I20-Scanning-X-ray spectroscopy (XAS/XES)
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Jingming
Zhao
,
Ying
Zhuo
,
Daniel E.
Diaz
,
Muralidharan
Shanmugam
,
Abbey J.
Telfer
,
Peter J.
Lindley
,
Daniel
Kracher
,
Takahiro
Hayashi
,
Lisa S.
Seibt
,
Florence J.
Hardy
,
Oliver
Manners
,
Tobias M.
Hedison
,
Katherine A.
Hollywood
,
Reynard
Spiess
,
Kathleen M.
Cain
,
Sofia
Diaz-Moreno
,
Nigel S.
Scrutton
,
Morten
Tovborg
,
Paul H.
Walton
,
Derren J.
Heyes
,
Anthony P.
Green
Diamond Proposal Number(s):
[28477]
Open Access
Abstract: Oxygenase and peroxygenase enzymes generate intermediates at their active sites which bring about the controlled functionalization of inert C–H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which can occur during both coupled and uncoupled turnover. Herein, we use a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, high-energy resolution fluorescence detection X-ray absorption spectroscopy, and electron paramagnetic resonance spectroscopy to study two transient intermediates that together form a protective pathway built into the active sites of copper-dependent lytic polysaccharide monooxygenases (LPMOs). First, a transient high-valent species is generated at the copper histidine brace active site following treatment of the LPMO with either hydrogen peroxide or peroxyacids in the absence of substrate. This intermediate, which we propose to be a CuII–(histidyl radical), then reacts with a nearby tyrosine residue in an intersystem-crossing reaction to give a ferromagnetically coupled (S = 1) CuII–tyrosyl radical pair, thereby restoring the histidine brace active site to its resting state and allowing it to re-enter the catalytic cycle through reduction. This process gives the enzyme the capacity to minimize damage to the active site histidine residues “on the fly” to increase the total turnover number prior to enzyme deactivation, highlighting how oxidative enzymes are evolved to protect themselves from deleterious side reactions during uncoupled turnover.
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Sep 2023
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Harshwardhan
Poddar
,
Ronald
Rios-Santacruz
,
Derren J.
Heyes
,
Muralidharan
Shanmugam
,
Adam
Brookfield
,
Linus O.
Johannissen
,
Colin W.
Levy
,
Laura N.
Jeffreys
,
Shaowei
Zhang
,
Michiyo
Sakuma
,
Jacques-Philippe
Colletier
,
Sam
Hay
,
Giorgio
Schirò
,
Martin
Weik
,
Nigel S.
Scrutton
,
David
Leys
Diamond Proposal Number(s):
[24447]
Open Access
Abstract: CarH is a coenzyme B12-dependent photoreceptor involved in regulating carotenoid biosynthesis. How light-triggered cleavage of the B12 Co-C bond culminates in CarH tetramer dissociation to initiate transcription remains unclear. Here, a series of crystal structures of the CarH B12-binding domain after illumination suggest formation of unforeseen intermediate states prior to tetramer dissociation. Unexpectedly, in the absence of oxygen, Co-C bond cleavage is followed by reorientation of the corrin ring and a switch from a lower to upper histidine-Co ligation, corresponding to a pentacoordinate state. Under aerobic conditions, rapid flash-cooling of crystals prior to deterioration upon illumination confirm a similar B12-ligand switch occurs. Removal of the upper His-ligating residue prevents monomer formation upon illumination. Combined with detailed solution spectroscopy and computational studies, these data demonstrate the CarH photoresponse integrates B12 photo- and redox-chemistry to drive large-scale conformational changes through stepwise Co-ligation changes.
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Aug 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[24447]
Abstract: The ability to programme new modes of catalysis into proteins would allow the development of enzyme families with functions beyond those found in nature. To this end, genetic code expansion methodology holds particular promise, as it allows the site-selective introduction of new functional elements into proteins as non-canonical amino acid side chains. Here, we exploit an expanded genetic code to develop a photoenzyme that operates via triplet energy transfer catalysis, a versatile mode of reactivity in organic synthesis that is currently not accessible to biocatalysis. Installation of a genetically encoded photosensitiser into the beta-propeller scaffold of DA_20_0013 converts a de novo Diels-Alderase into a photoenzyme for [2+2]-cycloadditions (EnT1.0). Subsequent development and implementation of a platform for photoenzyme evolution afforded an efficient and enantioselective enzyme (EnT1.3, up to 99% e.e.) that can promote intramolecular and bimolecular cycloadditions, including transformations that have proven challenging to achieve selectively with small molecule catalysts. EnT1.3 performs >300 turnovers and, in contrast to small molecule photocatalysts, can operate effectively under aerobic conditions and at ambient temperatures. An X-ray crystal structure of an EnT1.3-product complex shows how multiple functional components work in synergy to promote efficient and selective photocatalysis. This study opens up a wealth of new excited-state chemistry in protein active sites and establishes the framework for developing a new generation of enantioselective photocatalysts.
