Redox Cofactors Research Articles

NAD precursors cycle between host tissues and the gut microbiome

Nicotinamide adenine dinucleotide (NAD) is an essential redox cofactor in mammals and microbes. Here we use isotope tracing to investigate the precursors supporting NAD synthesis in the gut microbiome of mice. We find that dietary NAD precursors are absorbed in the proximal part of the gastrointestinal tract and not available to microbes in the distal gut. Instead, circulating host nicotinamide enters the gut lumen and supports microbial NAD synthesis. The microbiome converts host-derived nicotinamide into nicotinic acid, which is used for NAD synthesis in host tissues and maintains circulating nicotinic acid levels even in the absence of dietary consumption. Moreover, the main route from oral nicotinamide riboside, a widely used nutraceutical, to host NAD is via conversion into nicotinic acid by the gut microbiome. Thus, we establish the capacity for circulating host micronutrients to feed the gut microbiome, and in turn be transformed in a manner that enhances host metabolic flexibility.

Redox cofactor metabolism in Saccharomyces cerevisiae and its impact on the production of alcoholic fermentation end-products

The alcoholic fermentation of organic carbon sources by Saccharomyces cerevisiae produces many by-products, with the most abundant originating from central carbon metabolism. The production of these metabolites involves redox reactions and largely depends on the maintenance of redox homeostasis. Despite the metabolic pathways being mostly conserved across strains of S. cerevisiae, their production of various amounts of metabolic products suggests that their intracellular concentration of redox cofactors and/or redox balance differ. This study explored the redox status dynamics and NAD(H) and NADP(H) cofactor ratios throughout alcoholic fermentation in four S. cerevisiae strains that exhibit different carbon metabolic fluxes. This study focussed on the molecular end-products of fermentation, redox cofactor ratios and the impact thereof on redox homeostasis. Strain-dependent differences were identified in the redox cofactor levels, with NADP(H) ratios and levels remaining stable while NAD(H) levels decreased drastically as the fermentation progressed. Changes in the NAD+/NADH ratio were also observed. Total levels of NAD(H) decreased drastically as the fermentation progressed despite the cells remaining viable until the end of fermentation. NAD+ was found to be favoured initially while NADH was favoured towards the end of the fermentation. The change in the NAD+/NADH redox cofactor ratio during fermentation was linked with the production of end-products. The findings in this study could steer further research in the selection of S. cerevisiae wine strains for desirable aroma contributions based on their intracellular redox balance management.

Glucose-6-Phosphate Dehydrogenase, ZwfA, a Dual Cofactor-Specific Isozyme Is Predominantly Involved in the Glucose Metabolism of Pseudomonas bharatica CSV86T.

