Search

The nitrosonium ion-catalyzed dehydrogenative coupling of heteroarenes under mild reaction conditions is reported. The developed method utilizes ambient molecular oxygen as a terminal oxidant, and only water is produced as byproduct. Dehydrogenative coupling of heteroarenes translated into the rapid discovery of novel hedgehog signaling pathway inhibitors, emphasizing the importance of the developed methodology. [ABSTRACT FROM AUTHOR]

Understanding concentration distributions, release sites, and release dynamics of volatile organic compounds (VOCs) from the human is expected to lead to methods for noninvasive disease screening and assessment of metabolisms. In this study, we developed a visualization system (sniff-cam) that enabled one to identify a spatiotemporal change of gaseous acetaldehyde (AcH) in real-time. AcH sniff-cam was composed of a camera, a UV-LED array sheet, and an alcohol dehydrogenase (ADH)-immobilized mesh. A reverse reaction of ADH was employed for detection of gaseous AcH where a relationship between fluorescence intensity from nicotinamide adenine dinucleotide and the concentration of AcH was inversely proportional; thus, the concentration distribution of AcH was measured by detecting the fluorescence decrease. Moreover, the image differentiation method that calculated a fluorescence change rate was employed to visualize a real-time change in the concentration distribution of AcH. The dynamic range of the sniff-cam was 0.1-10 ppm which encompassed breath AcH concentrations after drinking. Finally, the sniff-cam achieved the visualization of the concentration distribution of AcH in breath and skin gas. A clear difference of breath AcH concentration was observed between aldehyde dehydrogenase type 2 active and inactive subjects, which was attributed to metabolic capacities of AcH. AcH in skin gas showed a similar time course of AcH concentration to the breath and a variety ofrelease concentration distribution. Using different NADH-dependent dehydrogenases in the sniff-cam could lead to a versatile method for noninvasive disease screening by acquiring spatiotemporal information on various VOCs in breath or skin gas. [ABSTRACT FROM AUTHOR]

An artificial Ca2+-regulated PQQ glucose dehydrogenase (PQQ-GDH) enzyme was electrically connected to conducting electrodes and semiconductor interfaces. Direct electron transfer from the enzyme to the conducting electrode support was stimulated by the addition of Ca2+ cations resulting in reversible enzyme activation. A signal-switchable biofuel cell and biomolecular release have been realized using the Ca2+-activated enzyme immobilized on conducting electrodes. Interfacing the signal-switchable enzyme with a semiconductor chip allowed electronic read out of the enzyme ON-OFF states. The developed approach based on the signal-regulated PQQ-GDH enables numerous bioelectrochemical/bioelectronic applications of the developed systems in signal-activated biosensors and biofuel cells, as well as in biomolecular computing/logic systems. [ABSTRACT FROM AUTHOR]

The synthesis, photophysics, and biochemical utility of a fluorescent NAD[SUP +] analogue based on an isothiazolo[4,3-d]pyrimidine core (NtzAD[SUP +]) are described. Enzymatic reactions, photophysically monitored in real time, show NtzAD+ and NtzADH to be substrates for yeast alcohol dehydrogenase and lactate dehydrogenase, respectively, with reaction rates comparable to that of the native cofactors. A drop in fluorescence is seen as NtzAD+ is converted to NtzADH, reflecting a complementary photophysical behavior to that of the native NAD+/NADH. NtzAD+ and NtzADH serve as substrates for NADase, which selectively cleaves the nicotinamide's glycosidic bond yielding tzADP-ribose. NtzAD+ also serves as a substrate for ribosyl transferases, including human adenosine ribosyl transferase 5 (ART5) and Cholera toxin subunit A (CTA), which hydrolyze the nicotinamide and transfer tzADP-ribose to an arginine analogue, respectively. These reactions can be monitored by fluorescence spectroscopy, in stark contrast to the corresponding processes with the nonemissive NAD+. [ABSTRACT FROM AUTHOR]

Previously, we reported a hypoxia-inducible factor (HIF)-1 inhibitor LW6 containing an (aryloxyacetylamino)benzoic acid moiety inhibits malate dehydrogenase 2 (MDH2) using a chemical biology approach. Structure-activity relationship studies on a series of (aryloxyacetylamino)benzoic acids identified selective MDH1, MDH2, and dual inhibitors, which were used to study the relationship between MDH enzyme activity and HIF-1 inhibition. We hypothesized that dual inhibition of MDH1 and MDH2 might be a powerful approach to target cancer metabolism and selected methyl-3-(3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanamido)-benzoate (16c) as the most potent dual inhibitor. Kinetic studies revealed that compound 16c competitively inhibited MDH1 and MDH2. Compound 16c inhibited mitochondrial respiration and hypoxia-induced HIF-1a accumulation. In xenograft assays using HCT116 cells, compound 16c demonstrated significant in vivo antitumor efficacy. This finding provides concrete evidence that inhibition of both MDH1 and MDH2 may provide a valuable platform for developing novel therapeutics that target cancer metabolism and tumor growth. [ABSTRACT FROM AUTHOR]

In this study, transcriptomic and Ingenuity Pathway Analysis (IPA) underlined that an ex-vivo TCDD treatment (0.1 nM) stimulated insulin-release in mouse pancreatic islets via the effect on the Akt-mTOR-p70S6K, AMPK and ERK1/2 pathways. Functional studies using both ex-vivo islets and the mouse β-cell-line (Min-6) validated the stimulatory effects of TCDD (0.1 and 1 nM) on basal-insulin secretion. At 0.1 nM TCDD treatment on Min-6, Western blot analysis showed activation of ERK1/2 and decreased expression of pyruvate dehydrogenase kinase (PDK). A reduction of PDK expression is associated with an increase of pyruvate dehydrogenase flux. This observation was supported by the detection of significantly higher cellular ATP levels, an increase of glucose-stimulated-insulin-secretion (GSIS), and an inhibition of the AMPK pathway. At 1 nM TCDD treatment on Min-6, significant inhibitions of the Akt-mTOR pathway, cellular ATP production, and GSIS were evident. The experimental studies in Min-6 supported the IPA of transcriptomic data in pancreatic islets. Collectively, TCDD treatment caused an elevated basal-insulin release in both islets and β-cell cultures. Moreover, our data revealed that the modulation of the Akt-mTOR-p70S6K, AMPK and ERK1/2 pathways might be an important component of the mechanism for the TCDD-perturbing effects on ATP production in β-cells in affecting insulin secretion. [ABSTRACT FROM AUTHOR]

