P450 BM3 Monooxygenase as an Efficient NAD(P)H‐Oxidase for Regeneration of Nicotinamide Cofactors in ADH‐Catalysed Preparative Scale Biotransformations

Enzymatic oxidations of primary and secondary alcohols catalysed by nicotinamide dependent alcohol dehydrogenases on the preparative scale require cofactor regeneration systems. Of critical value from an economic and ecological perspective is the application of NAD(P)H‐oxidases, which utilise molecular oxygen as a cost‐effective, atom‐efficient and environmentally benign oxidant to regenerate the cofactor NAD(P)⁺. Herein, the P450 BM3 monooxygenase from Bacillus megaterium is presented as an NAD(P)H‐oxidase for the successful regeneration of both NADP⁺ and NAD⁺ on the preparative scale. This enzyme was exemplarily applied for ADH‐catalysed oxidative kinetic resolutions of racemic secondary alcohols and the desymmetrisation of a meso‐diol leading to enantiomerically enriched secondary alcohols in both cases. Furthermore, the ADH‐catalysed oxidation of a primary alcohol targeting the corresponding aldehyde was performed. The obtained results significantly broaden the scope of feasible oxidative biotransformations, thereby increasing the number of synthetic reactions complying with key challenges of a modern and sustainable chemistry such as mild reaction conditions, environmentally benign solvents, and biodegradable non‐toxic catalysts.

     

Negative Cooperativity in NAD(P)H Quinone Oxidoreductase 1 (NQO1)

NAD(P)H quinone oxidoreductase-1 (NQO1) is a homodimeric protein that acts as a detoxifying enzyme or as a chaperone protein. Dicourmarol interacts with NQO1 at the NAD(P)H binding site and can both inhibit enzyme activity and modulate the interaction of NQO1 with other proteins. We show that the binding of dicoumarol and related compounds to NQO1 generates negative cooperativity between the monomers. This does not occur in the presence of the reducing cofactor, NAD(P)H, alone. Alteration of Gly150 (but not Gly149 or Gly174) abolished the dicoumarol-induced negative cooperativity. Analysis of the dynamics of NQO1 with the Gaussian network model indicates a high degree of collective motion by monomers and domains within NQO1. Ligand binding is predicted to alter NQO1 dynamics both proximal to the ligand binding site and remotely, close to the second binding site. Thus, drug-induced modulation of protein motion might contribute to the biological effects of putative inhibitors of NQO1.     

NAD(P)H:Quinone Oxidoreductase 1 (NQO1) P187S Polymorphism and Prostate Cancer Risk in Caucasians

NAD(P)H:quinone oxidoreductase 1 (NQO1) catalyses the reduction of quinoid compounds to hydroquinones, preventing the generation of free radicals and reactive oxygen. A “C” to “T” transversion at position 609 of NQO1, leading to a nonsynonymous amino acid change (Pro187Ser, P187S), results in an altered enzyme activity. No NQO1 protein activity was detected in NQO1 ⁶⁰⁹TT genotype, and low to intermediate activity was detected in NQO1 ⁶⁰⁹CT genotype compared with ⁶⁰⁹CC genotype. Thus, this polymorphism may result in altered cancer predisposition. For prostate cancer, only sparse data are available. We therefore analyzed the distribution of the NQO1 P187S SNP (single nucleotide polymorphism) in prostate cancer patients and a healthy control group. Allelic variants were determined using RFLP analysis. Overall, 232 patients without any malignancy and 119 consecutive prostate cancer patients were investigated. The genotype distribution in our cohorts followed the Hardy–Weinberg equilibrium in cases and controls. The distribution of the NQO1 codon 187 SNP did not differ significantly between prostate cancer patients and the control group (p = 0.242). There was also no association between the allelic variants and stage or Gleason score of the tumors. The NQO1 P187S SNP was not significantly associated with an increased prostate cancer risk in our cohorts. The SNP has also no influence on histopathological characteristics of the tumors. A combined analysis of all available data from published European studies also showed no significant differences in the genotype distribution between controls and prostate cancer patients. Our data suggest a minor role of the NQO1 nucleotide 609 polymorphism in prostate carcinogenesis.     