Glucose-6-phosphate dehydrogenase (Zwf) is an important enzyme in glucose metabolism via the Entner-Doudoroff pathway and the first enzyme in the oxidative pentose-phosphate pathway. It generates NAD(P)H during the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, thus aiding in anabolic processes, energy yield, and oxidative stress responses. Pseudomonas bharatica CSV86T preferentially utilized aromatic compounds over glucose and exhibited a significantly lower growth rate on glucose (0.24 h-1) with a prolonged lag phase (~10 h). In strain CSV86T, glucose was metabolized via the intracellular phosphorylative route only because it lacked an oxidative (gluconate and 2-ketogluconate) route. The genome harbored three genes zwfA, zwfB, and zwfC encoding three Zwf isozymes. The present study aimed to understand gene arrangement, gene expression profiling, and molecular and kinetic properties of the purified enzymes to unveil their physiological significance in the strain CSV86T. The zwfA was found to be a part of the zwfA-pgl-eda operon, which was proximal to other glucose transport and metabolic clusters. The zwfB was found to be arranged as a gnd-zwfB operon, while zwfC was present independently. Among the three, zwfA was transcribed maximally, and the purified ZwfA displayed the highest catalytic efficiency, cooperativity with respect to G6P, and dual cofactor specificity. Isozymes ZwfB and ZwfC were NADP+-preferring and NADP+-specific, respectively. Among other functionally characterized Zwfs, ZwfA from strain CSV86T displayed poor catalytic efficiency and the further absence of oxidative routes of glucose metabolism reflected its lower growth rate on glucose compared to P. putida KT2440 and could be probable reasons for the unique carbon source utilization hierarchy. IMPORTANCE Pseudomonas bharatica CSV86T metabolizes glucose exclusively via the intracellular phosphorylative Entner-Doudoroff pathway leading the entire glucose flux through Zwf as the strain lacks oxidative routes. This may lead to limiting the concentration of downstream metabolic intermediates. The strain CSV86T possesses three isoforms of glucose-6-phosphate dehydrogenase, ZwfA, ZwfB, and ZwfC. The expression profile and kinetic properties of purified enzymes will help to understand glucose metabolism. Isozyme ZwfA dominated in terms of expression and displayed cooperativity with dual cofactor specificity. ZwfB preferred NADP+, and ZwfC was NADP+ specific, which may aid in redox cofactor balance. Such beneficial metabolic flexibility facilitated the regulation of metabolic pathways giving survival/fitness advantages in dynamic environments. Additionally, multiple genes allowed the distribution of function among these isoforms where the primary function was allocated to one of the isoforms.

Direct wiring of redox cofactors onto gold electrodes via inorganic iron-sulfur clusters enhances charge transport

Inorganic iron-sulfur clusters (Fe–S) have emerged as potential electrode anchors and electron shuttles for enzymatic biosensor systems involving nicotinamide adenine dinucleotide (NAD) cofactor. In this work, we report how other cofactors that include nicotinamide adenine dinucleotide phosphate (NADP), flavin adenine dinucleotide (FAD), pyrroloquinoline quinone (PQQ), and coenzyme-Q10 (CoQ10), electrochemically respond to iron-sulfur mediated gold electrodes. Here, select redox cofactors were tethered onto gold electrode supports via inorganic Fe–S (FeS, FeS2, Fe2S3, Fe3S4) and organic Fe–S (Ferredoxin), using the layer-by-layer self-assembly. A combination of docking simulations, secondary ion mass spectrometry, and Fourier-transform infrared spectroscopy revealed the formation of iron carbonyl bonds between FeS and NAD, leading to electrochemical interactions between Fe–S and the redox cofactors. Electrochemical studies confirmed both inorganic and organic iron-sulfur molecules can anchor cofactors onto the gold electrode supports. Up to a 300% increase in anodic peak currents was observed when the inorganic Fe–S replaced the organic counterpart, Ferredoxin, to wire the cofactors onto the gold electrodes. Electrode kinetic measurements revealed the reversible and quasi-reversible behavior of the inorganic and organic Fe–S electrodes, respectively. Reductive desorption studies indicated Fe3S4 closely followed by FeS, forming robust wiring associations with gold by displaying the highest surface coverage, desorption power, and the lowest resistance. Consequently, the inorganic iron-sulfur clusters facilitate an effective method for fabricating enzymatic biosensors for many analytes based on the enzyme-cofactor combination.

Logistic Regression-Guided Identification of Cofactor Specificity-Contributing Residues in Enzyme with Sequence Datasets Partitioned by Catalytic Properties.

