I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[13587, 18598]
Abstract: Alternatives to petroleum-based chemicals are highly sought-after for on-going efforts to reduce the damaging effects of human activity on the environment. Copper radical oxidases from Auxiliary Activity Family 5/Subfamily 2 (AA5_2) are attractive biocatalysts because they oxidize primary alcohols in a chemo-selective manner without complex organic cofactors. However, despite numerous studies on canonical galactose oxidases (GalOx, EC 1.1.3.9) and engineered variants, and the recent discovery of a Colletotrichum graminicola copper radical alcohol oxidase (AlcOx, EC 1.1.3.13), the catalytic potentials of very few AA5_2 members have been characterized. Guided by sequence similarity network and phylogenetic analyses, in this study we targeted a distinct paralog from the fungus C. graminicola as a representative member of a large uncharacterized subgroup of AA5_2. Through recombinant production and detailed kinetic analysis, we demonstrated that this enzyme is weakly active towards carbohydrates, but efficiently catalyzes the oxidation of aryl alcohols to the corresponding aldehydes. As such, this represents the initial characterization of a demonstrable aryl alcohol oxidase (AAO, EC 1.1.3.7) in AA5, an activity which is classically associated with flavin-dependent glucose-methanol-choline (GMC) oxidoreductases of Auxiliary Activity Family 3 (AA3). X-ray crystallography revealed a distinct multidomain architecture comprising an N-terminal PAN domain abutting a canonical AA5 seven-bladed propeller catalytic domain. Of direct relevance to biomass processing, the wild-type enzyme exhibits the highest activity on the primary alcohol of 5-hydroxymethylfurfural (HMF), a product of significant interest in the lignocellulosic bio-refinery concept. Thus, the chemoselective oxidation of HMF to 2,5-diformylfuran (DFF) by C. graminicola aryl alcohol oxidase (CgrAAO) from AA5 provides a fundamental building block for chemistry via biotechnology.
|
Feb 2020
|
|
E02-JEM ARM 300CF
|
Diamond Proposal Number(s):
[23504]
Abstract: The emergence of nickel single-atoms on nitrogen-doped carbons as high-performance catalysts amenable to rationalization due to their well-defined structure could lead to applicable technologies for the electrocatalytic CO2 reduction reaction (eCO2RR). However, real materials are unlikely to display a uniform site structure, which limits the scope of current efforts focused on idealized models for future implementation. Here, we prepare distinct nickel entities (single atoms or nanoparticles) on nitrogen-doped carbons and evaluate them in eCO2RR. Single atoms demonstrate a characteristic high selectivity to CO. However, this is not altered by the presence of metal nanoparticles formed upon reducing the nitrogen content of the carrier. In contrast, nanoparticles incorporated via a colloidal route promote the parasitic hydrogen evolution reaction. In these systems, the CO selectivity evolves upon repeated exposure to potential, reaching values comparable to single atoms. By introducing CO stripping voltammetry as a characterization tool for this class of materials, we identify a decreased metallic surface, suggesting that the nanoparticle surface is altered by CO. The findings highlight the critical role of dynamic effects in catalyst design for eCO2RR.
|
Feb 2020
|
|
|
|
Abstract: The construction and manipulation of amine-containing architectures is of importance to academic and industrial development and discovery programs. The photochemical single-electron reduction of imine derivatives to generate α-amino radical intermediates has emerged as a powerful umpolung strategy for opening up underexplored routes to such amine motifs. Furthermore, these radicals have been shown to engage in a wide variety of chemistry, including radical–radical coupling, addition to electrophiles, and reductive amination chemistry. The concept has also begun to see application to iminium ion intermediates and the extension to enantiocontrolled C–C bond formation. This Perspective covers recent efforts in this synthetic strategy to simple and complex amine structures alike.
