Glutamate Side Chain Research Articles

Identification of a Polypeptide Inhibitor of O-GlcNAc Transferase with Picomolar Affinity.

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that binds thousands of different proteins, including substrates that it glycosylates and nonsubstrate interactors that regulate its biology. OGT also has one proteolytic substrate, the transcriptional coregulator host cell factor 1 (HCF-1), which it cleaves in a process initiated by glutamate side chain glycosylation at a series of central repeats. Although HCF-1 is OGT's most prominent binding partner, its affinity for the enzyme has not been quantified. Here, we report a time-resolved Förster resonance energy transfer assay to measure ligand binding to OGT and show that an HCF-1-derived polypeptide (HCF3R) binds with picomolar affinity to the enzyme (KD ≤ 85 pM). This high affinity is driven in large part by conserved asparagines in OGT's tetratricopeptide repeat domain, which form bidentate contacts to the HCF-1 peptide backbone; replacing any one of these asparagines with alanine reduces binding by more than 5 orders of magnitude. Because the HCF-1 polypeptide binds so tightly to OGT, we tested its ability to inhibit enzymatic function. We found that HCF3R potently inhibits OGT both in vitro and in cells and used this finding to develop a genetically encoded, inducible OGT inhibitor that can be degraded with a small molecule, allowing for reversible and tunable inhibition of OGT.

Insights into the function and regulation of the calcium-activated chloride channel TMEM16A

The TMEM16A channel, a member of the TMEM16 protein family comprising chloride (Cl−) channels and lipid scramblases, is activated by the free intracellular Ca2+ increments produced by inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release after GqPCRs or Ca2+ entry through cationic channels. It is a ubiquitous transmembrane protein that participates in multiple physiological functions essential to mammals' lives. TMEM16A structure contains two identical 10-segment monomers joined at their transmembrane segment 10. Each monomer harbours one independent hourglass-shaped pore gated by Ca2+ ligation to an orthosteric site adjacent to the pore and controlled by two gates. The orthosteric site is created by assembling negatively charged glutamate side chains near the pore´s cytosolic end. When empty, this site generates an electrostatic barrier that controls channel rectification. In addition, an isoleucine-triad forms a hydrophobic gate at the boundary of the cytosolic vestibule and the inner side of the neck. When the cytosolic Ca2+ rises, one or two Ca2+ ions bind to the orthosteric site in a voltage (V)-dependent manner, thus neutralising the electrostatic barrier and triggering an allosteric gating mechanism propagating via transmembrane segment 6 to the hydrophobic gate. These coordinated events lead to pore opening, allowing the Cl− flux to ensure the physiological response. The Ca2+-dependent function of TMEM16A is highly regulated. Anions with higher permeability than Cl− facilitate V dependence by increasing the Ca2+ sensitivity, intracellular protons can replace Ca2+ and induce channel opening, and phosphatidylinositol 4,5-bisphosphate bound to four cytosolic sites likely maintains Ca2+ sensitivity. Additional regulation is afforded by cytosolic proteins, most likely by phosphorylation and protein-protein interaction mechanisms.

Clues to the Design of Aggregation-Resistant Insulin from Proline Scanning of Highly Amyloidogenic Peptides Derived from the N-Terminal Segment of the A-Chain.

Insulin aggregation poses a significant problem in pharmacology and medicine as it occurs during prolonged storage of the hormone and in vivo at insulin injection sites. We have recently shown that dominant forces driving the self-assembly of insulin fibrils are likely to arise from intermolecular interactions involving the N-terminal segment of the A-chain (ACC1-13). Here, we study how proline substitutions within the pilot GIVEQ sequence of this fragment affect its propensity to aggregate in both neutral and acidic environments. In a reasonable agreement with in silico prediction based on the Cordax algorithm, proline substitutions at positions 3, 4, and 5 turn out to be very effective in preventing aggregation according to thioflavin T-fluorescence-based kinetic assay, infrared spectroscopy, and atomic force microscopy (AFM). Since the valine and glutamate side chains within this segment are strongly involved in the interactions with the insulin receptor, we have focused on the possible implications of the Q → P substitution for insulin's stability and interactions with the receptor. To this end, comparative molecular dynamics (MD) simulations of the Q5P mutant and wild-type insulin were carried out for both free and receptor-bound (site 1) monomers. The results point to a mild destabilization of the mutant vis à vis the wild-type monomer, as well as partial preservation of key contacts in the complex between Q5P insulin and the receptor. We discuss the implications of these findings in the context of the design of aggregation-resistant insulin analogues retaining hormonal activity.

