Abstract The W/SeCys-FdhAB formate dehydrogenase from Desulfovibrio vulgaris Hildenborough is a dimeric periplasmic enzyme that catalyzes the reversible oxidation of formate and reduction of CO2. It belongs to the group of metal-dependent FDHs, with a tungsten at the active site coordinated by two pyranopterin guanine dinucleotides, a selenocysteine, and one labile sulfur atom. FdhAB has a remarkably high activity and unusual tolerance to oxygen, making it an ideal model system to study biological CO2 reduction.

The biological activity of vanadium complexes, namely, as insulin enhancers, is well known. We report a combined X-ray crystallography, electron paramagnetic resonance, and density functional theory study of the interaction of vanadium picolinate complexes with hen egg white lysozyme (HEWL). We show that the VIVO(pic)2 complex covalently binds to the COO– group of the side chain of Asp52 of HEWL. The long VIV=O bond obtained in the X-ray study is explained to be due to reduction of VIV to VIII during exposure of the crystals to the intense X-ray beam.

AbstractVanadium is an element ubiquitously present in our planet's crust and thus there are several organisms that use vanadium for activity or function of proteins. Examples are the vanadium-dependent haloperoxidases and the vanadium-containing nitrogenases. Some organisms that use vanadium have extremely efficient and selective protein-dependent systems for uptake and transport of vanadium and are able to accumulate high levels of vanadium from seawater, vanabins being a unique family of vanadium binding proteins found in ascidians involved in this process. For all of the systems a discussion regarding the role of the V-containing proteins is provided, mostly centered on structural aspects of the vanadium site and, when possible or relevant, relating this to the mechanisms operating. Phosphate is very important in biological systems and is involved in an extensive number of biological recognition and bio-catalytic systems. Vanadate(V) is able to inhibit many of the enzymes involved in these processes, such as ATPases, phosphatases, ribonucleases, phosphodiesterases, phosphoglucomutase and glucose-6-phosphatase, and it appears clear that this is closely related to the analogous physicochemical properties of vanadate and phosphate. The ability of vanadium to interfere with the metabolic processes involving Ca2+ and Mg2+, connected with its versatility to undergo changes in coordination geometry, allow V to influence the function of a large variety of phosphate-metabolizing enzymes and vanadate(V) salts and compounds have been frequently used either as inhibitors of these enzymes, or as probes to study the mechanisms of their reactions and catalytic cycle. In this review we give an overview of the many examples so far reported, also disclosing that vanadate(IV) may also have an equally efficient inhibiting effect. The prospective application of vanadium compounds as therapeutics has also been an important topic of research. How vanadium may be transported in blood and up-taken by cells are particularly relevant issues, this being mainly dependent on transferrin (and albumin) present in blood plasma. The thousands of studies reported on the effects of vanadium compounds reflect the complexity of the interactions occurring. Although it is not easy to anticipate/determine if a particular effect observed in a test tube or in vitro is also going to take place in vivo, it is clear that vanadium ions may interfere with many metabolic processes at many distinct levels. Emphasis is given on structural and functional aspects of vanadium–protein interactions relevant for vanadium binding and/or for clarification of role of the metal center in the reaction mechanisms. The additional knowledge that the presence of vanadium can change the action of a protein, other than simply inhibiting it, may also be important to understand how vanadium affects biological systems. This possibility, together with the vanadate–phosphate analogy further potentiates the belief that vanadium probably has relevant functions in living beings, which may involve interaction or incorporation of the metal ion and/or its compounds with several proteins.

Gold nanoparticles (AuNPs) exhibit unique properties that have made them a very attractive material for application in biological assays. Given the potentially interesting interactions between AuNPs and biological macromolecules, we investigated AuNPs-induced protein crystal growth. Differently functionalized AuNPs were tested as additives in cocrystallization studies with model proteins (hen egg white lysozyme (HEWL), ribonuclease A (RNase A), and proteinase K) as well as with case studies where there were problems in obtaining well-diffracting crystals. Trials were performed considering different crystallization drawbacks, from total absence of crystals to improvement of crystal morphology, size, twinning, and number of crystals per drop. Improvement of some of these factors was observed in the cases of HEWL, RNase A, phenylalanine hydroxylase (PAR), myoglobin, native aldehyde oxidase (AOH), and human albumin. In these proteins, the presence of the AuNPs promoted an increase in the size and/or better crystal morphology. From the systematic trials and subsequent observations, it can be concluded that the introduction of AuNPs should definitely be considered in crystal optimization trials to improve previously determined crystallization conditions.