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Sep 2022
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I24-Microfocus Macromolecular Crystallography
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Samuel L.
Rose
,
Seiki
Baba
,
Hideo
Okumura
,
Svetlana V.
Antonyuk
,
Daisuke
Sasaki
,
Tobias M.
Hedison
,
Muralidharan
Shanmugam
,
Derren J.
Heyes
,
Nigel S.
Scrutton
,
Takashi
Kumasaka
,
Takehiko
Tosha
,
Robert R.
Eady
,
Masaki
Yamamoto
,
S. Samar
Hasnain
Open Access
Abstract: Many enzymes utilize redox-coupled centers for performing catalysis where these centers are used to control and regulate the transfer of electrons required for catalysis, whose untimely delivery can lead to a state incapable of binding the substrate, i.e., a dead-end enzyme. Copper nitrite reductases (CuNiRs), which catalyze the reduction of nitrite to nitric oxide (NO), have proven to be a good model system for studying these complex processes including proton-coupled electron transfer (ET) and their orchestration for substrate binding/utilization. Recently, a two-domain CuNiR from a Rhizobia species (Br2DNiR) has been discovered with a substantially lower enzymatic activity where the catalytic type-2 Cu (T2Cu) site is occupied by two water molecules requiring their displacement for the substrate nitrite to bind. Single crystal spectroscopy combined with MSOX (multiple structures from one crystal) for both the as-isolated and nitrite-soaked crystals clearly demonstrate that inter-Cu ET within the coupled T1Cu-T2Cu redox system is heavily gated. Laser-flash photolysis and optical spectroscopy showed rapid ET from photoexcited NADH to the T1Cu center but little or no inter-Cu ET in the absence of nitrite. Furthermore, incomplete reoxidation of the T1Cu site (∼20% electrons transferred) was observed in the presence of nitrite, consistent with a slow formation of NO species in the serial structures of the MSOX movie obtained from the nitrite-soaked crystal, which is likely to be responsible for the lower activity of this CuNiR. Our approach is of direct relevance for studying redox reactions in a wide range of biological systems including metalloproteins that make up at least 30% of all proteins.
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Jul 2022
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I04-Macromolecular Crystallography
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Mary
Ortmayer
,
Florence J.
Hardy
,
Matthew G.
Quesne
,
Karl
Fisher
,
Colin
Levy
,
Derren J.
Heyes
,
C. Richard A.
Catlow
,
Sam P.
De Visser
,
Stephen E. J.
Rigby
,
Sam
Hay
,
Anthony P.
Green
Diamond Proposal Number(s):
[12788]
Open Access
Abstract: Nature employs high-energy metal-oxo intermediates embedded within enzyme active sites to perform challenging oxidative transformations with remarkable selectivity. Understanding how different local metal-oxo coordination environments control intermediate reactivity and catalytic function is a long-standing objective. However, conducting structure–activity relationships directly in active sites has proven challenging due to the limited range of amino acid substitutions achievable within the constraints of the genetic code. Here, we use an expanded genetic code to examine the impact of hydrogen bonding interactions on ferryl heme structure and reactivity, by replacing the N–H group of the active site Trp51 of cytochrome c peroxidase by an S atom. Removal of a single hydrogen bond stabilizes the porphyrin π-cation radical state of CcP W191F compound I. In contrast, this modification leads to more basic and reactive neutral ferryl heme states, as found in CcP W191F compound II and the wild-type ferryl heme-Trp191 radical pair of compound I. This increased reactivity manifests in a >60-fold activity increase toward phenolic substrates but remarkably has negligible effects on oxidation of the biological redox partner cytc. Our data highlight how Trp51 tunes the lifetimes of key ferryl intermediates and works in synergy with the redox active Trp191 and a well-defined substrate binding site to regulate catalytic function. More broadly, this work shows how noncanonical substitutions can advance our understanding of active site features governing metal-oxo structure and reactivity.