Metabolic Fingerprinting for the Diagnosis of Clinically Similar Long COVID and Fibromyalgia Using a Portable FT-MIR Spectroscopic Combined with Chemometrics.

Post Acute Sequelae of SARS-CoV-2 infection (PASC or Long COVID) is characterized by lingering symptomatology post-initial COVID-19 illness that is often debilitating. It is seen in up to 30-40% of individuals post-infection. Patients with Long COVID (LC) suffer from dysautonomia, malaise, fatigue, and pain, amongst a multitude of other symptoms. Fibromyalgia (FM) is a chronic musculoskeletal pain disorder that often leads to functional disability and severe impairment of quality of life. LC and FM share several clinical features, including pain that often makes them indistinguishable. The aim of this study is to develop a metabolic fingerprinting approach using portable Fourier-transform mid-infrared (FT-MIR) spectroscopic techniques to diagnose clinically similar LC and FM. Blood samples were obtained from LC (n = 50) and FM (n = 50) patients and stored on conventional bloodspot protein saver cards. A semi-permeable membrane filtration approach was used to extract the blood samples, and spectral data were collected using a portable FT-MIR spectrometer. Through the deconvolution analysis of the spectral data, a distinct spectral marker at 1565 cm-1 was identified based on a statistically significant analysis, only present in FM patients. This IR band has been linked to the presence of side chains of glutamate. An OPLS-DA algorithm created using the spectral region 1500 to 1700 cm-1 enabled the classification of the spectra into their corresponding classes (Rcv > 0.96) with 100% accuracy and specificity. This high-throughput approach allows unique metabolic signatures associated with LC and FM to be identified, allowing these conditions to be distinguished and implemented for in-clinic diagnostics, which is crucial to guide future therapeutic approaches.

Characterisation of the heme aqua-ligand coordination environment in an engineered peroxygenase cytochrome P450 variant

The cytochrome P450 enzymes (CYPs) are heme-thiolate monooxygenases that catalyse the insertion of an oxygen atom into the C–H bonds of organic molecules. In most CYPs, the activation of dioxygen by the heme is aided by an acid-alcohol pair of residues located in the I-helix of the enzyme. Mutation of the threonine residue of this acid-alcohol pair of CYP199A4, from the bacterium Rhodospeudomonas palustris HaA2, to a glutamate residue induces peroxygenase activity. In the X-ray crystal structures of this variant an interaction of the glutamate side chain and the distal aqua ligand of the heme was observed and this results in this ligand not being readily displaced in the peroxygenase mutant on the addition of substrate. Here we use a range of bulky hydrophobic and nitrogen donor containing ligands in an attempt to displace the distal aqua ligand of the T252E mutant of CYP199A4. Ligand binding was assessed by UV–visible absorbance spectroscopy, native mass spectrometry, electron paramagnetic resonance and X-ray crystallography. None of the ligands tested, even the nitrogen donor ligands which bind directly to the iron in the wild-type enzyme, resulted in displacement of the aqua ligand. Therefore, modification of the I-helix threonine residue to a glutamate residue results in a significant strengthening of the ferric distal aqua ligand. This ligand was not displaced using any of the ligands during this study and this provides a rationale as to why this mutant can shutdown the monooxygenase pathway of this enzyme and switch to peroxygenase activity.