The three-dimensional nature of interactions between enzymes and their substrates often leads to exacting spatial binding orientations and stereoselectivity in chemical catalysis. Dehydrogenases that use NAD+ as a redox cofactor tend to show stereospecificity in transferring a hydride to the C4 of the nicotinamide moiety via either the re or the si face. The stereospecificity of this hydride transfer, which results in a prochiral C4 in the reduced NADH, may be determined using deuterated substrates and ¹H NMR spectroscopy. A biochemistry lab activity that combines analysis of the intermolecular interactions and spatial orientation between substrate, cofactor, and enzyme from a recent crystal structure of yeast alcohol dehydrogenase with improved in situ single-tube reaction conditions for elucidating the prochiral specificity of yeast alcohol dehydrogenase through ¹H NMR spectra analysis is presented. [ABSTRACT FROM AUTHOR]

Enzymes are biocatalysts that play key roles in diverse chemical reactions in living organisms and industrial conversion processes generating value-added products. Since wild-type enzymes obtained from nature are normally not optimal for various applications, enzyme engineering is usually required for enhanced or new properties. Site-specific incorporation of a non-natural amino acid became a powerful protein-engineering tool. In this short review, we briefly summarize our contribution to enzyme complex formation and active site design of enzymes using the technique of site-specific incorporation of a non-natural amino acid. First, site-specific incorporation of a non-natural amino acid at a permissive site of an enzyme led to bioconjugation to other molecules without compromising critical properties. Murine dihydrofolate reductase (mDHFR) was site-specifically conjugated to a biotin via click chemistry to achieve site-specific immobilization. Similarly, formate dehydrogenase (FDH) was site-specifically conjugated to an organometallic catalyst for cofactor regeneration. Furthermore, successive applications of two different click chemistries allowed site-specific coupling of FDH and mannitol dehydrogenase for the enhanced overall reaction efficiencies via substrate channeling effects and active site-orientation control. Besides enzyme bioconjugation, precise control of non-natural amino acid incorporation also allows for active site modification with a non-natural amino acid. Introduction of a bulky non-natural amino acid into the mDHFR active site lowered binding affinity to its inhibitor methotrexate without compromising binding affinity to its substrate dihydrofolate. Similarly, introduction of a bulky non-natural amino acid into the mDHFR active site led to the alteration of substrate specificity toward a poor substrate folate over a good substrate dihydrofolate. [ABSTRACT FROM AUTHOR]

Given the putative role of PHGDH in cancer, development of inhibitors is required to explore its function. In this context, we established and validated a straightforward enzymatic assay suitable for high-throughput screening and we identified inhibitors with similar chemical scaffolds. Through a convergent pharmacophore approach, we synthesized α-ketothioamides that exhibit interesting in vitro PHGDH inhibition and encouraging cellular results. These novel probes may be used to understand the emerging biology of this metabolic target. [ABSTRACT FROM AUTHOR]

Recent advances in the understanding of the relationship between cancer and metabolism have highlighted the relevance of the serine synthetic pathway (SSP), which consists of three successive enzymatic reactions. Enzymes of the SSP, such as phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase 1 (PSAT-1), were recently highlighted because they are amplified in a significant subset of human tumors, and their suppression by RNAi caused a decrease in cancer cell survival and growth. Currently, the discovery of drugs that inhibit these enzymes is still in its infancy, and the identification of suitable inhibitors could serve to understand the emerging biology of these metabolic enzymes. In this review, we present the SSP as a significant and novel emerging area for medicinal chemistry and we provide an overview of one of the key enzymes of the pathway, PHGDH. [ABSTRACT FROM AUTHOR]

Redox-active phenazinium salts bonded to amphiphilic polymer backbones are demonstrated to function as high-performance electron-transfer mediators in enzymatic bioanodes applicable to biofuel cells. The redox-active moieties could be easily tethered to the electrodes by physical adsorption of the hydrophobic regions of the polymer backbones onto the electrode surface. On the other hand, long hydrophilic chains were essential to ensure high mobility of the redox-active moieties in aqueous solutions and to enhance their electron-transfer properties. We found that an amphiphilic mediator with a linear polymer backbone exhibited stable adsorption behavior on the electrode surface and generated high bioelectrocatalytic current (>1.8 ± 0.32 mA/cm²) in the presence of pyrroloquinoline quinone-dependent glucose dehydrogenase and an aqueous solution of glucose fuel. This current was more than two times higher than that of an electrode treated with a low-molecular-weight phenazinium salt. Moreover, the bioelectrode modified with the polymer mediator retained the high electrocatalytic current after 10 exchanges of the glucose fuel. The mediator-modified bioelectrodes are expected to be useful for various bio-related energy and electronic devices. [ABSTRACT FROM AUTHOR]

The construction from scratch of synthetic cells by assembling molecular building blocks is unquestionably an ambitious goal from a scientific and technological point of view. To realize functional life-like systems, minimal enzymatic modules are required to sustain the processes underlying the out-of-equilibrium thermodynamic status hallmarking life, including the essential supply of energy in the form of electrons. The nicotinamide cofactors NAD(H) and NADP(H) are the main electron carriers fueling reductive redox reactions of the metabolic network of living cells. One way to ensure the continuous availability of reduced nicotinamide cofactors in a synthetic cell is to build a minimal enzymatic module that can oxidize an external electron donor and reduce NAD(P) + . In the diverse world of metabolism there is a plethora of potential electron donors and enzymes known from living organisms to provide reducing power to NAD(P) + coenzymes. This perspective proposes guidelines to enable the reduction of nicotinamide cofactors enclosed in phospholipid vesicles, while avoiding high burdens of or cross-talk with other encapsulated metabolic modules. By determining key requirements, such as the feasibility of the reaction and transport of the electron donor into the cell-like compartment, we select a shortlist of potentially suitable electron donors. We review the most convenient proteins for the use of these reducing agents, highlighting their main biochemical and structural features. Noting that specificity toward either NAD(H) or NADP(H) imposes a limitation common to most of the analyzed enzymes, we discuss the need for specific enzymes─transhydrogenases─to overcome this potential bottleneck.