Glucans are polymers of D-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes including immunomodulation, anti-cancer activities, pathogen virulence and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure-function studies and their exploitation. We describe construction of a glucome microarray, the first sequence-defined glycome-scale microarray, using a designer approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. The negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear homo and hetero and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or β-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signalling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.

The cooperative assembly of biopolymers and small molecules can yield functional materials with precisely tunable properties. Here, the fabrication, characterization, and use of multicomponent hybrid gels as selective gas sensors are reported. The gels are composed of liquid crystal droplets self-assembled in the presence of ionic liquids, which further coassemble with biopolymers to form stable matrices. Each individual component can be varied and acts cooperatively to tune gels' structure and function. The unique molecular environment in hybrid gels is explored for supramolecular recognition of volatile compounds. Gels with distinct compositions are used as optical and electrical gas sensors, yielding a combinatorial response conceptually mimicking olfactory biological systems, and tested to distinguish volatile organic compounds and to quantify ethanol in automotive fuel. The gel response is rapid, reversible, and reproducible. These robust, versatile, modular, pliant electro-optical soft materials possess new possibilities in sensing triggered by chemical and physical stimuli.

Metal-dependent formate dehydrogenases (Fdh) catalyze the reversible conversion of CO2 to formate, with unrivalled efficiency and selectivity. However, the key catalytic aspects of these enzymes remain unknown, preventing us from fully benefiting from their capabilities in terms of biotechnological applications. Here, we report a time-resolved characterization by X-ray crystallography of the Desulfovibrio vulgaris Hildenborough SeCys/W-Fdh during formate oxidation. The results allowed us to model five different intermediate structures and to chronologically map the changes occurring during enzyme reduction. Formate molecules were assigned for the first time to populate the catalytic pocket of a Fdh. Finally, the redox reversibility of DvFdhAB in crystals was confirmed by reduction and reoxidation structural studies.

Reducing CO2 is a challenging chemical transformation that biology solves easily, with high efficiency and specificity. In particular, formate dehydrogenases are of great interest since they reduce CO2 to formate, a valuable chemical fuel and hydrogen storage compound. The metal-dependent formate dehydrogenases of prokaryotes can show high activity for CO2 reduction. Here, we report an expression system to produce recombinant W/Sec-FdhAB from Desulfovibrio vulgaris Hildenborough fully loaded with cofactors, its cata-lytic characterization and crystal structures in oxidised and reduced states. The enzyme has very high activi-ty for CO2 reduction and displays remarkable oxygen stability. The crystal structure of the formate-reduced enzyme shows Sec still coordinating the tungsten, supporting a mechanism of stable metal coordination during catalysis. Comparison of the oxidised and reduced structures shows significant changes close to the active site. The DvFdhAB is an excellent model for studying catalytic CO2 reduction and probing the mecha-nism of this conversion.Reducing CO2 is a challenging chemical transformation that biology solves easily, with high efficiency and specificity. In particular, formate dehydrogenases are of great interest since they reduce CO2 to formate, a valuable chemical fuel and hydrogen storage compound. The metal-dependent formate dehydrogenases of prokaryotes can show high activity for CO2 reduction. Here, we report an expression system to produce recombinant W/Sec-FdhAB from Desulfovibrio vulgaris Hildenborough fully loaded with cofactors, its cata-lytic characterization and crystal structures in oxidised and reduced states. The enzyme has very high activi-ty for CO2 reduction and displays remarkable oxygen stability. The crystal structure of the formate-reduced enzyme shows Sec still coordinating the tungsten, supporting a mechanism of stable metal coordination during catalysis. Comparison of the oxidised and reduced structures shows significant changes close to the active site. The DvFdhAB is an excellent model for studying catalytic CO2 reduction and probing the mecha-nism of this conversion.

The plant cell-wall is constituted by structurally diverse polysaccharides. The biodegradation of these is a crucial process for life sustainability. Cellulolytic microorganisms are highly efficient in this process by assembling modular architectures of carbohydrate-active enzymes with appended non-catalytic carbohydrate-binding modules (CBMs). Carbohydrate microarrays offer high-throughput and sensitive tools for uncovering carbohydrate-binding specificities of CBMs{,} which is pivotal to understand the function of these modules in polysaccharide biodegradation mechanisms. Features of this technology will be here briefly reviewed with highlights of microarray approaches to study plant-carbohydrates and CBM-carbohydrate interactions{,} along with an overview of plant polysaccharides and microorganisms strategies for their recognition.