|
Jan 2020
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[18598]
Abstract: 2,3-Dihydroxypropanesulfonate (DHPS) is a major sulfur species in the biosphere. One important route for the production of DHPS is sulfoglycolytic catabolism of sulfoquinovose (SQ) through the Embden-Meyerhof-Parnas (sulfo-EMP) pathway. SQ is a sulfonated carbohydrate present in plant and cyanobacterial sulfolipids (sulfoquinovosyl diacylglyceride and its metabolites) and is biosynthesised globally at a rate of around 10 billion tonnes per annum. The final step in the bacterial sulfo-EMP pathway involves reduction of sulfolactaldehyde (SLA) to DHPS, catalysed by an NADH-dependent SLA reductase. Based on conserved sequence motifs, we assign SLA reductase to the β-hydroxyacid dehydrogenase (β-HAD) family, an example of a β-HAD enzyme that acts on a sulfonic acid substrate, rather than a carboxylic acid. We report crystal structures of the SLA reductase YihU from E. coli K-12 in its apo and cofactor-bound states, as well as a ternary complex YihU•NADH•DHPS with the cofactor and product bound in the active site. Conformational flexibility observed in these structures, combined with kinetic studies, confirm a sequential mechanism and provide evidence for dynamic domain movements that occur during catalysis. The ternary complex structure reveals a conserved sulfonate pocket in SLA reductase that recognises the sulfonate oxygens through hydrogen bonding to Asn174, Ser178, and the backbone amide of Arg123, along with an ordered water molecule. This triad of residues distinguishes these enzymes from classical β-HADs that act on carboxylate substrates. A comparison of YihU crystal structures with close structural homologues within the β-HAD family highlights key differences in the overall domain organization and identifies a peptide sequence that is predictive of SLA reductase activity.
|
Jan 2020
|
|
I04-Macromolecular Crystallography
|
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.
|
Jan 2020
|
|
|
|
Jeremy
Nugent
,
Carlos
Arroniz
,
Bethany R.
Shire
,
Alistair J.
Sterling
,
Helena D.
Pickford
,
Marie L. J.
Wong
,
Steven
Mansfield
,
Dimitri F. J.
Caputo
,
Benjamin
Owen
,
James J.
Mousseau
,
Fernanda
Duarte
,
Edward A.
Anderson
Abstract: Photoredox catalysis has transformed the landscape of radical-based synthetic chemistry. Additions of radicals generated through photoredox catalysis to carbon–carbon π-bonds are well-established; however, this approach has yet to be applied to the functionalization of carbon–carbon σ-bonds. Here, we report the first such use of photoredox catalysis to promote the addition of organic halides to the carbocycle [1.1.1]propellane; the product bicyclo[1.1.1]pentanes (BCPs) are motifs of high importance in the pharmaceutical industry and in materials chemistry. Showing broad substrate scope and functional group tolerance, this methodology results in the first examples of bicyclopentylation of sp2 carbon–halogen bonds to access (hetero)arylated BCPs, as well as the functionalization of nonstabilized sp3 radicals. Substrates containing alkene acceptors allow the single-step construction of polycyclic bicyclopentane products through unprecedented atom transfer radical cyclization cascades, while the potential to accelerate drug discovery is demonstrated through late-stage bicyclopentylations of natural productlike and druglike molecules. Mechanistic investigations demonstrate the importance of the photocatalyst in this chemistry and provide insight into the balance of radical stability and strain relief in the reaction cycle.
|
Sep 2019
|
|
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[17201]
Abstract: Norcoclaurine synthase (NCS) catalyzes a stereoselective
Pictet−Spengler reaction to give the key intermediate, (S)-norcoclaurine
in benzylisoquinoline alkaloid biosynthesis. This family of alkaloids
contains many bioactive molecules including morphine and berberine.
Recently, NCS has been demonstrated to accept a variety of aldehydes
and some ketones as substrates, leading to a range of chiral
tetrahydroisoquinoline (THIQ) products. Here, we report the unusual
acceptance of α-substituted aldehydes, in particular α-methyl-substituted
aldehydes, by wild-type Thalictrum flavum NCS (Δ33Tf NCS) to give
THIQ products. Moreover, the kinetic resolution of several α-
substituted aldehydes to give THIQs with two defined chiral centers
in a single step with high conversions was achieved. Several dopamine analogues were also accepted as substrates, and reactions
were amenable to scale up. Active site mutants of TfNCS were then used, which demonstrated the potential to enhance the
stereoselectivities in the reaction and improve yields. The rationale for the acceptance of these substrates and improved activity
with different mutants has been gained from a co-crystallized structure of Δ33TfNCS with a nonproductive mimic of a reaction
intermediate bound in the active site. Finally, molecular dynamics simulations were performed to study the binding of dopamine
and an α-substituted aldehyde and provided further insight into the reaction with these substrates.