Biosensors with direct electron output hold promise for nearly seamless integration with portable electronic devices. However, so far, they have been based on naturally occurring enzymes that significantly limit the spectrum of detectable analytes. Here, we present a novel biosensor architecture based on analyte-driven intermolecular recombination and activity reconstitution of a re-engineered component of glucometers: PQQ-glucose dehydrogenase. We demonstrate that this sensor architecture can be rapidly adopted for the detection of immunosuppressant drugs, α-amylase protein, or protease activity of thrombin and Factor Xa. The biosensors could be stored in dried form without appreciable loss of activity. We further show that ligand-induced activity of the developed biosensors could be directly monitored by chronoamperometry, enabling construction of disposable sensory electrodes. We expect that this architecture could be expanded to the detection of other biochemical activities, post-translational modifications, nucleic acids, and inorganic molecules. [ABSTRACT FROM AUTHOR]

Carbon dioxide accumulation is a major concern for the ecosystems, but its abundance and low cost make it an interesting source for the production of chemical feedstocks and fuels. However, the thermodynamic and kinetic stability of the carbon dioxide molecule makes its activation a challenging task. Studying the chemistry used by nature to functionalize carbon dioxide should be helpful for the development of new efficient (bio)catalysts for atmospheric carbon dioxide utilization. In this work, the ability of Desulfovibrio desulfuricans formate dehydrogenase (Dd FDH) to reduce carbon dioxide was kinetically and mechanistically characterized. The Dd FDH is suggested to be purified in an inactive form that has to be activated through a reduction-dependent mechanism. A kinetic model of a hysteretic enzyme is proposed to interpret and predict the progress curves of the Dd FDH-catalyzed reactions (initial lag phase and subsequent faster phase). Once activated, Dd FDH is able to efficiently catalyze, not only the formate oxidation (kcat of 543 s-1, Km of 57.1 μM), but also the carbon dioxide reduction (kcat of 46.6 s-1, Km of 15.7 μM), in an overall reaction that is thermodynamically and kinetically reversible. Noteworthy, both Dd FDH-catalyzed formate oxidation and carbon dioxide reduction are completely inactivated by cyanide. Current FDH reaction mechanistic proposals are discussed and a different mechanism is here suggested: formate oxidation and carbon dioxide reduction are proposed to proceed through hydride transfer and the sulfo group of the oxidized and reduced molybdenum center, Mo6+=S and Mo4+-SH, are suggested to be the direct hydride acceptor and donor, respectively. [ABSTRACT FROM AUTHOR]

The creation of electron transfer (ET) chains based on the defined arrangement of enzymes and redox proteins on electrode surfaces represents an interesting approach within the field of bioelectrocatalysis. In this study, we investigated the ET reaction of the flavin-dependent enzyme fructose dehydrogenase (FDH) with the redox protein cytochrome c (cyt c). Two different pH optima were found for the reaction in acidic and neutral solutions. When cyt c was adsorbed on an electrode surface while the enzyme remained in solution, ET proceeded efficiently in media of neutral pH. Interprotein ET was also observed in acidic media; however, it appeared to be less efficient. These findings suggest that two different ET pathways between the enzyme and cyt c may occur. Moreover, cyt c and FDH were immobilized in multiple layers on an electrode surface by means of another biomacromolecule: DNA (double stranded) using the layer-by-layer technique. The biprotein multilayer architecture showed a catalytic response in dependence on the fructose concentration, indicating that the ET reaction between both proteins is feasible even in the immobilized state. The electrode showed a defined response to fructose and a good storage stability. Our results contribute to the better understanding of the ET reaction between FDH and cyt c and provide the basis for the creation of all-biomolecule based fructose sensors the sensitivity of which can be controlled by the layer preparation. [ABSTRACT FROM AUTHOR]

The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system for studies on the role of protein dynamics in catalysis, but so far these studies have been limited by the availability of structural information. Second, CbFDH and its mutants can be used in various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in commercial development. Here, we report the crystallization and structural determination of both holo- and apo-CbFDH. The free-energy barrier for the catalyzed reaction was computed and indicates that this structure indeed represents a catalytically competent form of the enzyme. Complementing kinetic examinations demonstrate that the recombinant CbFDH has a well-organized reactive state. Finally, a fortuitous observation has been made: the apoenzyme crystal was obtained under cocrystallization conditions with a saturating concentration of both the cofactor (NAD+) and inhibitor (azide), which has a nanomolar dissociation constant. It was found that the fraction of the apoenzyme present in the solution is less than 1.7 × 10-7 (i.e., the solution is 99.9999% holoenzyme). This is an extreme case where the crystal structure represents an insignificant fraction of the enzyme in solution, and a mechanism rationalizing this phenomenon is presented. [ABSTRACT FROM AUTHOR]

Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal-containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pKa of the active site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable of reducing nitrate to nitrite. We propose a mechanistic scheme that combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase. [ABSTRACT FROM AUTHOR]

The α,β-epoxyketone proteasome inhibitor TMC-86A was discovered as a previously unreported metabolite of Streptomyces chromofuscus ATCC49982, and the gene cluster responsible for its biosynthesis was identified via genome sequencing. Incorporation experiments with [13C-methyl]L-methionine implicated an α-dimethyl-β-keto acid intermediate in the biosynthesis of TMC-86A. Incubation of the chemically synthesized α-dimethyl-β-keto acid with a purified recombinant flavin-dependent enzyme that is conserved in all known pathways for epoxyketone biosynthesis resulted in formation of the corresponding α-methyl-α,β-epoxyketone. This transformation appears to proceed via an unprecedented decarboxylation-dehydrogenation-monooxygenation cascade. The biosynthesis of the TMC-86A warhead is completed by cytochrome P450-mediated hydroxylation of the α-methyl-α,β-epoxyketone. [ABSTRACT FROM AUTHOR]

Here we report a self-powered-bipolar-electrochromic-electrode (termed SP-BP-EC-E) array for the displaying applications including catalyst screening, catalytic activity measurement, and enzyme substrate quantification. By replacing the directional (or active) power source with the isotropic chemical energy to drive the bipolar electrochemical reaction, the driving background signal, bipolar electrode (BPE) background signal, uneven reporting signal and the influence of electrolysis which commonly appear in traditional bipolar systems are effectively eliminated from origin. Thus, the reporting signals from the SP-BP-EC-E arrays can be more direct and reliable to reflect the target nature. Such a SP-BP-EC-E platform exhibits a sensitive response toward the fast analysis of commercial Pt black catalyst, NiPdAu hollow nanospheres, glucose dehydrogenase, and glucose. To our knowledge, this test paper-like SP-BP-EC-E is the simplest platform for high-throughput screening to date, which offers a very convenient approach for nonprofessional people to access the complicated screening and fast analysis of the electrocatalysts and biocatalyst activity and quantification of enzymatic substrates. [ABSTRACT FROM AUTHOR]