Exploring the Sequence Space of Antimicrobial Peptide Dendrimers

AbstractThere is an urgent need to develop new antibacterial agents against multidrug resistant bacteria. Herein we report our investigation of antimicrobial peptide dendrimers (AMPDs) active against Gram‐negative bacteria, whose sequences were designed using a genetic algorithm optimizing molecular similarity to the previously reported AMPD T7 with sequence (KL)8(KKL)4(KKLL)2KKKL. Our computational approach selected analogues unlikely to emerge from a systematic study, including AMPD X66 with a non‐conservative Leu→Glu mutation at the dendrimer core which proved compatible with antibacterial effects. Circular dichroism showed that this AMPD is α‐helical. Molecular dynamics suggest that its α‐helical structure is stabilized by an intramolecular salt bridge involving the core glutamate side chain and a lysine side chain in the dendrimer branches. More substantial variations at the dendrimer core were also tolerated such as the installation of the dianionic pegylated fatty acid side chain of the drug semaglutide potentially useful for in vivo studies.

Biomolecular Adsorprion at ZnS Nanomaterials: A Molecular Dynamics Simulation Study of the Adsorption Preferences, Effects of the Surface Curvature and Coating.

The understanding of interactions between nanomaterials and biological molecules is of primary importance for biomedical applications of nanomaterials, as well as for the evaluation of their possible toxic effects. Here, we carried out extensive molecular dynamics simulations of the adsorption properties of about 30 small molecules representing biomolecular fragments at ZnS surfaces in aqueous media. We computed adsorption free energies and potentials of mean force of amino acid side chain analogs, lipids, and sugar fragments to ZnS (110) crystal surface and to a spherical ZnS nanoparticle. Furthermore, we investigated the effect of poly-methylmethacrylate (PMMA) coating on the adsorption preferences of biomolecules to ZnS. We found that only a few anionic molecules: aspartic and glutamic acids side chains, as well as the anionic form of cysteine show significant binding to pristine ZnS surface, while other molecules show weak or no binding. Spherical ZnS nanoparticles show stronger binding of these molecules due to binding at the edges between different surface facets. Coating of ZnS by PMMA changes binding preferences drastically: the molecules that adsorb to a pristine ZnS surface do not adsorb on PMMA-coated surfaces, while some others, particularly hydrophobic or aromatic amino-acids, show high binding affinity due to binding to the coating. We investigate further the hydration properties of the ZnS surface and relate them to the binding preferences of biomolecules.

Controlling Helical Asymmetry in Supramolecular Copolymers by In Situ Chemical Modification.

Amplification of asymmetry in complex molecular systems results from a delicate interplay of chiral supramolecular structures and their chemical reactivity. In this work, we show how the helicity of supramolecular assemblies can be controlled by performing a non-stereoselective methylation reaction on comonomers. By methylating chiral glutamic acid side chains in benzene-1,3,5-tricarboxamide (BTA) derivatives to form methyl esters, the assembly properties are modulated. As reacted comonomers, the methyl ester-BTAs induce a stronger bias in the screw-sense of helical fibers predominantly composed of stacked achiral alkyl-BTA monomers. Hence, applying the in situ methylation in a system with the glutamic acid-BTA comonomer induces asymmetry amplification. Moreover, mixing small quantities of enantiomers of glutamic acid-BTA and glutamate methyl ester-BTA in the presence of the achiral alkyl-BTAs leads to deracemization and inversion of the helical structures in solution via the in situ reaction toward a thermodynamic equilibrium. Theoretical modeling suggests that the observed effects are caused by enhanced comonomer interactions after the chemical modification. Our presented methodology enables on-demand control over asymmetry in ordered functional supramolecular materials.

Chemoenzymatic and Synthetic Approaches To Investigate Aspartate- and Glutamate-ADP-Ribosylation.