Changing the substrate/cofactor specificity of an enzyme requires multiple mutations at spatially adjacent positions around the substrate pocket. However, this is challenging when solely based on crystal structure information because enzymes undergo dynamic conformational changes during the reaction process. Herein, we proposed a method for estimating the contribution of each amino acid residue to substrate specificity by deploying a phylogenetic analysis with logistic regression. Since this method can estimate the candidate amino acids for mutation by ranking, it is readable and can be used in protein engineering. We demonstrated our concept using redox cofactor conversion of the Escherichia coli malic enzyme as a model, which still lacks crystal structure elucidation. The use of logistic regression with amino acid sequences classified by cofactor specificity showed that the NADP+-dependent malic enzyme completely switched cofactor specificity to NAD+ dependence without the need for a practical screening step. The model showed that surrounding residues made a greater contribution to cofactor specificity than those in the interior of the substrate pocket. These residues might be difficult to identify from crystal structure observations. We show that a highly accurate and inferential machine learning model was obtained using amino acid sequences of structurally homologous and functionally distinct enzymes as input data.

Understand the antibacterial behavior and mechanism of hydrothermal wastewater

Unlocking the antibacterial potential is an emerging strategy to valorizing the toxic wastewater from hydrothermal liquefaction (HTL). Here, we investigated the response and biological mechanism of antibacterial properties of HTL wastewater. Four different biowastes i.e. microalgae, cornstalk, cow manure and swine manure were used as the feedstock of HTL to create wastewater with diverse molecule spectrum, whereas ten strains i.e. five gram-positive strains and five gram-negative strains were employed to represent typical pathogenic microorganism. HTL wastewater exhibited antibacterial potential and obvious reduction on cell viability at high inclusion ratio, although the minimum inhibitory concentration (MIC) and cell response intensity varied depending on different HTL feedstocks and strain species. The decreased ATP generation and increased H2O2 accumulation in treated cells further confirmed the inhibition of HTL wastewater on the cell metabolism. The antibacterial mechanism of HTL wastewater was confirmed, including damage to biomolecules or membranes, depletion of crucial components, disruption of metabolic circuits and imbalance of creation of redox cofactor. The complex compounds in HTL wastewater were probably attributed to the multiple inhibition pathways and the relationship among those multiple pathways was speculated. The present study contributes to the mechanism analysis of complex compound mixture and bactericide characteristics of HTL wastewater.

Pyrroloquinoline Quinone Administration Alleviates Allergic Airway Inflammation in Mice by Regulating the JAK-STAT Signaling Pathway.

The current asthma therapies are inadequate for many patients with severe asthma. Pyrroloquinoline quinone (PQQ) is a naturally-occurring redox cofactor and nutrient that can exert a multitude of physiological effects, including anti-inflammatory and antioxidative effects. We sought to explore the effects of PQQ on allergic airway inflammation and reveal the underlying mechanisms. In vitro, the effects of PQQ on the secretion of epithelial-derived cytokines by house dust mite- (HDM-) incubated 16-HBE cells and on the differentiation potential of CD4+ T cells were investigated. In vivo, PQQ was administered to mice with ovalbumin- (OVA-) induced asthma, and lung pathology and inflammatory cell infiltration were assessed. The changes in T cell subsets and signal transducers and activators of transcription (STATs) were evaluated by flow cytometry. Pretreatment with PQQ significantly decreased HDM-stimulated thymic stromal lymphopoietin (TSLP) production in a dose-dependent manner in 16-HBE cells and inhibited Th2 cell differentiation in vitro. Treatment with PQQ significantly reduced bronchoalveolar lavage fluid (BALF) inflammatory cell counts in the OVA-induced mouse model. PQQ administration also changed the secretion of IFN-γ and IL-4 as well as the percentages of Th1, Th2, Th17, and Treg cells in the peripheral blood and lung tissues, along with inhibition the phosphorylation of STAT1, STAT3, and STAT6 while promoting that of STAT4 in allergic airway inflammation model mice. PQQ can alleviate allergic airway inflammation in mice by improving the immune microenvironment and regulating the JAK-STAT signaling pathway. Our findings suggest that PQQ has great potential as a novel therapeutic agent for inflammatory diseases, including asthma.

S-Adenosylmethionine (SAM)-Dependent Methyltransferase MftM is Responsible for Methylation of the Redox Cofactor Mycofactocin.