Coimmobilization of pyranose dehydrogenase as an enzyme catalyst, osmium redox polymers [Os(4,4'-dimethoxy-2,2'-bipyridine)2(poly(vinylimidazole))10Cl]+ or [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly(vinylimidazole))10Cl]+ as mediators, and carbon nanotube conductive scaffolds in films on graphite electrodes provides enzyme electrodes for glucose oxidation. The recombinant enzyme and a deglycosylated form, both expressed in Pichia pastoris, are investigated and compared as biocatalysts for glucose oxidation using flow injection amperometry and voltammetry. In the presence of 5 mM glucose in phosphate-buffered saline (PBS) (50 mM phosphate buffer solution, pH 7.4, with 150 mM NaCl), higher glucose oxidation current densities, 0.41 mA cm-2, are obtained from enzyme electrodes containing the deglycosylated form of the enzyme. The optimized glucose-oxidizing anode, prepared using deglycosylated enzyme coimmobilized with [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly(vinylimidazole))10Cl]+ and carbon nanotubes, was coupled with an oxygen-reducing bilirubin oxidase on gold nanoparticle dispersed on gold electrode as a biocathode to provide a membraneless fully enzymatic fuel cell. A maximum power density of 275 μW cm-2 is obtained in 5 mM glucose in PBS, the highest to date under these conditions, providing sufficient power to enable wireless transmission of a signal to a data logger. When tested in whole human blood and unstimulated human saliva maximum power densities of 73 and 6 μW cm-2 are obtained for the same fuel cell configuration, respectively. [ABSTRACT FROM AUTHOR]

Three maturase enzymes--HydE, HydF, and HydG--synthesize and insert the organometallic component of the [FeFe]-hydrogenase active site (the H-cluster). HydG generates the first organometallic intermediates in this process, ultimately producing an [Fe(CO)2(CN)] complex. A limitation in understanding the mechanism by which this complex forms has been uncertainty regarding the precise metallocluster composition of HydG that comprises active enzyme. We herein show that the HydG auxiliary cluster must bind both l-cysteine and a dangler Fe in order to generate the [Fe(CO)2(CN)] product. These findings support a mechanistic framework in which a [(Cys)Fe(CO)2(CN)]- species is a key intermediate in H-cluster maturation. [ABSTRACT FROM AUTHOR]

The key active site residues K185, Y139, D217, D241, D245, and N102 of Thermus thermophilus 3-isopropylmalate dehydrogenase (Tt-IPMDH) have been replaced, one by one, with Ala. A drastic decrease in the kcat value (0.06% compared to that of the wild-type enzyme) has been observed for the K185A and D241A mutants. Similarly, the catalytic interactions (Km values) of these two mutants with the substrate IPM are weakened by more than 1 order of magnitude. The other mutants retained some (1-13%) of the catalytic activity of the wild-type enzyme and do not exhibit appreciable changes in the substrate Km values. The pH dependence of the wild-type enzyme activity (pK = 7.4) is shifted toward higher values for mutants K185A and D241A (pK values of 8.4 and 8.5, respectively). For the other mutants, smaller changes have been observed. Consequently, K185 and D241 may constitute a proton relay system that can assist in the abstraction of a proton from the OH group of IPM during catalysis. Molecular dynamics simulations provide strong support for the neutral character of K185 in the resting state of the enzyme, which implies that K185 abstracts the proton from the substrate and D241 assists the process via electrostatic interactions with K185. Quantum mechanics/molecular mechanics calculations revealed a significant increase in the activation energy of the hydride transfer of the redox step for both D217A and D241A mutants. Crystal structure analysis of the molecular contacts of the investigated residues in the enzyme-substrate complex revealed their additional importance (in particular that of K185, D217, and D241) in stabilizing the domain-closed active conformation. In accordance with this, small-angle X-ray scattering measurements indicated the complete absence of domain closure in the cases of D217A and D241A mutants, while only partial domain closure could be detected for the other mutants. This suggests that the same residues that are important for catalysis are also essential for inducing domain closure. [ABSTRACT FROM AUTHOR]

It is remarkable how nature has been able to construct enzymesthat, despite sharing many similarities, have simple but key differencesthat tune them for completely different functions in living cells.Periplasmic nitrate reductase (Nap) and formate dehydrogenase (Fdh)from the DMSOr family are representative examples of this. Both enzymesshare almost identical three-dimensional protein foldings and activesites, in terms of coordination number, geometry and nature of theligands. The substrates of both enzymes (nitrate and formate) arepolyatomic anions that also share similar charge and stereochemistry.In terms of the catalytic mechanism, both enzymes have a common activationmechanism (the sulfur-shift mechanism) that ensures a constant coordinationnumber around the metal ion during the catalytic cycle. In spite ofthese similarities, they catalyze very different reactions: Nap abstractsan oxygen atom from nitrate releasing nitrite, whereas FdH catalyzesa hydrogen atom transfer from formate and releases carbon dioxide. [ABSTRACT FROM AUTHOR]

Protein film electrochemistry hasbeen used to investigate reactionsof highly active nickel-containing carbon monoxide dehydrogenases(CODHs). When attached to a pyrolytic graphite electrode, these enzymesbehave as reversible electrocatalysts, displaying CO2reductionor CO oxidation at minimal overpotential. The O2sensitivityof CODH is suppressed by adding cyanide, a reversible inhibitor ofCO oxidation, or by raising the electrode potential. Reduction ofN2O, isoelectronic with CO2, is catalyzed byCODH, but the reaction is sluggish, despite a large overpotential,and results in inactivation. Production of H2and formateunder highly reducing conditions is consistent with calculations predictingthat a nickel-hydrido species might be formed, but the very low ratessuggest that such a species is not on the main catalytic pathway. [ABSTRACT FROM AUTHOR]

Ribosomally synthesized peptides are generally limited to l-amino acid building blocks. Given the advantageous properties of peptides containing d-amino acids such as stabilization of certain turns and against proteolytic degradation, methods to introduce d-stereocenters are valuable. Here we report the first in vitro reconstitution and characterization of a dehydrogenase that carries out the asymmetric reduction of dehydroalanine. NpnJA reduces dehydroalanine to d-Ala using NAPDH as cosubstrate. The enzyme displays high substrate tolerance allowing introduction of d-Ala into a range of non-native substrates. In addition to the in vitro reactions, we describe five examples of using Escherichia coli as biosynthetic host for d-alanine introduction into ribosomal peptides. A deuterium-label-based coupled-enzyme assay was used to rapidly determine the stereochemistry of the newly installed alanine. [ABSTRACT FROM AUTHOR]

Primary biliary cirrhosis is an immune-mediatedchronic liver diseasewhose diagnosis relies on the detection of serum antimitochondrialantibodies directed against a complex set of proteins, among whichpyruvate dehydrogenase complex is considered the main autoantigen.We studied the immunological role of the lipoyl domain of this proteinusing synthetic lipoylated peptides, showing that the lipoyl chainchirality does not affect autoantibody recognition and, most importantly,confirming that both lipoylated and unlipoylated peptides are ableto recognize specific autoantibodies in patients sera. In fact, 74%of patients sera recognize at least one of the tested peptides butvery few positive sera recognized exclusively the lipoylated peptide,suggesting that the lipoamide moiety plays a marginal role withinthe autoreactive epitope. These results are supported by a conformationalanalysis showing that the lipoyl moiety of pyruvate dehydrogenasecomplex appears to be involved in hydrophobic interactions, whichmay limit its exposition and thus its contribution to the complexantigenic epitope. A preliminary analysis of the specificity of thetwo most active peptides indicates that they could be part of a panelof synthetic antigens collectively able to mimic in a simple immunoenzymaticassay the complex positivity pattern detected in immunofluorescence. [ABSTRACT FROM AUTHOR]