Probes and nano-delivery systems targeting NAD(P)H:quinone oxidoreductase 1: a mini-review

The two-electron cytoplasmic reductase NAD(P)H:quinone oxidoreductase 1 is expressed in many tissues. NAD(P)H:quinone oxidoreductase 1 is well-known for being highly expressed in most cancers. Therefore, it could be a target for cancer therapy. Because it is a quinone reductase, many bioimaging probes based on quinone structures target NAD(P)H:quinone oxidoreductase 1 to diagnose tumours. Its expression is higher in tumours than in normal tissues, and using target drugs such as β-lapachone to reduce side effects in normal tissues can help. However, the physicochemical properties of β-lapachone limit its application. The problem can be solved by using nanosystems to deliver β-lapachone. This mini-review summarizes quinone-based fluorescent, near-infrared and two-photon fluorescent probes, as well as nanosystems for delivering the NAD(P)H:quinone oxidoreductase 1-activating drug β-lapachone. This review provides valuable information for the future development of probes and nano-delivery systems that target NAD(P)H:quinone oxidoreductase 1.     

Aerobic Cytotoxicity of Aromatic N-Oxides: The Role of NAD(P)H:Quinone Oxidoreductase (NQO1)

Derivatives of tirapazamine and other heteroaromatic N-oxides (ArN→O) exhibit tumoricidal, antibacterial, and antiprotozoal activities, which are typically attributed to bioreductive activation and free radical generation. In this work, we aimed to clarify the role of NAD(P)H:quinone oxidoreductase (NQO1) in ArN→O aerobic cytotoxicity. We synthesized 9 representatives of ArN→O with uncharacterized redox properties and examined their single-electron reduction by rat NADPH:cytochrome P-450 reductase (P-450R) and Plasmodium falciparum ferredoxin:NADP⁺ oxidoreductase (PfFNR), and by rat NQO1. NQO1 catalyzed both redox cycling and the formation of stable reduction products of ArN→O. The reactivity of ArN→O in NQO1-catalyzed reactions did not correlate with the geometric average of their activity towards P-450R- and PfFNR, which was taken for the parameter of their redox cycling efficacy. The cytotoxicity of compounds in murine hepatoma MH22a cells was decreased by antioxidants and the inhibitor of NQO1, dicoumarol. The multiparameter regression analysis of the data of this and a previous study (DOI: 10.3390/ijms20184602) shows that the cytotoxicity of ArN→O (n = 18) in MH22a and human colon carcinoma HCT-116 cells increases with the geometric average of their reactivity towards P-450R and PfFNR, and with their reactivity towards NQO1. These data demonstrate that NQO1 is a potentially important target of action of heteroaromatic N-oxides.     

Guidelines for the Application of NAD(P)H Regenerating Glucose Dehydrogenase in Synthetic Processes

Glucose dehydrogenase (GDH) is frequently used for the reduction of NAD⁺ and NADP⁺ in bench‐ and industrial‐scale syntheses because the coenzyme regenerating system GDH is easy to apply, robust and relatively inexpensive. To optimize the application of this long known coenzyme regeneration system we investigated the commonly applied Bacillus GDH and characterized this enzyme by its kinetic features in the presence of substrates and products at pH 6.4 and 8.0. Three substrates/products were found to inhibit GDH considerably: (i) the reaction product glucono‐1,5‐lactone, (ii) the reduced coenzyme NAD(P)H and (iii) the oxidized coenzyme NAD(P)⁺. The inhibition of GDH under several process conditions was modeled using the determined kinetic constants. It was found that the GDH regeneration system is strongly inhibited by the usually applied conditions. This study provides the rate equation of the GDH reaction and simulations of this coenzyme regenerating system leading to an improved prediction and, thus, to a faster scale‐up and increased efficiency of NAD(P)H‐dependent synthetic processes.     