Mycobacteria produce several unusual cofactors that contribute to their metabolic versatility and capability to survive in different environments. Mycofactocin (MFT) is a redox cofactor involved in ethanol metabolism. The redox-active core moiety of mycofactocin is derived from the short precursor peptide MftA, which is modified by several maturases. Recently, it has been shown that the core moiety is decorated by a β-1,4-glucan chain. Remarkably, the second glucose moiety of the oligosaccharide chain was found to be 2-O-methylated in Mycolicibacterium smegmatis. The biosynthetic gene responsible for this methylation, however, remained elusive, and no methyltransferase gene was part of the MFT biosynthetic gene cluster. Here, we applied reverse genetics to identify the gene product of MSMEG_6237 (mftM) as the SAM-dependent methyltransferase was responsible for methylation of the cofactor in M. smegmatis. According to metabolic analysis and comparative genomics, the occurrence of methylated MFT species was correlated with the presence of mftM homologues in the genomes of mycofactocin producers. This study revealed that the pathogen Mycobacterium tuberculosis does not methylate mycofactocins. Interestingly, mftM homologues co-occur with both mycofactocin biosynthesis genes as well as the putative mycofactocin-dependent alcohol dehydrogenase Mdo. We further showed that mftM knock-out mutants of M. smegmatis suffer from a prolonged lag phase when grown on ethanol as a carbon source. In addition, in vitro digestion of the glucose chain by cellulase suggested a protective function of glucan methylation. These results close an important knowledge gap and provide a basis for future studies into the physiological functions of this unusual cofactor modification.

Purification and structural characterization of the Na+-translocating ferredoxin: NAD+ reductase (Rnf) complex of Clostridium tetanomorphum

Various microbial metabolisms use H+/Na+-translocating ferredoxin:NAD+ reductase (Rnf) either to exergonically oxidize reduced ferredoxin by NAD+ for generating a transmembrane electrochemical potential or reversely to exploit the latter for producing reduced ferredoxin. For cryo-EM structural analysis, we elaborated a quick four-step purification protocol for the Rnf complex from Clostridium tetanomorphum and integrated the homogeneous and active enzyme into a nanodisc. The obtained 4.27 Å density map largely allows chain tracing and redox cofactor identification complemented by biochemical data from entire Rnf and single subunits RnfB, RnfC and RnfG. On this basis, we postulated an electron transfer route between ferredoxin and NAD via eight [4Fe-4S] clusters, one Fe ion and four flavins crossing the cell membrane twice related to the pathway of NADH:ubiquinone reductase. Redox-coupled Na+ translocation is provided by orchestrating Na+ uptake/release, electrostatic effects of the assumed membrane-integrated FMN semiquinone anion and accompanied polypeptide rearrangements mediated by different redox steps.

Tailorable Tetrahelical Bundles as a Toolkit for Redox Studies.

Oxidoreductases have evolved over millions of years to perform a variety of metabolic tasks crucial for life. Understanding how these tasks are engineered relies on delivering external electron donors or acceptors to initiate electron transfer reactions. This is a challenge. Small-molecule redox reagents can act indiscriminately, poisoning the cell. Natural redox proteins are more selective, but finding the right partner can be difficult due to the limited number of redox potentials and difficulty tuning them. De novo proteins offer an alternative path. They are robust and can withstand mutations that allow for tailorable changes. They are also devoid of evolutionary artifacts and readily bind redox cofactors. However, no reliable set of engineering principles have been developed that allow for these proteins to be fine-tuned so their redox midpoint potential (Em) can form donor/acceptor pairs with any natural oxidoreductase. This work dissects protein-cofactor interactions that can be tuned to modulate redox potentials of acceptors and donors using a mutable de novo designed tetrahelical protein platform with iron tetrapyrrole cofactors as a test case. We show a series of engineered heme b-binding de novo proteins and quantify their resulting effect on Em. By focusing on the surface charge and buried charges, as well as cofactor placement, chemical modification, and ligation of cofactors, we are able to achieve a broad range of Em values spanning a range of 330 mV. We anticipate this work will guide the design of proteinaceous tools that can interface with natural oxidoreductases inside and outside the cell while shedding light on how natural proteins modulate Em values of bound cofactors.