In this work, an aerobic copper-catalyzed intramolecular C(sp³)-H amidation has been disclosed, which presents a rare example of copper-catalyzed functionalization of an unactivated C(sp³)-H bond with O2 as the sole oxidant. In addition, a new protocol for the preparation of a removable 5-methoxyquinolyl moiety has been documented. [ABSTRACT FROM AUTHOR]

A palladium-catalyzed efficient C-H acylation reaction of biaryl-2-amines and aromatic aldehydes is developed. This dehydrogenative cross-coupling protocol could furnish monoacylation and diacylation products in moderate to good yields with a broad substrate scope and good regioselectivity. [ABSTRACT FROM AUTHOR]

The human enzyme dihydroorotate dehydrogenase(HsDHODH) has been studied for being a target fordevelopment of newantineoplasic and antiproliferative drugs. The synthetic peptide N-t(DH)represents the N-terminal microdomain of this enzyme, responsiblefor anchoring it to the inner mitochondrial membrane. Also, it isknown to harbor quinones that are essential for enzyme catalysis.Here we report structural features of the peptide/membrane interactionsobtained by using CD and DEER spectroscopic techniques, both in micellesand in lipid vesicles. The data revealed different peptide conformationalstates in micelles and liposomes, which could suggest that this microdomainacts in specific regions or areas of the mitochondria, which can berelated with the control of the quinone access to the HsDHODH active site. This is the first study to report on conformationalchanges of the HsDHODH N-terminal microdomain througha combination of CD and DEER spectroscopic techniques. [ABSTRACT FROM AUTHOR]

Despite a lack of convincing in vitro evidence and a number of sound refutations, it is widely accepted that renalase is an enzyme unique to animals that catalyzes the oxidative degradation of catecholamines in blood in order to lower vascular tone. Very recently, we identified isomers of β- NAD(P)H as substrates for renalase (Beaupre, B. A. et al. (2015) Biochemistry, 54, 795-806). These molecules carry the hydride equivalent on the 2 or 6 position of the nicotinamide base and presumably arise in nonspecific redox reactions of nicotinamide dinucleotides. Renalase serves to rapidly oxidize these isomers to form β-NAD(P)+ and then pass the electrons to dioxygen, forming H2O2. We have also shown that these substrate molecules are highly inhibitory to dehydrogenase enzymes and thus have proposed an intracellular metabolic role for this enzyme. Here, we identify a renalase from an organism without a circulatory system. This bacterial form of renalase has the same substrate specificity profile as that of human renalase but, in terms of binding constant (Kd), shows a marked preference for substrates derived from β-NAD+. 2-dihydroNAD(P) substrates reduce the enzyme with rate constants (fcred) that greatly exceed those for 6-dihydroNAD(P) substrates. Taken together, kred/Kd values indicate a minimum 20-fold preference for 2DHNAD. We also offer the first structures of a renalase in complex with catalytically relevant ligands β-NAD+ and β-NADH (the latter being an analogue of the substrate(s)). These structures show potential electrostatic repulsion interactions with the product and a unique binding orientation for the substrate nicotinamide base that is consistent with the identified activity. [ABSTRACT FROM AUTHOR]

In this study, the influence of two quinones (1,2- and 1,4-benzoquinone) on the operation and mechanism of electron transfer in PQC--dependent glucose dehydrogenase (PQQ--sGDH) anodes has been determined. Benzoquinones were experimentally explored as mediators present in the electrolyte. The electrochemical performance of the PQQ7s--GDH anodes with and without the mediators was examined and for the first time molecular docking simulations were used to gain a fundamental understanding to explain the role of the mediator molecules in the design and operation of the enzymatic electrodes. It was proposed that the higher performance of the PQQ--sGDH anodes in the presence of 1,2- and 1,4-benzoquinones introduced in the solution is due to the shorter distance between these molecules and PQQ in the enzymatic molecule. It was also hypothesized that when 1,4-benzoquinone is adsorbed on a carbon support, it would play the dual role of a mediator and an orienting agent. At the same time, when 1,2-benzoquinone and ubiquinone are adsorbed on the electrode surface, the enzyme would transfer the electrons directly to the support, and these molecules would primarily play the role of an orienting agent. [ABSTRACT FROM AUTHOR]

A rhodium-catalyzed [2 + 2 + 2] cycloaddition of cyano-yne-allene scaffolds followed by a dehydrogenative process enabling the direct synthesis of unsaturated pyridine-containing compounds that can be conveniently converted to 2,6-naphthyridine derivatives is reported. [ABSTRACT FROM AUTHOR]

Methylenetetrahydrofolate dehydrogenase-cyclohydrolase (FolD) catalyzes interconversion of 5,10-methylene-tetrahydrofolate and 10-formyltetrahydrofolate in the one-carbon metabolic pathway. In some organisms, the essential requirement of 10-formyltetrahydrofolate may also be fulfilled by formyltetrahydrofolate synthetase (Fhs). Recently, we developed an Escherichia coli strain in which the folD gene was deleted in the presence of Clostridium perfringens fhs (E. coli ΔfolD/p-fhs) and used it to purify FolD mutants (free from the host-encoded FolD) and determine their biological activities. Mutations in the key residues of E. coli FolD, as identified from three-dimensional structures (D121A, Q98K, K54S, Y50S, and R191E), and a genetic screen (G122D and C58Y) were generated, and the mutant proteins were purified to determine their kinetic constants. Except for the R191E and K54S mutants, others were highly compromised in terms of both dehydrogenase and cyclohydrolase activities. While the R191E mutant showed high cyclohydrolase activity, it retained only a residual dehydrogenase activity. On the other hand, the K54S mutant lacked the cyclohydrolase activity but possessed high dehydrogenase activity. The D121A and G122D (in a loop between two helices) mutants were highly compromised in terms of both dehydrogenase and cyclohydrolase activities. In vivo and in vitro characterization of wild-type and mutant (R191E, G122D, D121A, Q98K, C58Y, K54S, and Y50S) FolD together with three-dimensional modeling has allowed us to develop a better understanding of the mechanism for substrate binding and catalysis by E. coli FolD. [ABSTRACT FROM AUTHOR]