The functional and biological significance of the selected CASP12 targets are described by the authors of the structures. The crystallographers discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP12 experiment. This article is protected by copyright. All rights reserved.

Abstract Relative humidity is simultaneously a sensing target and a contaminant in gas and volatile organic compound (VOC) sensing systems, where strategies to control humidity interference are required. An unmet challenge is the creation of gas-sensitive materials where the response to humidity is controlled by the material itself. Here, humidity effects are controlled through the design of gelatin formulations in ionic liquids without and with liquid crystals as electrical and optical sensors, respectively. In this design, the anions [DCA]− and [Cl]− of room temperature ionic liquids from the 1-butyl-3-methylimidazolium family tailor the response to humidity and, subsequently, sensing of VOCs in dry and humid conditions. Due to the combined effect of the materials formulations and sensing mechanisms, changing the anion from [DCA]− to the much more hygroscopic [Cl]−, leads to stronger electrical responses and much weaker optical responses to humidity. Thus, either humidity sensors or humidity-tolerant VOC sensors that do not require sample preconditioning or signal processing to correct humidity impact are obtained. With the wide spread of 3D- and 4D-printing and intelligent devices, the monitoring and tuning of humidity in sustainable biobased materials offers excellent opportunities in e-nose sensing arrays and wearable devices compatible with operation at room conditions.

A new and never yet reported hetero arylidene-9(10H)-anthrone structure (4) was unexpectedly isolated on reaction of 1,2-dimethyl-3-ethylimidazolium iodide (2) and 9-anthracenecarboxaldehyde (3) under basic conditions. Its structure was unequivocally attributed by X-ray crystallography. No cytotoxicity in human healthy fibroblasts and in two different cancer cell lines was observed indicating its applicability in biological systems. Compound 4 interacts with CT-DNA by intercalation between the adjacent base pairs of DNA with a high binding affinity (Kb = 2.0(± 0.20) x 105 M-1) which is 10x higher than that described for doxorubicin (Kb = 3.2 (±0.23) × 104 M-1). Furthermore, compound 4 quenches the fluorescence emission of GelRed-CT-DNA system with a quenching constant (KSV) of 3.3(±0.3) x 103 M-1 calculated by the Stern-Volmer equation.

Silk fibroin is a biobased material with excellent biocompatibility and mechanical properties, but its use in bioelectronics is hampered by the difficult dissolution and low intrinsic conductivity. Some ionic liquids are known to dissolve fibroin but removed after fibroin processing. However, ionic liquids and fibroin can cooperatively give rise to functional materials, and there are untapped opportunities in this combination. The dissolution of fibroin, followed by gelation, in designer ionic liquids from the imidazolium chloride family with varied alkyl chain lengths (2–10 carbons) is shown here. The alkyl chain length of the anion has a large impact on fibroin secondary structure which adopts unconventional arrangements, yielding robust gels with distinct hierarchical organization. Furthermore, and due to their remarkable air-stability and ionic conductivity, fibroin ionogels are exploited as active electrical gas sensors in an electronic nose revealing the unravelled possibilities of fibroin in soft and flexible electronics.

Metal-dependent formate dehydrogenases are very promising targets for enzyme optimization and design of bio-inspired catalysts for CO2 reduction, towards novel strategies for climate change mitigation. For effective application of these enzymes, the catalytic mechanism must be fully understood, and the molecular determinants clarified. Despite numerous studies, several doubts persist, namely regarding the role played by the possible dissociation of the SeCys ligand from the Mo/W active site. Additionally, the O2 sensitivity of these enzymes must also be understood as it poses an important obstacle for biotechnological applications. Here we present a combined biochemical, spectroscopic, and structural characterization of Desulfovibrio vulgaris FdhAB (DvFdhAB) when exposed to oxygen in the presence of a substrate (formate or CO2). This study reveals that O2 inactivation is promoted by the presence of either substrate and involves forming a new species in the active site, captured in the crystal structures, where the SeCys ligand is displaced from tungsten coordination and replaced by a dioxygen or peroxide molecule. This new form was reproducibly obtained and supports the conclusion that, although W-DvFdhAB can catalyze the oxidation of formate in the presence of oxygen for some minutes, it gets irreversibly inactivated after prolonged O2 exposure in the presence of either substrate. These results reveal that oxidative inactivation does not require reduction of the metal, as widely assumed, as it can also occur in the oxidized state in the presence of CO2.Competing Interest StatementThe authors have declared no competing interest.AORAldehyde Oxido-reductaseDTTDithiothreitolDvDesulfovibrio vulgarisEPRElectron Paramagnetic ResonanceFdhFormate dehydrogenaseHPHigh PressureMGDMolybdopterin Guanine DinucleotidesNDNew dropROSReactive Oxygen SpeciesSODSuperoxide dismutaseTSAThermal Shift Assay