(Invited) Electroreduction of Nitrogen to Ammonia at Iron Catalytic Sites Generated at Interfaces Utilizing Iron Phosphides and Heme-Type Complexes

The feasibility of performing nitrogen electroreduction reaction, or nitrogen fixation, particularly in aqueous solutions, constitutes an attractive prospect to produce ammonia under ambient, or near ambient, conditions. Development of durable, specific and reasonably efficient low-cost catalysts remains a great challenge for electrochemical science and technology. Currently, most of electrochemical approaches to N2-fixation suffers from slow kinetics due to the difficulty of achieving the appropriate adsorption and activation of dinitrogen leading to cleavage of the strong, triple N≡N bond. This symposium will feature presentations on research dealing with the fundamental and applied aspects of nitrogen reduction reactions of relevance various aspects of science and existing technologies.Transition metal phosphides are promising non-noble earth-abundant catalysts with broad electrocatalytic properties, in particular when it comes to electroreduction of such inert reactants as carbon dioxide. Herein, we report the successful electrocatalytic reduction of nitrogen (N2) using different phases of iron phosphide activated at 450 °C. For example, the FeP and Fe2P phases have been found to act as efficient catalysts for the formation of NH3 in alkaline and semineutral media. Detection of in-situ formed product has been achieved by probing the electrooxidation of NH3 to nitrogen (N2) using the additional working electrode modified with Pt nanoparticles. On mechanistic grounds, the iron (Fe0) sites seem to be electrocatalytic active during the reduction of nitrogenThe iron sites can also be generated within the porphyrin rings and related coordination structures. Their high electrocatalytic activity was historically demonstrated for the CO2-reduction. The results obtained here indicate the feasibility of horseradish peroxidase (HRP) to act as the biocatalyst, when deposited on the glassy carbon (an inert electrode substrate), capable of inducing electroduction of not only CO2 but also N2. HRP is a metalloenzyme, in which a large alpha-helical protein which binds heme as a redox cofactor. The actual electrocatalytic properties shall be attributed to the existence of heme groups, i.e. complexes of iron ions coordinated to porphyrin units. On mechanistic grounds, the nitrogen molecules seems to be chemisorbed or attracted to the heme centers during the activation step. Indeed, only “adsorbates” of N2 are catalytically reduced. In other words, the HRP-surface-attached N2 molecules, rather than the bulk reactant, are reduced at the electrocatalytic interface. The reduction reaction of nitrogen is a multielectron and multiproton transfer process suffering from high over-potentials and limited selectivity The N2 molecule with triple bond requires special means of activation through strong adsorption at the electrocatalytic interface. Obviously most of electrocatalysts will be more active toward hydrogen evolution than ammonia production. Consequently, ammonia is produced at trace levels, and the reaction efficiency is very low. On the whole, the presence of the α-helix HRP secondary structure (composed of backbone N−H groups that hydrogen-bonded to the backbone C=O groups of the amino acid network) is likely to contribute to the system’s good stability and selectivity (e.g., with respect to the competing hydrogen evolution reaction).

Changes in the Electron Transfer Symmetry in the Photosystem I Reaction Centers upon Removal of Iron-Sulfur Clusters.