A versatile and general route has been developed for the asymmetric synthesis of a wide family of 3-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazines bearing different pattern substitutions in the aromatic ring. Whereas hydrolases were not suitable for resolution of these racemic cyclic nitrogenated amines, alternative chemoenzymatic strategies were designed through independent pathways leading to both amine antipodes. On one hand, bioreduction of 1-(2-nitrophenoxy)-propan-2-ones allowed the recovery of the enantiopure (S)-alcohols in high yields using the alcohol dehydrogenase from Rhodococcus ruber (ADH-A), whereas evo-1.1.200 ADH led to their counterpart (R)-enantiomers also with complete selectivity and quantitative conversion. Alternatively, lipase-catalyzed acetylation of these racemic alcohols, and the complementary hydrolysis of the acetate analogues, gave access to the corresponding optically enriched products with high stereodiscrimination. Particularly attractive was the design of a chemoenzymatic strategy in six steps for the production of (S)-(--)-7,8-difluoro-3-methyl-3,4'dihydro-2H-benzo-[b][1,4]oxazine, which is a key precursor of the antimicrobial agent Levofloxacin. [ABSTRACT FROM AUTHOR]

In washing-free electrochemical detection, various redox and reactive species cause significant interference. To minimize this interference, we report a washing-free electrochemical immunosensor using flavin adenine dinucleotide (FAD)-dependent glycerol- 3-phosphate dehydrogenase (GPDH) and glycerol-3-phosphate (GP) as an enzyme label and its substrate, respectively, because the reaction of FAD-dependent dehydrogenases with dissolved O2 is slow and the level of GP preexisting in blood is low (<0.1 mM). A combination of a low electrocatalytic indium--tin oxide (ITO) electrode and fast electronmediating Ru(NH3)63+ is employed to obtain a high signal-to-background ratio via proximitydependent electron mediation of Ru(NH3)63+ between the ITO electrode and the GPDH label. Electrochemical oxidation of GPDH-generated Ru(NH3)62+ is performed at 0.05 V vs Ag/AgCl, at which point the electrochemical interference is very low. When a washing-free immunosensor is applied to cardiac troponin I detection in human serum, the calculated detection limit is approximately 10 pg/mL, indicating that the immunosensor is very sensitive in spite of the use of washing-free detection with a short detection period (10 min for incubation and 100 s for electrochemical measurement). The low-interference washing-free electrochemical immunosensor shows good promise for fast and simple point-of-care testing. [ABSTRACT FROM AUTHOR]

Phenyllactic adds are found in numerous natural products as well as in active substances used in medicine or plant protection. Enantiomerically pure phenyllactic adds are available by transition-metal-catalyzed hydrogenations or chemoenzymatic reductions of the corresponding 3-aryl-2-oxopropanoic acids. We show here that D-lactate dehydrogenase from Staphylococcus epidermidis reduces a broad spectrum of 2-oxo adds, which are difficult substrates for transition-metal-catalyzed reactions, with excellent enantioselectivities in a simple experimental setup. [ABSTRACT FROM AUTHOR]

By using catalytic amount of CoCl2with dmgH (dimethylglyoxime)as ligand to form a photosensitizer in situ, a highly selective, efficient,and environmentally benign visible light mediated cross-dehydrogenativecoupling (CDC) reaction has been developed in aqueous medium. Thedesired cross-coupling C–C bonds that involve Csp3with Csp, Csp2, and Csp3, respectively,were achieved exclusively in high yields without formation of anyother byproduct. [ABSTRACT FROM AUTHOR]

The kinetic parameters for activation of yeast triosephosphate isomerase (ScTIM), yeast orotidine monophosphate decarboxylase (ScOMPDC), and human liver glycerol 3-phosphate dehydrogenase (hlGPDH) for catalysis of reactions of their respective phosphodianion truncated substrates are reported for the following oxydianions: HPO32-, FPO32-, S2O32-, SO42- and HOPO32-. Oxydianions bind weakly to these unliganded enzymes and tightly to the transition state complex (E·S‡), with intrinsic oxydianion Gibbs binding free energies that range from -8.4 kcal/mol for activation of hlGPDH-catalyzed reduction of glycolaldehyde by FPO32- to -3.0 kcal/mol for activation of ScOMPDC-catalyzed decarboxylation of 1-β-d-erythrofuranosyl)orotic acid by HOPO32-. Small differences in the specificity of the different oxydianion binding domains are observed. We propose that the large -8.4 kcal/mol and small -3.8 kcal/mol intrinsic oxydianion binding energy for activation of hlGPDH by FPO32- and S2O32-, respectively, compared with activation of ScTIM and ScOMPDC reflect stabilizing and destabilizing interactions between the oxydianion -F and -S with the cationic side chain of R269 for hlGPDH. These results are consistent with a cryptic function for the similarly structured oxydianion binding domains of ScTIM, ScOMPDC and hlGPDH. Each enzyme utilizes the interactions with tetrahedral inorganic oxydianions to drive a conformational change that locks the substrate in a caged Michaelis complex that provides optimal stabilization of the different enzymatic transition states. The observation of dianion activation by stabilization of active caged Michaelis complexes may be generalized to the many other enzymes that utilize substrate binding energy to drive changes in enzyme conformation, which induce tight substrate fits. [ABSTRACT FROM AUTHOR]

Chaperone machinery in living systemscan catch denatured enzymesand induce their reactivation. Chaperone mimics are beneficial forapplying enzymatic reactions in vitro. In this work,the affinity between liposomes and thermally denatured enzymes wascontrolled to stabilize the enzyme activity. The model enzyme is formatedehydrogenase from Candida boidinii(CbFDH) whichis a homodimer and negatively charged in the phosphate buffer solution(pH 7.2) used. The activity of free CbFDH readily decreased at 58°C following the first-order kinetics with the half-life t1/2of 27 min. The turbidity measurements showedthat the denatured enzyme molecules formed aggregates. The liposomescomposed of zwitterionic phosphatidylcholines (PCs) stabilized theCbFDH activity at 58 °C, as revealed with six different PCs.The PC liposomes were indicated to bind to the aggregate-prone enzymemolecules, allowing reactivation at 25 °C. The cofactor β-reducednicotinamide adenine dinucleotide (NADH) also stabilized the enzymeactivity. The affinity between liposomes and denatured CbFDH couldbe modulated by incorporating cationic 1,2-dioleoyloxy-3-trimethylammoniumpropane chloride (DOTAP) in PC membranes. The t1/2values significantly increased in the presence of liposomes([lipid] = 1.5 mM) composed of PC and DOTAP at the mole fraction fDof 0.1. On the other hand, the DOTAP-richliposomes (fD≥ 0.7) showed strongaffinity toward denatured CbFDH, accelerating its deactivation. Theliposomes with low charge density function as chaperone mimics thatcan efficiently catch the denatured enzymes without interfering withtheir intramolecular interaction for reactivation. [ABSTRACT FROM AUTHOR]