TheBacteroides thetaiotaomicronhas developed a consortium of enzymes capable of overcoming steric constraints and degrading, in a sequential manner, the complex rhamnogalacturonan II (RG-II) polysaccharide. BT0996 protein acts in the initial stages of the RGII depolymerisation, where its two catalytic modules remove the terminal monosaccharides from RG-II side chains A and B. BT0996 is modular and has three putative carbohydrate-binding modules (CBMs) for which the roles in the RG-II degradation are unknown. Here, we present the characterisation of themoduleat the C-terminal domain, which we designated BT0996C. The high-resolution structure obtained by X-ray crystallography reveals that the protein displays a typical β-sandwich fold with structural similarity to CBMs assigned to families 6 and 35. The distinctive features are: 1) the presence of several charged residues at the BT0996-C surface creating a large, broad positive lysine-rich patch that encompasses the putative binding site; and 2) the absence of the highly conserved binding-site signatures observed in CBMs from families 6 and 35, such as region A tryptophan and region C asparagine. These findings hint at a binding mode of BT0996-C not yet observed in its homologues. In line with this, carbohydrate microarrays and microscale thermophoresis show the ability of BT0996-C to bind α1-4-linked polygalacturonic acid, and that electrostatic interactions are essential for the recognition of the anionic polysaccharide. The results support the hypothesis that BT0996-C may have evolved to potentiate the action of BT0996 catalytic modules on the complex structure of RG-II by binding to the polygalacturonic acid backbone sequence.

Neisseria gonorrhoeae is an obligate human pathogenic bacterium responsible for gonorrhea, a sexually transmitted disease. The bacterial peroxidase, an enzyme present in the periplasm of this bacterium, detoxifies the cells against hydrogen peroxide and constitutes one of the primary defenses against exogenous and endogenous oxidative stress in this organism. The 38 kDa heterologously produced bacterial peroxidase was crystallized in the mixed-valence state, the active state, at pH 6.0, and the crystals were soaked with azide, producing the first azide-inhibited structure of this family of enzymes. The enzyme binds exogenous ligands such as cyanide and azide, which also inhibit the catalytic activity by coordinating the P heme iron, the active site, and competing with its substrate, hydrogen peroxide. The inhibition constants were estimated to be 0.4 ± 0.1 µM and 41 ± 5 mM for cyanide and azide, respectively. Imidazole also binds and inhibits the enzyme in a more complex mechanism by binding to P and E hemes, which changes the reduction potential of the latest heme. Based on the structures now reported, the catalytic cycle of bacterial peroxidases is revisited. The inhibition studies and the crystal structure of the inhibited enzyme comprise the first platform to search and develop inhibitors that target this enzyme as a possible new strategy against N. gonorrhoeae.

Deconstruction of cellulose, the most abundant plant cell wall polysaccharide, requires the cooperative activity of a large repertoire of microbial enzymes. Modular cellulases contain non-catalytic type A Carbohydrate-Binding Modules (CBMs) that specifically bind to the crystalline regions of cellulose, thus promoting enzyme efficacy through proximity and targeting effects. Although type A CBMs play a critical role in cellulose recycling, their mechanism of action remains poorly understood. Here we produced a library of recombinant CBMs representative of the known diversity of type A modules. The binding properties of 40 CBMs, in fusion with an N-terminal green fluorescence protein (GFP) domain, revealed that type A CBMs possess the ability to recognize different crystalline forms of cellulose and chitin over a wide range of temperatures, pHs and ionic strengths. A Spirochaeta thermophila CBM64, in particular, displayed plasticity in its capacity to bind both crystalline and soluble carbohydrates under a wide range of extreme conditions. The structure of S. thermophila StCBM64C revealed an untwisted, flat, carbohydrate-binding interface comprising the side chains of four tryptophan residues in a coplanar linear arrangement. Significantly, two highly conserved asparagine side chains, each one located between two tryptophan residues, are critical to insoluble and soluble glucan recognition but not to bind xyloglucan. Thus, CBM64 compact structure and its extended and versatile ligand interacting platform illustrates how type A CBMs target their appended plant cell wall degrading enzymes to a diversity of recalcitrant carbohydrates under a wide range of environmental conditions.