We report here chemoenzymatic and fully synthetic methodologies to modify aspartate and glutamate side chains with ADP-ribose at specific sites on peptides. Structural analysis of aspartate and glutamate ADP-ribosylated peptides reveals near-quantitative migration of the side chain linkage from the anomeric carbon to the 2″- or 3″-ADP-ribose hydroxyl moieties. We find that this linkage migration pattern is unique to aspartate and glutamate ADP-ribosylation and propose that the observed isomer distribution profile is present in biochemical and cellular environments. After defining distinct stability properties of aspartate and glutamate ADP-ribosylation, we devise methods to install homogenous ADP-ribose chains at specific glutamate sites and assemble glutamate-modified peptides into full-length proteins. By implementing these technologies, we show that histone H2B E2 tri-ADP-ribosylation is able to stimulate the chromatin remodeler ALC1 with similar efficiency to histone serine ADP-ribosylation. Our work reveals fundamental principles of aspartate and glutamate ADP-ribosylation and enables new strategies to interrogate the biochemical consequences of this widespread protein modification.

Biochemical and mechanistic analysis of the cleavage of branched DNA by human ANKLE1.

ANKLE1 is a nuclease that provides a final opportunity to process unresolved junctions in DNA that would otherwise create chromosomal linkages blocking cell division. It is a GIY-YIG nuclease. We have expressed an active domain of human ANKLE1 containing the GIY-YIG nuclease domain in bacteria, that is monomeric in solution and when bound to a DNA Y-junction, and unilaterally cleaves a cruciform junction. Using an AlphaFold model of the enzyme we identify the key active residues, and show that mutation of each leads to impairment of activity. There are two components in the catalytic mechanism. Cleavage rate is pH dependent, corresponding to a pKa of 6.9, suggesting an involvement of the conserved histidine in proton transfer. The reaction rate depends on the nature of the divalent cation, likely bound by glutamate and asparagine side chains, and is log-linear with the metal ion pKa. We propose that the reaction is subject to general acid-base catalysis, using a combination of tyrosine and histidine acting as general base and water directly coordinated to the metal ion as general acid. The reaction is temperature dependent; activation energy Ea=37 kcal mol-1, suggesting that cleavage is coupled to opening of DNA in the transition state.

Voltage-gated sodium channels (Na<sub>V</sub>) in GtoPdb v.2023.1

Sodium channels are voltage-gated sodium-selective ion channels present in the membrane of most excitable cells. Sodium channels comprise of one pore-forming α subunit, which may be associated with either one or two β subunits [179]. α-Subunits consist of four homologous domains (I-IV), each containing six transmembrane segments (S1-S6) and a pore-forming loop. The positively charged fourth transmembrane segment (S4) acts as a voltage sensor and is involved in channel gating. The crystal structure of the bacterial NavAb channel has revealed a number of novel structural features compared to earlier potassium channel structures including a short selectivity filter with ion selectivity determined by interactions with glutamate side chains [278]. Interestingly, the pore region is penetrated by fatty acyl chains that extend into the central cavity which may allow the entry of small, hydrophobic pore-blocking drugs [278]. Auxiliary β1, β2, β3 and β4 subunits consist of a large extracellular N-terminal domain, a single transmembrane segment and a shorter cytoplasmic domain.The nomenclature for sodium channels was proposed by Goldin et al., (2000) [146] and approved by the NC-IUPHAR Subcommittee on sodium channels (Catterall et al., 2005, [53]).

Phosphatidyl serine preconditions the A2AAR for G-protein coupling via a conserved motif on TM6 and TM7.