Performance Evaluation of the UVAPS in Measuring Biological Aerosols: Fluorescence Spectra from NAD(P)H Coenzymes and Riboflavin

This article presents the results of the performance evaluation of the Ultraviolet Aerodynamic Particle Sizer (UVAPS, model 3312, TSI Inc., St. Paul, MN, USA), the novel instrument for real-time monitoring of biological aerosols. The main objective of the study was to compare the UVAPS response in measuring aerosols containing NADH, NADPH, or riboflavin particles. At the excitation and emission wavelengths at which the UVAPS operates, these compounds are the primary intrinsic fluorophores specific to biological particles. In addition, the study was focused on determining the detection limits of the UVAPS for these fluorophores. This information is important for the interpretation of UVAPS data while measuring bacterial aerosols. Fluorescence measurements were initially taken with a Varian Cary Eclipse Fluorescence Spectrophotometer for all three fluorophores. The samples were then aerosolized with the 6-jet Collison nebulizer. Riboflavin was found to be a stronger fluorophore than both NAD(P)H coenzymes. The fluorescence signals were considerably weaker for the NADPH samples compared to the NADH samples. The sensitivity of the UVAPS was found to be sufficiently high to detect the NADH and riboflavin at the concentrations characteristic of bacterial cells. The results of this study are discussed in a context of the results previously reported for the bacterial aerosols. It can be concluded that the amount of fluorophores detectable in uniformly mixed particles is equal to or less than the fluorophores expected to be present in the individual bacterial particles.     

FLIM of NAD(P)H in Lymphatic Nodes Resolves T-Cell Immune Response to the Tumor

Assessment of T-cell response to the tumor is important for diagnosis of the disease and monitoring of therapeutic efficacy. For this, new non-destructive label-free methods are required. Fluorescence lifetime imaging (FLIM) of metabolic coenzymes is a promising innovative technology for the assessment of the functional status of cells. The purpose of this work was to test whether FLIM can resolve metabolic alterations that accompany T-cell reactivation to the tumors. The study was carried out on C57Bl/6 FoxP3-EGFP mice bearing B16F0 melanoma. Autofluorescence of the immune cells in fresh lymphatic nodes (LNs) was investigated. It was found that fluorescence lifetime parameters of nicotinamide adenine dinucleotide (phosphate) NAD(P)H are sensitive to tumor development. Effector T-cells in the LNs displayed higher contribution of free NADH, the form associated with glycolysis, in all tumors and the presence of protein-bound NADPH, associated with biosynthetic processes, in the tumors of large size. Flow cytometry showed that the changes in the NADH fraction of the effector T-cells correlated with their activation, while changes in NADPH correlated with cell proliferation. In conclusion, FLIM of NAD(P)H in fresh lymphoid tissue is a powerful tool for assessing the immune response to tumor development.     

无电荷超滤膜酶膜反应器中的辅酶截留

构建了无电荷超滤膜酶膜反应器,考察了影响辅酶截留率的多种因素.实验结果表明,连续超滤膜反应器中加入聚乙烯亚胺(PEI,50 kDa),NAD 能够有效地被超滤膜截留.在Tris-HCl缓冲液中,PEI/NAD 摩尔比为5:1时,达到较高的辅酶截留率(R=0.823).溶液离子强度的增加减少辅酶的截留率,但可增加超滤膜超滤流速.在含盐溶液中,添加0.5%或1.O%的牛血清白蛋白可增加NAD 截留率.本装置对NADH的截留率高于NAD 的截留率,因此PEI适用于辅酶再生体系.同时还考察了1,3-丙二醇酶催化体系中PEI对甘油脱氢酶和1,3-丙二醇氧化还原酶酶活力的影响.     

Simultaneous Probing of Metabolism and Oxygenation of Tumors In Vivo Using FLIM of NAD(P)H and PLIM of a New Polymeric Ir(III) Oxygen Sensor

Tumor cells are well adapted to grow in conditions of variable oxygen supply and hypoxia by switching between different metabolic pathways. However, the regulatory effect of oxygen on metabolism and its contribution to the metabolic heterogeneity of tumors have not been fully explored. In this study, we develop a methodology for the simultaneous analysis of cellular metabolic status, using the fluorescence lifetime imaging microscopy (FLIM) of metabolic cofactor NAD(P)H, and oxygen level, using the phosphorescence lifetime imaging (PLIM) of a new polymeric Ir(III)-based sensor (PIr3) in tumors in vivo. The sensor, derived from a polynorbornene and cyclometalated iridium(III) complex, exhibits the oxygen-dependent quenching of phosphorescence with a 40% longer lifetime in degassed compared to aerated solutions. In vitro, hypoxia resulted in a correlative increase in PIr3 phosphorescence lifetime and free (glycolytic) NAD(P)H fraction in cells. In vivo, mouse tumors demonstrated a high degree of cellular-level heterogeneity of both metabolic and oxygen states, and a lower dependence of metabolism on oxygen than cells in vitro. The small tumors were hypoxic, while the advanced tumors contained areas of normoxia and hypoxia, which was consistent with the pimonidazole assay and angiographic imaging. Dual FLIM/PLIM metabolic/oxygen imaging will be valuable in preclinical investigations into the effects of hypoxia on metabolic aspects of tumor progression and treatment response.     