In photosynthetic reaction centers of intact photosystem I (PSI) complexes from cyanobacteria, electron transfer at room temperature occurs along two symmetrical branches of redox cofactors A and B at a ratio of ~3 : 1 in favor of branch A. Previously, this has been indirectly demonstrated using pulsed absorption spectroscopy and more directly by measuring the decay modulation frequencies of electron spin echo signals (electron spin echo envelope modulation, ESEEM), which allows to determine the distance between the separated charges of the primary electron donor P700+ and phylloquinone acceptors A1A- and A1B- in the symmetric redox cofactors branches A and B. In the present work, these distances were determined using ESEEM in PSI complexes lacking three 4Fe-4S clusters, FX, FA, and FB, and the PsaC protein subunit (the so-called P700-A1 core), in which phylloquinone molecules A1A and A1B serve as the terminal electron acceptors. It was shown that in the P700-A1 core preparations, the average distance between the centers of the P700+A1- ion-radical pair at a temperature of 150 K in an aqueous glycerol solution and in a dried trehalose matrix, as well as in a trehalose matrix at 280 K, is ~25.5 Å, which corresponds to the symmetrical electron transfer along the A and B branches of redox cofactors at a ratio of 1 : 1. Possible reasons for the change in the electron transfer symmetry in PSI upon removal of the PsaC subunit and 4Fe-4S clusters FX, FA, and FB are discussed.

(Invited) Electroreduction of Nitrogen to Ammonia at Iron Catalytic Sites Generated at Interfaces Utilizing Iron Phosphides and Heme-Type Complexes

There has been growing interest in the development of durable, specific and reasonably efficient low-cost catalysts for nitrogen (N2) electroreduction reaction, or nitrogen fixation, particularly in aqueous solutions capable of producing ammonia under ambient, or near ambient, conditions. The successful electrocatalytic reduction of nitrogen (N2) and formation of NH3 in alkaline an medium has been reported here using the Fe3P phase of iron phosphide. Detection of in-situ formed product has been achieved by probing the electrooxidation of NH3 to nitrogen (N2) using the additional working electrode modified with Pt nanoparticles. On mechanistic grounds, the iron (Fe0) sites seem to be electrocatalytic active during the reduction of nitrogen. The iron sites can also be generated within the phtalocyanine ring binding metal ions through four inwardly projecting nitrogen centers. Furthermore, horseradish peroxidase metalloenzyme, in which a large alpha-helical protein binds heme as a redox cofactor, is capable of inducing electroduction of N2.

Two-component carnitine monooxygenase from Escherichia coli: functional characterization, inhibition and mutagenesis of the molecular interface.

Gut microbial production of trimethylamine (TMA) from l-carnitine is directly linked to cardiovascular disease. TMA formation is facilitated by carnitine monooxygenase, which was proposed as a target for the development of new cardioprotective compounds. Therefore, the molecular understanding of the two-component Rieske-type enzyme from Escherichia coli was intended. The redox cofactors of the reductase YeaX (FMN, plant-type [2Fe-2S] cluster) and of the oxygenase YeaW (Rieske-type [2Fe-2S] and mononuclear [Fe] center) were identified. Compounds meldonium and the garlic-derived molecule allicin were recently shown to suppress microbiota-dependent TMA formation. Based on two independent carnitine monooxygenase activity assays, enzyme inhibition by meldonium or allicin was demonstrated. Subsequently, the molecular interplay of the reductase YeaX and the oxygenase YeaW was addressed. Chimeric carnitine monooxygenase activity was efficiently reconstituted by combining YeaX (or YeaW) with the orthologous oxygenase CntA (or reductase CntB) from Acinetobacter baumannii. Partial conservation of the reductase/oxygenase docking interface was concluded. A structure guided mutagenesis approach was used to further investigate the interaction and electron transfer between YeaX and YeaW. Based on AlphaFold structure predictions, a total of 28 site-directed variants of YeaX and YeaW were kinetically analyzed. Functional relevance of YeaX residues Arg271, Lys313 and Asp320 was concluded. Concerning YeaW, a docking surface centered around residues Arg83, Lys104 and Lys117 was hypothesized. The presented results might contribute to the development of TMA-lowering strategies that could reduce the risk for cardiovascular disease.

