially repaired. During steady-state H2 oxidation at neutral pH (i.e., when the barrier to H+ exit via Q28 is almost totally closed), the catalytic cycle is dominated by the reduced states “Nia-R” and “Nia-C”, even under highly oxidizing conditions. Hence, E28 is not involved in the initial activation/deprotonation of H2, but facilitates H+ exit later in the catalytic cycle to regenerate the initial oxidized active state, assumed to be Nia-SI. Accordingly, the oxidized inactive resting state, “Ni-B”, is not produced by E28Q in the presence of H2 at high potential because Nia-SI (the precursor for Ni-B) cannot accumulate. The results have important implications for understanding the catalytic mechanism of [NiFe]-hydrogenases and the control of long-range proton-coupled electron transfer in hydrogenases and other enzymes.
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Aug 2018
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12346]
Abstract: Under anaerobic conditions Escherichia coli is able to metabolize molecular hydrogen via the action of several [NiFe]-hydrogenase enzymes. Hydrogenase-2, which is typically present in cells at low levels during anaerobic respiration, is a periplasmic-facing membrane-bound complex that functions as a proton pump to convert energy from H2 oxidation into a proton gradient; consequently, its structure is of great interest. Empirically, the complex consists of a tightly-bound core catalytic module, comprising large (HybC) and small (HybO) subunits, which is attached to an Fe-S protein (HybA) and an integral membrane protein, HybB. To date, efforts to gain a more detailed picture have been thwarted by low native expression levels of hydrogenase-2 and the labile interaction between HybOC and HybA/HybB subunits. In this paper we describe a new over-expression system that has facilitated determination of high-resolution crystal structures of HybOC and, hence, a prediction of the quaternary structure of the HybOCAB complex.
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Mar 2018
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B22-Multimode InfraRed imaging And Microspectroscopy
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Philip A.
Ash
,
Stephen B.
Carr
,
Holly A.
Reeve
,
Aiste
Skorupskaite
,
Jack
Rowbotham
,
Rebecca
Shutt
,
Mark
Frogley
,
Rhiannon Mari
Evans
,
Gianfelice
Cinque
,
Fraser A.
Armstrong
,
Kylie A.
Vincent
Diamond Proposal Number(s):
[13879]
Open Access
Abstract: We describe an approach to generating and verifying well-defined redox states in metalloprotein single crystals by combining electrochemical control with synchtroton infrared microspectroscopic imaging. For NiFe hydrogenase 1 from Escherichia coli we demonstrate fully reversible and uniform electrochemical reduction from the oxidised inactive to the fully reduced state, and temporally resolve steps during this reduction.
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May 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[12346]
Abstract: The active site of Hyd-1, an oxygen-tolerant membrane-bound [NiFe]-hydrogenase from Escherichia coli, contains four highly conserved residues that form a “canopy” above the bimetallic center, closest to the site at which exogenous agents CO and O2 interact, substrate H2 binds, and a hydrido intermediate is stabilized. Genetic modification of the Hyd-1 canopy has allowed the first systematic and detailed kinetic and structural investigation of the influence of the immediate outer coordination shell on H2 activation. The central canopy residue, arginine 509, suspends a guanidine/guanidinium side chain at close range above the open coordination site lying between the Ni and Fe atoms (N–metal distance of 4.4 Å): its replacement with lysine lowers the H2 oxidation rate by nearly 2 orders of magnitude and markedly decreases the H2/D2 kinetic isotope effect. Importantly, this collapse in rate constant can now be ascribed to a very unfavorable activation entropy (easily overriding the more favorable activation enthalpy of the R509K variant). The second most important canopy residue for H2 oxidation is aspartate 118, which forms a salt bridge to the arginine 509 headgroup: its mutation to alanine greatly decreases the H2 oxidation efficiency, observed as a 10-fold increase in the potential-dependent Michaelis constant. Mutations of aspartate 574 (also salt-bridged to R509) to asparagine and proline 508 to alanine have much smaller effects on kinetic properties. None of the mutations significantly increase sensitivity to CO, but neutralizing the expected negative charges from D118 and D574 decreases O2 tolerance by stabilizing the oxidized resting NiIII–OH state (“Ni-B”). An extensive model of the catalytic importance of residues close to the active site now emerges, whereby a conserved gas channel culminates in the arginine headgroup suspended above the Ni and Fe.
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Dec 2016
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