Self‐Immolative Bioluminogenic Quinone Luciferins for NAD(P)H Assays and Reducing Capacity‐Based Cell Viability Assays

Highly sensitive self‐cleavable trimethyl lock quinone‐luciferin substrates for diaphorase were designed and synthesized to measure NAD(P)H in biological samples and monitor viable cells via NAD(P)H‐dependent cellular oxidoreductase enzymes and their NAD(P)H cofactors.     

NAD(H) Regulates the Permeability Transition Pore in Mitochondria through an External Site

The opening of the permeability transition pore (mPTP) in mitochondria initiates cell death in numerous diseases. The regulation of mPTP by NAD(H) in the mitochondrial matrix is well established; however, the role of extramitochondrial (cytosolic) NAD(H) is still unclear. We studied the effect of added NADH and NAD⁺ on: (1) the Ca²⁺-retention capacity (CRC) of isolated rat liver, heart, and brain mitochondria; (2) the Ca²⁺-dependent mitochondrial swelling in media whose particles can (KCl) or cannot (sucrose) be extruded from the matrix by mitochondrial carriers; (3) the Ca²⁺-dependent mitochondrial depolarization and the release of entrapped calcein from mitochondria of permeabilized hepatocytes; and (4) the Ca²⁺-dependent mitochondrial depolarization and subsequent repolarization. NADH and NAD⁺ increased the CRC of liver, heart, and brain mitochondria 1.5–2.5 times, insignificantly affecting the rate of Ca²⁺-uptake and the free Ca²⁺ concentration in the medium. NAD(H) suppressed the Ca²⁺-dependent mitochondrial swelling both in KCl- and sucrose-based media but did not induce the contraction and repolarization of swollen mitochondria. By contrast, EGTA caused mitochondrial repolarization in both media and the contraction in KCl-based medium only. NAD(H) delayed the Ca²⁺-dependent depolarization and the release of calcein from individual mitochondria in hepatocytes. These data unambiguously demonstrate the existence of an external NAD(H)-dependent site of mPTP regulation.     

Synthesis of block copolymers with poly(methyl methacrylate): P(B-b-MMA), P(EB-b-MMA), P(S-b-B-b-MMA) and P(S-b-EB-b-MMA)

Summary Well-defined diblock copolymers poly(butadiene-b-methyl methacrylate) (=P(B-b-MMA)) and triblock copolymers poly(styrene-b-butadiene-b-methyl methacrylate) (=P(S-b-B-b-MMA)) have been prepared by sequential anionic polymerization in THF. The synthesis of P(B-b-MMA) and P(S-b-B-b-MMA) was most efficient in the presence of lithium alkoxides. By this method side reactions are suppressed and the polymerization can be performed at higher temperatures. The resulting triblock copolymers have narrow molecular weight distribution. The 1,2-PB midblock was quantitatively hydrogenated with tosylhydrazide to enhance thermal stability. Alternatively the hydrogenation can be performed at elevated pressure using hydrogen and Wilkinson-catalyst in butanone. Heterogeneous catalyst systems based on Palladium did not yield quantitative hydrogenation.     