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May 2021
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[22724]
Open Access
Abstract: Protochlorophyllide oxidoreductase (POR) catalyses reduction of protochlorophyllide (Pchlide) to chlorophyllide, a light‐dependent reaction of chlorophyll biosynthesis. POR is also important in plant development as it is the main constituent of prolamellar bodies in etioplast membranes. Prolamellar bodies are highly organised, paracrystalline structures comprising aggregated oligomeric structures of POR–Pchlide–NADPH complexes. How these oligomeric structures are formed and the role of Pchlide in oligomerisation remains unclear. POR crystal structures highlight two peptide regions that form a ‘lid’ to the active site, and undergo conformational change on binding Pchlide. Here, we show that Pchlide binding triggers formation of large oligomers of POR using size exclusion chromatography. A POR ‘octamer’ has been isolated and its structure investigated by cryo‐electron microscopy at 7.7 Å resolution. This structure shows that oligomer formation is most likely driven by the interaction of amino acid residues in the highly conserved lid regions. Computational modelling indicates that Pchlide binding stabilises exposure of hydrophobic surfaces formed by the lid regions, which supports POR dimerisation and ultimately oligomer formation. Studies with variant PORs demonstrate that lid residues are involved in substrate binding and photocatalysis. These highly conserved lid regions therefore have a dual function. The lid residues position Pchlide optimally to enable photocatalysis. Following Pchlide binding, they also enable POR oligomerisation – a process that is reversed through subsequent photocatalysis in the early stages of chloroplast development.
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Aug 2020
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I04-Macromolecular Crystallography
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Mary
Ortmayer
,
Karl
Fisher
,
Jaswir
Basran
,
Emmanuel M.
Wolde-Michael
,
Derren J.
Heyes
,
Colin
Levy
,
Sarah
Lovelock
,
J. L. Ross
Anderson
,
Emma L.
Raven
,
Sam
Hay
,
Stephen E. J.
Rigby
,
Anthony P.
Green
Diamond Proposal Number(s):
[12788]
Abstract: Nature employs a limited number of genetically encoded axial ligands to control diverse heme enzyme activities. Deciphering the functional significance of these ligands requires a quantitative understanding of how their electron donating capabilities modulate the structures and reactivities of the iconic ferryl intermediates compounds I and II. However, probing these relationships experimentally has proven challenging as ligand substitutions accessible via conventional mutagenesis do not allow fine tuning of electron donation and typically abolish catalytic function. Here we exploit engineered translation components to replace the histidine ligand of cytochrome c peroxidase (CcP) by a less electron donating Nδ-methyl histidine (Me-His) with little effect on enzyme structure. The rate of formation (k1) and the reactivity (k2) of compound I are unaffected by ligand substitution. In contrast, proton coupled electron transfer to compound II (k3) is 10-fold slower in CcP Me-His, providing a direct link between electron do-nation and compound II reactivity which can be explained by weaker electron donation from the Me-His ligand (‘the push’) affording an electron deficient ferryl-oxygen with reduced proton affinity (‘the pull’). The deleterious effects of the Me-His ligand can be fully compensated by introducing a W51F mutation, designed to increase ‘the pull’ by removing a hydrogen bond to the ferryl-oxygen. Analogous substitutions in ascorbate peroxidase (APX) lead to similar activity trends to those observed in CcP, suggesting a common mechanistic strategy is employed by enzymes using distinct electron transfer pathways. Our study highlights how non-canonical active site substitutions can be used to directly probe and deconstruct highly evolved bioinorganic mechanisms.
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Jan 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Shaowei
Zhang
,
Derren J.
Heyes
,
Lingling
Feng
,
Wenli
Sun
,
Linus O.
Johannissen
,
Huanting
Liu
,
Colin
Levy
,
Xuemei
Li
,
Ji
Yang
,
Xiaolan
Yu
,
Min
Lin
,
Samantha J. O.
Hardman
,
Robin
Hoeven
,
Michiyo
Sakuma
,
Sam
Hay
,
David
Leys
,
Zihe
Rao
,
Aiwu
Zhou
,
Qi
Cheng
,
Nigel S.