19F NMR of A2AAR in nanodiscs of defined composition indicates that negative charge modulates activation of the receptor. The fully active state is obtained either when bound to G-protein, or without G-protein but in a membrane environment containing negatively charged headgroups. Mutagenesis indicates that a trio of residues on the intracellular ends of TM6 and TM7 are responsible for the lipid-dependent activity. In order to determine the mechanism, a series of molecular dynamics simulations were performed. Comparison of simulations of the inactive and the active, G-protein bound state shows that a glutamic acid on the helix of the G-protein coordinates interactions between these three residues. This suggested a hypothesis in which a negatively charged headgroup can stand-in for the glutamic acid side chain, coordinating the ends of TM6 and TM7 and preconditioning the receptor for G-protein coupling. To test this idea, simulations of the fully active state were performed with and without phosphatidyl serine headgroups, but without the G-protein. Consistent with our hypothesis, the TM6 -- TM7 interactions are similar to the G-protein coupled state in the presence of PS, but similar to the inactive state in the absence of PS. Moreover, the interaction of a PS headgroup with a key residue on TM6 recapitulates the same interaction with the glutamic acid, but only when the receptor is in the active state.

Chlorpyrifos Oxon Activates Glutamate and Lysine for Protein Cross-linking.

Chronic low-dose exposure to organophosphorus (OP) toxicants is correlated with an increase in the risk of impaired cognition and neurodegenerative diseases. A mechanism to explain this relationship is needed. We suggest that the formation of organophosphate-induced high-molecular-weight protein aggregates that disrupt cell function may be the missing link. It has been demonstrated that such aggregation can be promoted by OP-labeled lysine. Alternatively, OP-labeled glutamate may be the initiator. To test this hypothesis, we treated MAP-rich tubulin Sus scrofa and human transglutaminase with chlorpyrifos oxon. Trypsin-digested proteins were subjected to liquid chromatography-tandem mass spectrometry followed by Protein Prospector searches to identify diethyl phosphate adducts and cross-linked peptides. We report the presence of diethyl phosphate adducts on the side chains of glutamate, lysine, and tyrosine, as well as cross-links between glutamate and lysine. Glutamate-lysine cross-linking could be initiated either by diethyl phosphate-activated glutamate or by diethyl phosphate-activated lysine to form stable isopeptide bonds between and within proteins. It was concluded that organophosphate-induced high-molecular-weight protein aggregates could promote brain dysfunction.

Metal coordination to solute binding proteins - exciting chemistry with potential biological meaning.

Zn(II) is essential for bacterial survival and virulence. In host cells, its abundance is extremely limited, thus, bacteria have evolved transport mechanisms that enable them to take up this essential metal nutrient. Paracoccus denitrificans encodes two solute binding proteins (SBPs) - ZnuA and AztC, which are responsible for zinc acquisition from the host cells. We focus on understanding the interactions of Zn(II) and Ni(II) (zinc's potential competitor, which is a biologically relevant metal ion essential for various bacterial enzymes) with the extracellular ZnuA and AztC's loops from P. denitrificans that are expected to be possible Zn(II) binding sites. In the case of Zn(II) complexes with ZnuA outercellular loop regions, the numerous histidines act as anchoring donors, forming complexes with up to four coordinated His residues, while in the AztC region, three imidazole nitrogens and one water molecule are involved in Zn(II) binding. In Zn(II) complexes with ZnuA His-rich loop regions, so-called polymorphic binding sites are observed. The large number of available imidazoles and carboxylic side chains also strongly affects the structure of Ni(II) complexes; the more histidines in the studied peptide, the higher the affinity to bind Ni(II) and the higher the pH value at which amide nitrogens start to participate in Ni(II) binding. Additionally, for Ni(II)-ZnuA complexes, a more rare octahedral geometry is observed and such complexes are more stable than the corresponding Zn(II) ones, in contrast to what was observed in the AztC region, suggesting that the numerous histidyl and glutamic acid side chains are more tempting for Ni(II) than for Zn(II).The general strong affinity of Zn(II)-zincophore complexes is also discussed.

