I03-Macromolecular Crystallography
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Florence J.
Hardy
,
Matthew G.
Quesne
,
Emilie F.
Gérard
,
Jingming
Zhao
,
Mary
Ortmayer
,
Christopher J.
Taylor
,
Hafiz S.
Ali
,
Jeffrey W.
Slater
,
Colin W.
Levy
,
Derren J.
Heyes
,
J. Martin
Bollinger
,
Sam P.
De Visser
,
Anthony P.
Green
Diamond Proposal Number(s):
[24447]
Open Access
Abstract: The ability to introduce noncanonical amino acids as axial ligands in heme enzymes has provided a powerful experimental tool for studying the structure and reactivity of their FeIV═O (“ferryl”) intermediates. Here, we show that a similar approach can be used to perturb the conserved Fe coordination environment of 2-oxoglutarate (2OG) dependent oxygenases, a versatile class of enzymes that employ highly-reactive ferryl intermediates to mediate challenging C–H functionalizations. Replacement of one of the cis-disposed histidine ligands in the oxygenase VioC with a less electron donating Nδ-methyl-histidine (MeHis) preserves both catalytic function and reaction selectivity. Significantly, the key ferryl intermediate responsible for C–H activation can be accumulated in both the wildtype and the modified protein. In contrast to heme enzymes, where metal-oxo reactivity is extremely sensitive to the nature of the proximal ligand, the rates of C–H activation and the observed large kinetic isotope effects are only minimally affected by axial ligand replacement in VioC. This study showcases a powerful tool for modulating the coordination sphere of nonheme iron enzymes that will enhance our understanding of the factors governing their divergent activities.
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Jul 2024
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I03-Macromolecular Crystallography
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Amy E.
Hutton
,
Jake
Foster
,
Rebecca
Crawshaw
,
Florence J.
Hardy
,
Linus O.
Johannissen
,
Thomas M.
Lister
,
Emilie F.
Gérard
,
Zachary
Birch-Price
,
Richard
Obexer
,
Sam
Hay
,
Anthony P.
Green
Diamond Proposal Number(s):
[24447]
Open Access
Abstract: Directed evolution of computationally designed enzymes has provided new insights into the emergence of sophisticated catalytic sites in proteins. In this regard, we have recently shown that a histidine nucleophile and a flexible arginine can work in synergy to accelerate the Morita-Baylis-Hillman (MBH) reaction with unrivalled efficiency. Here, we show that replacing the catalytic histidine with a non-canonical Nδ-methylhistidine (MeHis23) nucleophile leads to a substantially altered evolutionary outcome in which the catalytic Arg124 has been abandoned. Instead, Glu26 has emerged, which mediates a rate-limiting proton transfer step to deliver an enzyme (BHMeHis1.8) that is more than an order of magnitude more active than our earlier MBHase. Interestingly, although MeHis23 to His substitution in BHMeHis1.8 reduces activity by 4-fold, the resulting His containing variant is still a potent MBH biocatalyst. However, analysis of the BHMeHis1.8 evolutionary trajectory reveals that the MeHis nucleophile was crucial in the early stages of engineering to unlock the new mechanistic pathway. This study demonstrates how even subtle perturbations to key catalytic elements of designed enzymes can lead to vastly different evolutionary outcomes, resulting in new mechanistic solutions to complex chemical transformations.
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Mar 2024
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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
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Open Access
Abstract: The catalytic versatility of pentacoordinated iron is highlighted by the broad range of natural and engineered activities of heme enzymes such as cytochrome P450s, which position a porphyrin cofactor coordinating a central iron atom below an open substrate binding pocket. This catalytic prowess has inspired efforts to design de novo helical bundle scaffolds that bind porphyrin cofactors. However, such designs lack the large open substrate binding pocket of P450s, and hence, the range of chemical transformations accessible is limited. Here, with the goal of combining the advantages of the P450 catalytic site geometry with the almost unlimited customizability of de novo protein design, we design a high-affinity heme-binding protein, dnHEM1, with an axial histidine ligand, a vacant coordination site for generating reactive intermediates, and a tunable distal pocket for substrate binding. A 1.6 Å X-ray crystal structure of dnHEM1 reveals excellent agreement to the design model with key features programmed as intended. The incorporation of distal pocket substitutions converted dnHEM1 into a proficient peroxidase with a stable neutral ferryl intermediate. In parallel, dnHEM1 was redesigned to generate enantiocomplementary carbene transferases for styrene cyclopropanation (up to 93% isolated yield, 5000 turnovers, 97:3 e.r.) by reconfiguring the distal pocket to accommodate calculated transition state models. Our approach now enables the custom design of enzymes containing cofactors adjacent to binding pockets with an almost unlimited variety of shapes and functionalities.