Scrutton
Diamond Proposal Number(s):
[8997, 12788]
Abstract: The enzyme protochlorophyllide oxidoreductase (POR) catalyses a light-dependent step in chlorophyll biosynthesis that is essential to photosynthesis and, ultimately, all life on Earth1,2,3. POR, which is one of three known light-dependent enzymes4,5, catalyses reduction of the photosensitizer and substrate protochlorophyllide to form the pigment chlorophyllide. Despite its biological importance, the structural basis for POR photocatalysis has remained unknown. Here we report crystal structures of cyanobacterial PORs from Thermosynechococcus elongatus and Synechocystis sp. in their free forms, and in complex with the nicotinamide coenzyme. Our structural models and simulations of the ternary protochlorophyllide–NADPH–POR complex identify multiple interactions in the POR active site that are important for protochlorophyllide binding, photosensitization and photochemical conversion to chlorophyllide. We demonstrate the importance of active-site architecture and protochlorophyllide structure in driving POR photochemistry in experiments using POR variants and protochlorophyllide analogues. These studies reveal how the POR active site facilitates light-driven reduction of protochlorophyllide by localized hydride transfer from NADPH and long-range proton transfer along structurally defined proton-transfer pathways.
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Oct 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Shaowei
Zhang
,
Michiyo
Sakuma
,
Girdhar S.
Deora
,
Colin
Levy
,
Alex
Klausing
,
Carlo
Breda
,
Kevin D.
Read
,
Chris D.
Edlin
,
Benjamin P.
Ross
,
Marina
Wright Muelas
,
Philip J.
Day
,
Stephen
O’hagan
,
Douglas B.
Kell
,
Robert
Schwarcz
,
David
Leys
,
Derren J.
Heyes
,
Flaviano
Giorgini
,
Nigel S.
Scrutton
Diamond Proposal Number(s):
[8997, 12788]
Open Access
Abstract: Dysregulation of the kynurenine pathway (KP) leads to imbalances in neuroactive metabolites associated with the pathogenesis of several neurodegenerative disorders, including Huntington’s disease (HD). Inhibition of the enzyme kynurenine 3-monooxygenase (KMO) in the KP normalises these metabolic imbalances and ameliorates neurodegeneration and related phenotypes in several neurodegenerative disease models. KMO is thus a promising candidate drug target for these disorders, but known inhibitors are not brain permeable. Here, 19 new KMO inhibitors have been identified. One of these (1) is neuroprotective in a Drosophila HD model but is minimally brain penetrant in mice. The prodrug variant (1b) crosses the blood–brain barrier, releases 1 in the brain, thereby lowering levels of 3-hydroxykynurenine, a toxic KP metabolite linked to neurodegeneration. Prodrug 1b will advance development of targeted therapies against multiple neurodegenerative and neuroinflammatory diseases in which KP likely plays a role, including HD, Alzheimer’s disease, and Parkinson’s disease.
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Jul 2019
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Tobias
Hedison
,
Rajesh
Shenoy
,
Andreea Iulia
Iorgu
,
Derren
Heyes
,
Karl
Fisher
,
Gareth
Wright
,
Sam
Hay
,
Robert Roy
Eady
,
Svetlana
Antonyuk
,
S. Samar
Hasnain
,
Nigel S.
Scrutton
Diamond Proposal Number(s):
[11740]
Open Access
Abstract: It is generally assumed that tethering enhances rates of electron harvesting and delivery to active sites in multi-domain enzymes by proximity and sampling mechanisms. Here, we explore this idea in a tethered 3-domain, trimeric copper-containing nitrite reductase. By reverse engineering, we find that tethering does not enhance the rate of electron delivery from its pendant cytochrome c to the catalytic copper-containing core. Using a linker that harbors a gatekeeper tyrosine in a nitrite access channel, the tethered haem domain enables catalysis by other mechanisms. Tethering communicates the redox state of the haem to the distant T2Cu center that helps initiate substrate binding for catalysis. It also tunes copper reduction potentials, suppresses reductive enzyme inactivation, enhances enzyme affinity for substrate and promotes inter-copper electron transfer. Tethering has multiple unanticipated beneficial roles, the combination of which fine-tunes function beyond simplistic mechanisms expected from proximity and restrictive sampling models.
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May 2019
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