Applications of Mesenchymal Stem Cells in Skin Regeneration and Rejuvenation

Mesenchymal stem cells (MSCs) are multipotent stem cells derived from adult stem cells. Primary MSCs can be obtained from diverse sources, including bone marrow, adipose tissue, and umbilical cord blood. Recently, MSCs have been recognized as therapeutic agents for skin regeneration and rejuvenation. The skin can be damaged by wounds, caused by cutting or breaking of the tissue, and burns. Moreover, skin aging is a process that occurs naturally but can be worsened by environmental pollution, exposure to ultraviolet radiation, alcohol consumption, tobacco use, and undernourishment. MSCs have healing capacities that can be applied in damaged and aged skin. In skin regeneration, MSCs increase cell proliferation and neovascularization, and decrease inflammation in skin injury lesions. In skin rejuvenation, MSCs lead to production of collagen and elastic fibers, inhibition of metalloproteinase activation, and promote protection from ultraviolet radiation-induced senescence. In this review, we focus on how MSCs and MSC-derived molecules improve diseased and aged skin. Additionally, we emphasize that induced pluripotent stem cell (iPSC)-derived MSCs are potentially advanced MSCs, which are suitable for cell therapy.     

NAD(H)-PEG Swing Arms Improve Both the Activities and Stabilities of Modularly-Assembled Transhydrogenases Designed with Predictable Selectivities

Protein engineering has been used to enhance the activities, selectivities, and stabilities of enzymes. Frequently tradeoffs are observed, where improvements in some features can come at the expense of others. Nature uses modular assembly of active sites for complex, multi-step reactions, and natural “swing arm” mechanisms have evolved to transfer intermediates between active sites. Biomimetic polyethylene glycol (PEG) swing arms modified with NAD(H) have been explored to introduce synthetic swing arms into fused oxidoreductases. Here we report that increasing NAD(H)-PEG swing arms can improve the activity of synthetic formate:malate oxidoreductases as well as the thermal and operational stabilities of the biocatalysts. The modular assembly approach enables the K_M values of new enzymes to be predictable, based on the parental enzymes. We describe four unique synthetic transhydrogenases that have no native homologs, and this platform could be easily extended for the predictive design of additional synthetic cofactor-independent transhydrogenases.     

Regeneration and biosynthesis of dytiscid defensive agents (Coleoptera: Dytiscidae)

The defensive secretions of the dytiscid species,Agabus seriatus (Say) andAgabus obtusatus (Say), were qualitatively and quantitatively analyzed by high-pressure liquid chromatography. The intrinsic ability ofA. Seriatus andA. Obtusatus to regenerate their prothoracic gland defensive secretions under laboratory conditions was determined by analyzing the secretions every seventh day for five weeks. Both beetles regenerated ～ 80% of their prothoracic gland components within two weeks.A. seriatus was injected with [4-¹⁴C]cholesterol and after a three-week regeneration period 7.5% of the¹⁴Clabel was found in the steroidal defensive secretion from the prothoracic glands.     

Enhanced Sulfur Tolerance of Ceria-Promoted NO x Storage Reduction (NSR) Catalysts: Sulfur Uptake, Thermal Regeneration and Reduction with H2(g)

SO _x uptake, thermal regeneration and the reduction of SO _x via H₂(g) over ceria-promoted NSR catalysts were investigated. Sulfur poisoning and desulfation pathways of the complex BaO/Pt/CeO₂/Al₂O₃ NSR system was investigated using a systematic approach where the functional sub-components such as Al₂O₃, CeO₂/Al₂O₃, BaO/Al₂O₃, BaO/CeO₂/Al₂O₃, and BaO/Pt/Al₂O₃ were studied in a comparative fashion. Incorporation of ceria significantly increases the S-uptake of Al₂O₃ and BaO/Al₂O₃ under both moderate and extreme S-poisoning conditions. Under moderate S-poisoning conditions, Pt sites seem to be the critical species for SO _x oxidation and SO _x storage, where BaO/Pt/Al₂O₃ and BaO/Pt/CeO₂/Al₂O₃ catalysts reveal a comparable extent of sulfation. After extreme S-poisoning due to the deactivation of most of the Pt sites, ceria domains are the main SO _x storage sites on the BaO/Pt/CeO₂/Al₂O₃ surface. Thus, under these conditions, BaO/Pt/CeO₂/Al₂O₃ surface stores more sulfur than that of BaO/Pt/Al₂O₃. BaO/Pt/CeO₂/Al₂O₃ reveals a significantly improved thermal regeneration behavior in vacuum with respect to the conventional BaO/Pt/Al₂O₃ catalyst. Ceria promotion remarkably enhances the SO _x reduction with H₂(g).