Backbone amides are determinants of Cl− selectivity in CLC ion channels

Chloride homeostasis is regulated in all cellular compartments. CLC-type channels selectively transport Cl− across biological membranes. It is proposed that side-chains of pore-lining residues determine Cl− selectivity in CLC-type channels, but their spatial orientation and contributions to selectivity are not conserved. This suggests a possible role for mainchain amides in selectivity. We use nonsense suppression to insert α-hydroxy acids at pore-lining positions in two CLC-type channels, CLC-0 and bCLC-k, thus exchanging peptide-bond amides with ester-bond oxygens which are incapable of hydrogen-bonding. Backbone substitutions functionally degrade inter-anion discrimination in a site-specific manner. The presence of a pore-occupying glutamate side chain modulates these effects. Molecular dynamics simulations show backbone amides determine ion energetics within the bCLC-k pore and how insertion of an α-hydroxy acid alters selectivity. We propose that backbone-ion interactions are determinants of Cl− specificity in CLC channels in a mechanism reminiscent of that described for K+ channels.

Acidic pH Promotes Refolding and Macroscopic Assembly of Amyloid β (16-22) Peptides at the Air-Water Interface.

Assembly by amyloid-beta (Aβ) peptides is vital for various neurodegenerative diseases. The process can be accelerated by hydrophobic interfaces such as the cell membrane interface and the air–water interface. Elucidating the assembly mechanism for Aβ peptides at hydrophobic interface requires knowledge of the microscopic structure of interfacial peptides. Here we combine scanning force microscopy, sum-frequency generation spectroscopy, and metadynamics simulations to probe the structure of the central fragment of Aβ peptides at the air–water interface. We find that the structure of interfacial peptides depends on pH: at neutral pH, the peptides adopt a less folded, bending motif by forming intra-hydrogen bonds; at acidic pH, the peptides refold into extended β-strand fibril conformation, which further promotes their macroscopic assembly. The conformational transition of interfacial peptides is driven by the reduced hydrogen bonds, both with water and within peptides, resulting from the protonation of acidic glutamic acid side chains.

Cysteine-Based Mimic of Arginylation Reproduces Neuroprotective Effects of the Authentic Post-Translational Modification on α-Synuclein.

Arginylation is an understudied post-translational modification (PTM) involving the transfer of arginine to aspartate or glutamate sidechains in a protein. Among the targets of this PTM is α-synuclein (αS), a neuronal protein involved in regulating synaptic vesicles. The aggregation of αS is implicated in neurodegenerative diseases, particularly in Parkinson's disease, and arginylation has been found to protect against this pathological process. Arginylated αS has been studied through semisynthesis involving multipart native chemical ligation (NCL), but this can be very labor-intensive with low yields. Here, we present a facile way to introduce a mimic of the arginylation modification into a protein of interest, compatible with orthogonal installation of labels such as fluorophores. We synthesize bromoacetyl arginine and react it with recombinant, site-specific cysteine mutants of αS. We validate the mimic by testing the vesicle binding affinity of mimic-arginylated αS, as well as its aggregation kinetics and monomer incorporation into fibrils, and comparing these results to those of authentically arginylated αS produced through NCL. In cultured neurons, we compare the fibril seeding capabilities of preformed fibrils carrying a small percentage of arginylated αS. We find that, consistent with authentically arginylated αS, mimic-arginylated αS does not perturb the protein's native function but alters aggregation kinetics and monomer incorporation. Both mimic and authentically modified αS suppress aggregation in neuronal cells. Our results provide further insight into the neuroprotective effects of αS arginylation, and our alternative strategy to generate arginylated αS enables the study of this PTM in proteins not accessible through NCL.