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Jul 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12788]
Open Access
Abstract: The availability of an expanded genetic code opens exciting new opportunities in enzyme design and engineering. In this regard histidine analogues have proven particularly versatile, serving as ligands to augment metalloenzyme function and as catalytic nucleophiles in designed enzymes. The ability to genetically encode multiple functional residues could greatly expand the range of chemistry accessible within enzyme active sites. Here we develop mutually orthogonal translation components to selectively encode two structurally similar histidine analogues. Transplanting known mutations from a promiscuous Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRSIFGFF) into a single domain PylRS from Methanomethylophilus alvus (MaPylRSIFGFF) provided a variant with improved efficiency and specificity for 3-methyl-L-histidine (MeHis) incorporation. The MaPylRSIFGFF clone was further characterized using in vitro biochemical assays and X-ray crystallography. We subsequently engineered the orthogonal MmPylRS for activity and selectivity for 3-(3-pyridyl)-L-alanine (3-Pyr), which was used in combination with MaPylRSIFGFF to produce proteins containing both 3-Pyr and MeHis. Given the versatile roles played by histidine in enzyme mechanisms, we anticipate that the tools developed within this study will underpin the development of enzymes with new and enhanced functions.
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Apr 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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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Elizabeth L.
Bell
,
Ross
Smithson
,
Siobhan
Kilbride
,
Jake
Foster
,
Florence J.
Hardy
,
Saranarayanan
Ramachandran
,
Aleksander A.
Tedstone
,
Sarah J.
Haigh
,
Arthur A.
Garforth
,
Philip J. R.
Day
,
Colin
Levy
,
Michael P.
Shaver
,
Anthony P.
Green
Diamond Proposal Number(s):
[12788, 17773]
Abstract: The recent discovery of IsPETase, a hydrolytic enzyme that can deconstruct poly(ethylene terephthalate) (PET), has sparked great interest in biocatalytic approaches to recycle plastics. Realization of commercial use will require the development of robust engineered enzymes that meet the demands of industrial processes. Although rationally engineered PETases have been described, enzymes that have been experimentally optimized via directed evolution have not previously been reported. Here, we describe an automated, high-throughput directed evolution platform for engineering polymer degrading enzymes. Applying catalytic activity at elevated temperatures as a primary selection pressure, a thermostable IsPETase variant (HotPETase, Tm = 82.5 °C) was engineered that can operate at the glass transition temperature of PET. HotPETase can depolymerize semicrystalline PET more rapidly than previously reported PETases and can selectively deconstruct the PET component of a laminated multimaterial. Structural analysis of HotPETase reveals interesting features that have emerged to improve thermotolerance and catalytic performance. Our study establishes laboratory evolution as a platform for engineering useful plastic degrading enzymes.
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Aug 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[17773]
Abstract: The combination of computational design and directed evolution could offer a general strategy to create enzymes with new functions. So far, this approach has delivered enzymes for a handful of model reactions. Here we show that new catalytic mechanisms can be engineered into proteins to accelerate more challenging chemical transformations. Evolutionary optimization of a primitive design afforded an efficient and enantioselective enzyme (BH32.14) for the Morita–Baylis–Hillman (MBH) reaction. BH32.14 is suitable for preparative-scale transformations, accepts a broad range of aldehyde and enone coupling partners and is able to promote selective monofunctionalizations of dialdehydes. Crystallographic, biochemical and computational studies reveal that BH32.14 operates via a sophisticated catalytic mechanism comprising a His23 nucleophile paired with a judiciously positioned Arg124. This catalytic arginine shuttles between conformational states to stabilize multiple oxyanion intermediates and serves as a genetically encoded surrogate of privileged bidentate hydrogen-bonding catalysts (for example, thioureas). This study demonstrates that elaborate catalytic devices can be built from scratch to promote demanding multi-step processes not observed in nature.
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Dec 2021
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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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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]
Open Access
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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