|
Sep 2019
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[10515]
Abstract: Hydrogenases are metalloenzymes that catalyze the redox conversion between H2 and protons. The so-called [NiFeSe] hydrogenases are highly active for both H2 production and oxidation, but like all hydrogenases, they are inhibited by O2. In the [NiFeSe] enzyme from Desulfovibrio vulgaris Hildenborough this results from the oxidation of an active site cysteine ligand. We designed mutations that constrict a hydrophilic channel that connects the protein surface to this active site cysteine. Two of the variants show markedly increased tolerance to O2 inactivation, while retaining high catalytic activities in both directions of the reaction, and structural studies confirm that these mutations prevent the oxidation of the cysteine. Our results indicate that the diffusion of O2 or ROS to the active site can occur through a hydrophilic water channel, in contrast to the widely held assumption that only hydrophobic channels are involved in active site inactivation. This provides an original strategy for optimizing the enzyme by protein engineering.
|
Aug 2019
|
|
I03-Macromolecular Crystallography
|
Emma Zsófia Aletta
Nagy
,
Souad Diana
Tork
,
Pauline A.
Lang
,
Alina
Filip
,
Florin Dan
Irimie
,
László
Poppe
,
Monica Ioana
Toşa
,
Christopher J.
Schofield
,
Jurgen
Brem
,
Csaba
Paizs
,
Laszlo Csaba
Bencze
Diamond Proposal Number(s):
[19458]
Abstract: Modification of the hydrophobic binding pocket of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) enables increased activity and selectivity towards phenylalanines and cinnamic acids mono-substituted with both electron donating (-CH3, -OCH3) and electron withdrawing (-CF3, -Br) groups at all positions (o-, m-, p-) of their aromatic ring. The results reveal specific residues involved in accommodating substituents at o-, m-, p-positions of the substrate’s phenyl ring. The predicted interactions were validated by crystallographic analysis of the binding mode of para-methoxy cinnamic acid complexed at the active site of PcPAL. The biocatalytic utility of the tailored PcPAL mutants was demonstrated by the efficient preparative scale synthesis of (S)-m-bromo-phenylalanine (ee: > 99%, yield: 60%) and (R)-p-methyl-phenylalanine (ee: 97%, yield: 49%), using the corresponding ammonia addition and ammonia elimination reactions catalyzed by the L134A and I460V PcPAL variants, respectively. Overall, the results reveal the potential for structure based protein engineering of PALs to provide enzymes with enhanced catalytic properties and which are specifically tailored for differently substituted phenylalanine analogues of high synthetic value.
|
Aug 2019
|
|
B18-Core EXAFS
|
Diamond Proposal Number(s):
[16250]
Abstract: Energy storage solutions are a vital component of the global transition towards renewable energy sources. The Power-to-Gas (PtG) concept, which stores surplus renewable energy in the form of methane, has therefore become increasingly relevant in recent years. At present, supported Ni nanoparticles are preferred as industrial catalysts for CO2 methanation due to their low cost and high methane selectivity. However, commercial Ni catalysts are not active enough in CO2 methanation to reach the high CO2 conversion (>99 %) required by the specifications for injection in the natural gas grid. Herein we demonstrate the promise of promotion of Ni by Mn, another low-cost base metal, for obtaining very active CO2 methanation catalysts, with results comparable to more expensive precious metal-based catalysts. The origin of this improved performance is revealed by a combined approach of nanoscale characterization, mechanistic study, and density functional theory calculations. Nanoscale characterization with STEM-EDX and X-ray absorption spectroscopy shows that NiMn catalysts consist of metallic Ni particles decorated by oxidic Mn2+ species. A mechanistic study combining IR spectroscopy of surface adsorbates, transient kinetic analysis with isotopically labelled CO2, density functional theory calculations and microkinetics simulations ascertain that the MnO clusters enhance CO2 adsorption and facilitate CO2 activation. A macroscale perspective was achieved by simulating the Ni and NiMn catalytic activity in a Sabatier reactor, which revealed that NiMn catalysts have the potential to meet the demanding PtG catalyst performance requirements, and can largely replace the need for expensive and scarce noble metal catalysts.
|
Jul 2019
|
|