Agrobacterium tumefaciens (At) strain C58 contains an oxidative enzyme pathway that can function on both D-glucuronic and D-galacturonic acid. The corresponding gene coding for At keto-deoxy-D-galactarate (KDG) dehydratase is located in the same gene cluster as those coding for uronate dehydrogenase (At Udh) and galactarolactone cycloisomerase (At Gci) which we have previously characterized. Here, we present the kinetic characterization and crystal structure of At KDG dehydratase, which catalyzes the next step, the decarboxylating hydrolyase reaction of KDG to produce α-ketoglutaric semialdehyde (α-KGSA) and carbon dioxide. The crystal structures of At KDG dehydratase and its complexes with pyruvate and 2-oxoadipic acid, two substrate analogues, were determined to 1.7 Å, 1.5 Å, and 2.1 Å resolution, respectively. Furthermore, mass spectrometry was used to confirm reaction end-products. The results lead us to propose a structure-based mechanism for At KDG dehydratase, suggesting that while the enzyme belongs to the Class I aldolase protein family, it does not follow a typical retro-aldol condensation mechanism. [ABSTRACT FROM AUTHOR]

Cryptosporidiuminosine 5′-monophosphatedehydrogenase (CpIMPDH) has emerged as a therapeutictarget for treating Cryptosporidiumparasites because it catalyzes a critical step in guanine nucleotidebiosynthesis. A 4-oxo-[1]benzopyrano[4,3-c]pyrazolederivative was identified as a moderately potent (IC50=1.5 μM) inhibitor of CpIMPDH. We report a SARstudy for this compound series resulting in 8k(IC50= 20 ± 4 nM). In addition, an X-ray crystal structureof CpIMPDH·IMP·8kis alsopresented. [ABSTRACT FROM AUTHOR]

Steadily growing demands for identification and 1 .Chip quantification of cellular metabolites in higher throughput have brought a need for new analytical technologies. Here, we developed a synthetic biological sensor system for quantifying metabolites from biological cell samples. For this, bacterial transcription factors were exploited, which bind to or dissociate from regulatory DNA elements in response to physiological changes in the cellular metabolite concentration range. Representatively, the bacterial pyruvate dehydrogenase (PdhR), trehalose (TreR), and L-arginine (ArgR) repressor proteins were functionalized to detect pyruvate, trehalose-6-phosphate (T6P), and arginine concentration in solution. For each transcription factor the mutual binding behavior between metabolite and DNA, their working range, and othogonality were determined. High-throughput, parallel processing, and automation were achieved through integration of the metabolic sensor system on a microfluidic large-scale integration (mLSI) chip platform. To demonstrate the functionality of the integrated metabolic sensor system, we measured diurnal concentration changes of pyruvate and the plant signaling molecule T6P within cell etxracts of Arabidopsis thaliana rosettes. The transcription factor sensor system is of generic nature and extendable on the microfluidic chip. [ABSTRACT FROM AUTHOR]

CO2 and formate are rapidly, selectively, and efficiently interconverted by tungsten-containing formate dehydrogenases that surpass current synthetic catalysts. However, their mechanism of catalysis is unknown, and no tractable system is available for study. Here, we describe the catalytic properties of the molybdenum-containing formate dehydrogenase H from the model organism Escherichia coli (EcFDH-H). We use protein film voltammetry to demonstrate that EcFDH-H is a highly active, reversible electrocatalyst. In each voltammogram a single point of zero net current denotes the CO2 reduction potential that varies with pH according to the Nernst equation. By quantifying formate production we show that electrocatalytic CO2 reduction is specific. Our results reveal the capabilities of a Mo-containing catalyst for reversible CO2 reduction and establish EcFDH-H as an attractive model system for mechanistic investigations and a template for the development of synthetic catalysts. [ABSTRACT FROM AUTHOR]

The flavin-mediated enzymatic oxidation of a CN bond in amino adds can occur through hydride transfer, carbanion, or polar nudeophilic mechanisms. Previous results with D-arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) using multiple deuterium kinetic isotope effects (KlEs) and computational studies established preferred binding of the substrate protonated on the α-amino group, with cleavages of the NH and CH bonds occurring in asynchronous fashion, consistent with the three possible mechanisms. The hydroxyl groups of YS3 and Y249 are ⩽4 Å from the imino and carboxylate groups of the reaction product iminoarginine, suggesting participation in binding and catalysis. In this study, we have investigated the reductive half-reactions of the Y53F and Y249F variants of PaDADH using substrate and solvent deuterium KlEs, solvent viscosity and pH effects, and quantum mechanical/molecular mechanical computational approaches to gain insights into the catalytic roles of the tyrosines and evaluate whether their mutations affect the transition state for substrate oxidation. Both Y53F and Y249F enzymes oxidized d - arginine with steady-state kinetic parameters similar to those of the wild-type enzyme. Rate constants for flavin reduction (kred ) with D-leucine, a slow substrate amenable to rapid kinetics, were 3-fold smaller than the wild-type value with similar pKa values for an unprotonated group of ∼10.0. Similar piC, values were observed for appKd in the variant and wild-type enzymes. However, cleavage of the substrate NH and CH bonds in the enzyme variants occurred in synchronous fashion, as suggested by multiple deuterium KlEs on fcred. These data can be reconciled with a hydride transfer mechanism, but not with carbanion and polar nucleophilic mechanisms. [ABSTRACT FROM AUTHOR]

Density functional theory is used for periodic models of monomeric vanadia species deposited on the CeO2(111) surface to study dissociative adsorption of methanol and its subsequent dehydrogenation to formaldehyde. Dispersion-corrected PBE+U calculations are performed and compared with HSE and B3LYP results. Dissociative adsorption of methanol at different sites on VO2•CeO2 (111) is highly exothermic with adsorption energies of 1.8 to 1.9 eV (HSE+D). Two relevant pathways for desorption of formaldehyde are found with intrinsic barriers for the redox step of 1.0 and 1.4 eV (HSE+D). The calculated desorption temperatures (370 and 495 K) explain the peaks observed in temperature-programmed desorption experiments. Different sites of the supported catalyst system are involved in the two pathways: (i) methanol can chemisorb on the CeO2 surface filling a so-called pseudovacancy and the H atom is transferred to an V—O—Ce interphase bond or (ii) CH3OH may chemisorb at the V—O—Ce interphase bond and form a V— OCH3 species from which H is transferred to the ceria surface, providing evidence for true cooperativity. In both cases, ceria is directly involved in the redox process, as two electrons are accommodated in Ce f states forming two Ce3+ ions whereas vanadium remains fully oxidized (V5+). [ABSTRACT FROM AUTHOR]