The TonB-dependent uptake of pyrroloquinoline-quinone (PQQ) and secretion of gluconate by Escherichia coli K-12.

Glucose is taken up by Escherichia coli through the phosphotransferase system (PTS) as the preferred carbon source. PTS mutants grow with glucose as a carbon source only in the presence of pyrroloquinoline quinone (PQQ), which is needed as a redox cofactor for the glucose dehydrogenase Gcd. The membrane-anchored Gcd enzyme oxidises glucose to gluconolactone in the periplasm. For this reaction to occur, external supply of PQQ is required as E. coli is unable to produce PQQ de novo. Growth experiments show that PqqU (previously YncD) is the TonB-ExbBD-dependent transporter for PQQ through the outer membrane. PQQ protected the cells from the PqqU-dependent phage IsaakIselin (Bas10) by competition for the receptor protein. As a high affinity uptake system, PqqU allows E. coli to activate Gcd even at surrounding PQQ concentrations of about 1nmoL/L. At about 30-fold higher PQQ concentrations, the activation of Gcd gets PqqU independent. Due to its small size, Pqq may also pass the outer membrane through porins. The PQQ-dependent production of gluconate has been demonstrated in many plant growth-promoting bacteria that solubilise phosphate minerals in the soil by secreting this acid. Under phosphate limiting conditions also E. coli induces the glucose dehydrogenase and secretes gluconate, even in absence of PTS, that is, even when the bacterium is unable to grow on glucose without PQQ.

An RNA aptamer that shifts the reduction potential of metabolic cofactors

The discovery of ribozymes has inspired exploration of RNA’s potential to serve as primordial catalysts in a hypothesized RNA world. Modern oxidoreductase enzymes employ differential binding between reduced and oxidized forms of redox cofactors to alter cofactor reduction potential and enhance the enzyme’s catalytic capabilities. The utility of differential affinity has been underexplored as a chemical strategy for RNA. Here we show an RNA aptamer that preferentially binds oxidized forms of flavin over reduced forms and markedly shifts flavin reduction potential by −40 mV, similar to shifts for oxidoreductases. Nuclear magnetic resonance structural analysis revealed π–π and donor atom–π interactions between the aptamer and flavin that cause unfavorable contacts with the electron-rich reduced form, suggesting a mechanism by which the local environment of the RNA-binding pocket drives the observed shift in cofactor reduction potential. It seems likely that primordial RNAs could have used similar strategies in RNA world metabolisms.

Declining muscle NAD+ in a hyperandrogenism PCOS mouse model: Possible role in metabolic dysregulation

Polycystic ovary syndrome (PCOS) is a common endocrine disorder, defined by reproductive and endocrine abnormalities, with metabolic dysregulation including obesity, insulin resistance and hepatic steatosis. Recently, it was found that skeletal muscle insulin sensitivity could be improved in obese, post-menopausal, pre-diabetic women through treatment with nicotinamide mononucleotide (NMN), a precursor to the prominent redox cofactor nicotinamide adenine dinucleotide (NAD+). Given that PCOS patients have a similar endocrine profile to these patients, we hypothesised that declining NAD levels in muscle might play a role in the pathogenesis of the metabolic syndrome associated with PCOS, and that this could be normalized through NMN treatment. Here, we tested the impact of NMN treatment on the metabolic syndrome of the dihydrotestosterone (DHT) induced mouse model of PCOS. We observed lower NAD levels in the muscle of PCOS mice, which was normalized by NMN treatment. PCOS mice were hyperinsulinaemic, resulting in increased adiposity and hepatic lipid deposition. Strikingly, NMN treatment completely normalized these aspects of metabolic dysfunction. We propose that addressing the decline in skeletal muscle NAD levels associated with PCOS can normalize insulin sensitivity, preventing compensatory hyperinsulinaemia, which drives obesity and hepatic lipid deposition, though we cannot discount an impact of NMN on other tissues to mediate these effects. These findings support further investigation into NMN treatment as a new therapy for normalizing the aberrant metabolic features of PCOS.