Metal-dependent formate dehydrogenases are important enzymes due to their activity of CO2 reduction to formate. The tungsten-containing FdhAB formate dehydrogenase from Desulfovibrio vulgaris Hildenborough is a good example displaying high activity, simple composition, and a notable structural and catalytic robustness. Here, we report the first spectroscopic redox characterization of FdhAB metal centers by EPR. Titration with dithionite or formate leads to reduction of three [4Fe–4S]1+ clusters, and full reduction requires Ti(III)–citrate. The redox potentials of the four [4Fe–4S]1+ centers range between −250 and −530 mV. Two distinct WV signals were detected, WDV and WFV, which differ in only the g2-value. This difference can be explained by small variations in the twist angle of the two pyranopterins, as determined through DFT calculations of model compounds. The redox potential of WVI/V was determined to be −370 mV when reduced by dithionite and −340 mV when reduced by formate. The crystal structure of dithionite-reduced FdhAB was determined at high resolution (1.5 Å), revealing the same structural alterations as reported for the formate-reduced structure. These results corroborate a stable six-ligand W coordination in the catalytic intermediate WV state of FdhAB.Metal-dependent formate dehydrogenases are important enzymes due to their activity of CO2 reduction to formate. The tungsten-containing FdhAB formate dehydrogenase from Desulfovibrio vulgaris Hildenborough is a good example displaying high activity, simple composition, and a notable structural and catalytic robustness. Here, we report the first spectroscopic redox characterization of FdhAB metal centers by EPR. Titration with dithionite or formate leads to reduction of three [4Fe–4S]1+ clusters, and full reduction requires Ti(III)–citrate. The redox potentials of the four [4Fe–4S]1+ centers range between −250 and −530 mV. Two distinct WV signals were detected, WDV and WFV, which differ in only the g2-value. This difference can be explained by small variations in the twist angle of the two pyranopterins, as determined through DFT calculations of model compounds. The redox potential of WVI/V was determined to be −370 mV when reduced by dithionite and −340 mV when reduced by formate. The crystal structure of dithionite-reduced FdhAB was determined at high resolution (1.5 Å), revealing the same structural alterations as reported for the formate-reduced structure. These results corroborate a stable six-ligand W coordination in the catalytic intermediate WV state of FdhAB.

The SOUL, or heme-binding protein HBP/SOUL, family represents a group of evolutionary conserved putative heme-binding proteins that contains a number of members in animal, plant andbacterial species. The structures of the murine form of HEBP1, or p22HBP, and the human form of HEBP2, or SOUL, have been determined in 2006 and 2011 respectively. In this work we discuss the structures of HEBP1 and HEBP2 in light of new X-ray data for heme bound murine HEBP1. The interaction between tetrapyrroles and HEBP1, initially proven to be hydrophobic in nature, was thought to also involve electrostatic interactions between heme propionate groups and positively charged amino acid side chains. However, the new X-ray structure, and results from murine HEBP1 variants and human HEBP1, confirm the hydrophobic nature of the heme-HEBP1 interaction, resulting in Kd values in the low nanomolar range, and rules out any electrostatic stabilization. Results from NMR relaxation time measurements for human HEBP1 describe a rigid globular protein with no change in motional regime upon heme binding. X-ray structures deposited in the PDB for human HEBP2 are very similar to each other and to the new heme-bound murine HEBP1 X-ray structure (backbone rmsd ca. 1 Å). Results from a HSQC spectrum centred on the histidine side chain Nδ-proton region for HEBP2 confirm that HEBP2 does not bind heme via H42 as no chemical shift differences were observed upon heme addition for backbone NH and Nδ protons. A survey of the functions attributed to HEBP1 and HEBP2 over the last 20 years span a wide range of cellular pathways. Interestingly, many of them are specific to higher eukaryotes, particularly mammals and a potential link between heme release under oxidative stress and human HEBP1 is also examined using recent data. However, at the present moment, trying to relate function to the involvement of heme or tetrapyrrole binding, specifically, makes little sense with our current biological knowledge and can only be applied to HEBP1, as HEBP2 does not interact with heme. We suggest that it may not be justified to call this very small family of proteins, heme-binding proteins. The family may be more correctly called “the SOUL family of proteins related to cellular fate” as, even though only HEBP1 binds heme tightly, both proteins may be involved in cell survival and/or proliferation.