Temperature effects on calcium binding to caseins

The kinetics of binding of calcium ions in molar excess to individual caseins and casein ingredients was studied in pH 6.4 aqueous solutions using stopped-flow absorption spectroscopy. An initial second-order reaction, faster for β-casein than for αs-casein due to lower energy of activation (ΔEa1,β = 8.2 kJ∙mol−1; ΔEa1,α = 18.1 kJ∙mol−1, respectively), is followed by a slower first-order reaction with similar energies of activation (ΔEa2,β = 25.3 kJ∙mol−1 and ΔEa2,α = 20.7 kJ∙mol−1) as determined from temperature dependence of rate between 25 °C and 50 °C. Sodium caseinate reacts faster with calcium than both αs-casein and β-casein in the first reaction of the two consecutive reactions, while the rate of the second falls between αs-casein and β-casein. Global spectral analysis showed the UV–visible spectra of the reaction intermediates of the caseins to be more similar to the final products than to the initial casein reactants. Dynamic and static light scattering indicated decreasing particle sizes and increasing particle surface upon calcium-binding most significantly at low temperatures. The calcium binding to casein was found endothermic by isothermal titration calorimetry. Calcium binding seems to be controlled by enthalpy/entropy compensation corresponding to an isoequilibrium temperature of 38 °C in agreement with binding of calcium to o-phosphoserine rather than to aspartate or glutamate side chains of the caseins. Binding capacity and affinity for calcium to αs-casein and sodium caseinate both increased with increasing temperature in agreement with the endothermic nature of the binding. Decreasing enthalpy of binding for each calcium indicating a decrease in heat capacity of the caseins upon calcium-binding. The small difference between binding enthalpy and energy of activation for association of calcium to αs-casein lead to the conclusion that calcium dissociation goes through an early transition state. The rate of calcium dissociation hardly depends on temperature also explaining why calcium binding to caseins is important for calcium bioaccessibility.

Structure of a monomeric photosystem II core complex from a cyanobacterium acclimated to far-red light reveals the functions of chlorophylls d and f

Far-red light (FRL) photoacclimation in cyanobacteria provides a selective growth advantage for some terrestrial cyanobacteria by expanding the range of photosynthetically active radiation to include far-red/near-infrared light (700–800 nm). During this photoacclimation process, photosystem II (PSII), the water:plastoquinone photooxidoreductase involved in oxygenic photosynthesis, is modified. The resulting FRL-PSII is comprised of FRL-specific core subunits and binds chlorophyll (Chl) d and Chl f molecules in place of several of the Chl a molecules found when cells are grown in visible light. These new Chls effectively lower the energy canonically thought to define the “red limit” for light required to drive photochemical catalysis of water oxidation. Changes to the architecture of FRL-PSII were previously unknown, and the positions of Chl d and Chl f molecules had only been proposed from indirect evidence. Here, we describe the 2.25 Å resolution cryo-EM structure of a monomeric FRL-PSII core complex from Synechococcus sp. PCC 7335 cells that were acclimated to FRL. We identify one Chl d molecule in the ChlD1 position of the electron transfer chain and four Chl f molecules in the core antenna. We also make observations that enhance our understanding of PSII biogenesis, especially on the acceptor side of the complex where a bicarbonate molecule is replaced by a glutamate side chain in the absence of the assembly factor Psb28. In conclusion, these results provide a structural basis for the lower energy limit required to drive water oxidation, which is the gateway for most solar energy utilization on earth.

Discovery, Biocatalytic Exploration and Structural Analysis of a 4-Ethylphenol Oxidase from Gulosibacter chungangensis.

The vanillyl‐alcohol oxidase (VAO) family is a rich source of biocatalysts for the oxidative bioconversion of phenolic compounds. Through genome mining and sequence comparisons, we found that several family members lack a generally conserved catalytic aspartate. This finding led us to study a VAO‐homolog featuring a glutamate residue in place of the common aspartate. This 4‐ethylphenol oxidase from Gulosibacter chungangensis (Gc4EO) shares 42 % sequence identity with VAO from Penicillium simplicissimum, contains the same 8α‐N3‐histidyl‐bound FAD and uses oxygen as electron acceptor. However, Gc4EO features a distinct substrate scope and product specificity as it is primarily effective in the dehydrogenation of para‐substituted phenols with little generation of hydroxylated products. The three‐dimensional structure shows that the characteristic glutamate side chain creates a closely packed environment that may limit water accessibility and thereby protect from hydroxylation. With its high thermal stability, well defined structural properties and high expression yields, Gc4EO may become a catalyst of choice for the specific dehydrogenation of phenolic compounds bearing small substituents.