Bacterial systems are being extensively studied and modified for energy, sensors, and industrial chemistry; yet, their molecular scale structure and activity are poorly understood. Designing efficient bioengineered bacteria requires cellular understanding of enzyme expression and activity. An atomic force microscope (AFM) was modified to detect and analyze the activity of redox active enzymes expressed on the surface of E. coli. An insulated gold-coated metal microwire with only the tip conducting was used as an AFM cantilever and a working electrode in a three-electrode electrochemical cell. Bacteria were engineered such that alcohol dehydrogenase II (ADHII) was surface displayed. A quinone, an electron transfer mediator, was covalently attached site specifically to the displayed ADHII. The AFM probe was used to lift a single bacterium off the surface for electrochemical analysis in a redox-free buffer. An electrochemical comparison between two quinone containing mutants with different distances from the NAD+ binding site in alcohol dehydrogenase II was performed. Electron transfer in redox active proteins showed increased efficiency when mediators are present closer to the NAD+ binding site. This study suggests that an integrated conducting AFM used for single cell electrochemical analysis would allow detailed understanding of enzyme electron transfer processes to electrodes, the processes integral to creating efficiently engineered biosensors and biofuel cells. [ABSTRACT FROM AUTHOR]

Sulfite dehydrogenase from the soilbacterium Sinorhizobiummeliloti(SorT) is a periplasmic, homodimeric molybdoenzymewith a molecular mass of 78 kDa. It differs from most other well studiedsulfite oxidizing enzymes, as it bears no heme cofactor. SorT doesnot readily reduce ferrous horse heart cytochrome cwhich is the preferred electron acceptor for vertebrate sulfiteoxidases. In the present study, ferrocene methanol (FM) (in its oxidizedferrocenium form) was utilized as an artificial electron acceptorfor the catalytic SorT sulfite oxidation reaction. Cyclic voltammetryof FM was used to generate the active form of the mediator at theelectrode surface. The FM-mediated catalytic sulfite oxidation bySorT was investigated by two different voltammetric methods, namely,(i) SorT freely diffusing in solution and (ii) SorT confined to athin layer at the electrode surface by a semipermeable dialysis membrane.A single set of rate and equilibrium constants was used to simulatethe catalytic voltammograms performed under different sweep ratesand with various concentrations of sulfite and FM which provides newinsights into the kinetics of the SorT catalytic mechanism. Further,we were able to model the role of the dialysis membrane in the kineticsof the overall catalytic system. [ABSTRACT FROM AUTHOR]

A series of four [S2Ni(µ-S)2FeCp*Cl] compounds with different tetradentate thiolate/thioether ligands bound to the Ni(II) ion is reported (Cp* = C5Me5). The {S2Ni(µ-S)2Fe} core of these compounds resembles structural features of the active site of [NiFe] hydrogenases. Detailed analyses of the electronic structures of these compounds by Mössbauer and electron paramagnetic resonance spectroscopy, magnetic measurements, and density functional theory calculations reveal the oxidation states Ni(II) low spin and Fe(II) high spin for the metal ions. The same electronic configurations have been suggested for the Cred1 state of the C-cluster [NiFeu] subsite in carbon monoxide dehydrogenases (CODH). The Ni-Fe distance of ~3 Å excludes a metal-metal bond between nickel and iron, which is in agreement with the computational results. Electrochemical experiments show that iron is the redox active site in these complexes, performing a reversible one-electron oxidation. The four complexes are discussed with regard to their similarities and differences both to the [NiFe] hydrogenases and the C-cluster of Ni-containing CODH. [ABSTRACT FROM AUTHOR]

The power output and stability of enzyme-based biofuel cells (BFCs) is greatly dependent on the properties of both the biocathode and bioanode, which may be adapted for portable power production. In this paper, a novel highly uniform three-dimensional (3D) macroporous gold (MP-Au) film was prepared by heating the gold "supraspheres", which were synthesized by a bottom-up protein templating approach, and followed by modification of laccase on the MP-Au film by covalent immobilization. The as-prepared laccase/MP-Au biocathode exhibited an onset potential of 0.62 V versus saturated calomel electrode (SCE, or 0.86 V vs NHE, normal hydrogen electrode) toward O2 reduction and a high catalytic current of 0.61 mAcm-2. On the other hand, mutated glucose dehydrogenase (GDH) surface displayed bacteria (GDH-bacteria) were used to improve the stability of the glucose oxidation at the bioanode. The as-assembled membraneless glucose/O2 fuel cell showed a high power output of 55.8 ± 2.0 pW cm-2 and open circuit potential of 0.80 V, contributing to the improved electrocatalysis toward O2 reduction at the laccase/MP-Au biocathode. Moreover, the BFC retained 84% of its maximal power density even after continuous operation for 55 h because of the high stability of the bacterial surface displayed GDH mutant toward glucose oxidation. Our findings may be promising for the development of more efficient glucose BFC for portable battery or self-powered device applications. [ABSTRACT FROM AUTHOR]

The L-lyxonate dehydratase (LyxD) in vitro enzymatic activity and in vivo metabolic function were assigned to members of an isofunctional family within the mandelate racemase (MR) subgroup of the enolase superfamily. This study combined in vitro and in vivo data to confirm that the dehydration of L-lyxonate is the biological role of the members of this family. In vitro kinetic experiments revealed catalytic efficiencies of ~104 M-1 s-1 as previously observed for members of other families in the MR subgroup. Growth studies revealed that L-lyxonate is a carbon source for Pseudomonas aeruginosa PAO1; transcriptomics using qRT-PCR established that the gene encoding LyxD as well as several other conserved proximal genes were upregulated in cells grown on L-lyxonate. The proximal genes were shown to be involved in a pathway for the degradation of L-lyxonate, in which the first step is dehydration by LyxD followed by dehydration of the 2-keto-3-deoxy-L-lyxonate product by 2-keto-3-deoxy-L-lyxonate dehydratase to yield α-ketoglutarate semialdehyde. In the final step, α- ketoglutarate semialdehyde is oxidized by a dehydrogenase to α-ketoglutarate, an intermediate in the citric acid cycle. An X-ray structure for the LyxD from Labrenzia aggregata IAM 12614 with Mg2+ in the active site was determined that confirmed the expectation based on sequence alignments that LyxDs possess a conserved catalytic His-Asp dyad at the end of seventh and sixth β-strands of the (β/α)7β-barrel domain as well as a conserved KxR motif at the end of second β-strand; substitutions for His 316 or Arg 179 inactivated the enzyme. This is the first example of both the LyxD function in the enolase superfamily and a pathway for the catabolism of L-lyxonate. [ABSTRACT FROM AUTHOR]