Conformational dynamics modulating electron transfer.

Diffusional dynamics of the donor-acceptor distance are responsible for the appearance of a new time scale of diffusion over the distance of electronic tunneling in electron-transfer reactions. The distance dynamics compete with the medium polarization dynamics in the dynamics-controlled electron-transfer kinetics. The pre-exponential factor of the electron-transfer rate constant switches, at the crossover distance, between a distance-independent, dynamics-controlled plateau and exponential distance decay. The crossover between two regimes is controlled by an effective relaxation time slowed down by a factor exponentially depending on the variance of the donor-acceptor displacement. Flexible donor-acceptor complexes must show a greater tendency for dynamics-controlled electron transfer. Energy chains based on electron transport are best designed by placing the redox cofactors near the crossover distance.

Electron Transfer in Binary Hemin-Modified Alkanethiol Self-Assembled Monolayers on Gold: Hemin's Lateral and Interfacial Interactions.

Orientated coupling of redox enzymes to electrodes by their reconstitution onto redox cofactors, such as hemin conjugated to self-assembled monolayers (SAMs) formed on the electrodes, poses the requirements for a SAM design enabling reconstitution. We show that the kinetics of electron transfer (ET) in binary SAMs of alkanethiols on gold composed of in situ hemin-conjugated 11-amino-1-undecanethiol (AUT) and diluting OH-terminated alkanethiols with 11, 6, and 2 methylene groups (MC11OH, MC6OH, and MC2OH) depends on both the SAM composition and surface density of hemin, Γheme. In AUT/MC11OH SAMs composed of equal linker/diluent lengths, the heterogeneous ET rate constant ks decreased with the Γheme and varied between 70 and 500 s-1. For shorter diluents, the ks of 245-330 s-1 (C6) and 300-340 s-1 (C2) showed a little (if any) Γheme dependence. In AUT/MC11OH SAMs, the increasing Γheme resulted in the steric crowding of hemin species and their neighboring lateral interactions in the plane of hemin localization, affecting the potential distribution at the SAM/electrode interface and inducing local electrostatic effects interfering with hemin oxidation. In AUT/MC6OH and AUT/MC2OH SAMs, hemin discharged at the plane of the closest approach to the gold surface, equal to the diluent length and permeable to electrolyte ions, which lessened those effects. All studied binary SAMs provided steric hindrance for protein reconstitution on the hemin cofactor conjugated to the extended AUT linker. Further use of SAM-modified electrodes with the covalently attached hemin as interfaces for heme proteins' reconstitution should consider SAMs with loosely dispersed redox centers terminating more rigid molecular wires. Such wires place hemin at fixed distances from the electrode surface and thus ensure the interfacial properties required for the effective on-surface reconstitution of proteins and enzymes.

Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform

Noncanonical redox cofactors are attractive low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) in biotransformation. However, engineering enzymes to utilize them is challenging. Here, we present a high-throughput directed evolution platform which couples cell growth to the in vivo cycling of a noncanonical cofactor, nicotinamide mononucleotide (NMN+). We achieve this by engineering the life-essential glutathione reductase in Escherichia coli to exclusively rely on the reduced NMN+ (NMNH). Using this system, we develop a phosphite dehydrogenase (PTDH) to cycle NMN+ with ~147-fold improved catalytic efficiency, which translates to an industrially viable total turnover number of ~45,000 in cell-free biotransformation without requiring high cofactor concentrations. Moreover, the PTDH variants also exhibit improved activity with another structurally deviant noncanonical cofactor, 1-benzylnicotinamide (BNA+), showcasing their broad applications. Structural modeling prediction reveals a general design principle where the mutations and the smaller, noncanonical cofactors together mimic the steric interactions of the larger, natural cofactors NAD(P)+.