Switching the Mechanism of NADH Photooxidation by Supramolecular Interactions

A series of three Ru(II) polypyridine complexes was investigated for the selective photocatalytic oxidation of NAD(P)H to NAD(P)⁺ in water. A combination of (time-resolved) spectroscopic studies and photocatalysis experiments revealed that ligand design can be used to control the mechanism of the photooxidation: For prototypical Ru(II) complexes a ¹O₂ pathway was found. Rudppz ([(tbbpy)₂Ru(dppz)]Cl₂, tbbpy=4,4'-di-tert-butyl-2,2'-bipyridine, dppz=dipyrido[3,2-a:2′,3′-c]phenazine), instead, initiated the cofactor oxidation by electron transfer from NAD(P)H enabled by supramolecular binding between substrate and catalyst. Expulsion of the photoproduct NAD(P)⁺ from the supramolecular binding site in Rudppz allowed very efficient turnover. Therefore, Rudppz permits repetitive selective assembly and oxidative conversion of reduced naturally occurring nicotinamides by recognizing the redox state of the cofactor under formation of H₂O₂ as additional product. This photocatalytic process can fuel discontinuous photobiocatalysis.     

The Mitochondrial Protein MitoNEET as a Probe for the Allostery of Glutamate Dehydrogenase

The proteins glutamate dehydrogenase (GDH) and mitoNEET are both targets of drug development efforts to treat metabolic disorders, cancer, and neurodegenerative diseases. However, these two proteins differ starkly in the current knowledge about ligand binding sites. MitoNEET is a [2Fe-2S]-containing protein with no obvious binding site for small ligands observed in its crystal structures. In contrast, GDH is known to have a variety of ligands at multiple allosteric sites thereby leading to complex regulation in activity. In fact, while GDH can utilize either NAD(H) or NADP(H) for catalysis at the active site, only NAD(H) binds at a regulatory site to inhibit GDH activity. Previously, we found that mitoNEET forms a covalent bond with GDH in vitro and increases the catalytic activity of the enzyme. In this study we evaluated the effects of mitoNEET binding on the allosteric control of GDH conferred by inhibitors. We examined all effectors using NAD or NADP as the coenzyme to determine allosteric linkage by the NAD-binding regulatory site. We found that GDH activity, in the presence of the inhibitory palmitoyl-CoA and EGCG, can be rescued by mitoNEET, regardless of the coenzyme used. This suggests that mitoNEET rescues GDH by stabilizing the open conformation.     

Integrated, electrically contacted NAD(P)+-dependent enzyme-carbon nanotube electrodes for biosensors and biofuel cell applications

Integrated, electrically contacted beta-nicotinamide adenine dinucleotide- (NAD(+)) or beta-nicotinamide adenine dinucleotide phosphate- (NADP(+)) dependent enzyme electrodes were prepared on single-walled carbon nanotube (SWCNT) supports. The SWCNTs were functionalized with Nile Blue (1), and the cofactors NADP(+) and NAD(+) were linked to 1 through a phenyl boronic acid ligand. The affinity complexes of glucose dehydrogenase (GDH) with the NADP(+) cofactor or alcohol dehydrogenase (AlcDH) with the NAD(+) cofactor were crosslinked with glutaric dialdehyde and the biomolecule-functionalized SWCNT materials were deposited on glassy carbon electrodes. The integrated enzyme electrodes revealed bioelectrocatalytic activities, and they acted as amperometric electrodes for the analysis of glucose or ethanol. The bioelectrocatalytic response of the systems originated from the biocatalyzed oxidation of the respective substrates by the enzyme with the concomitant generation of NAD(P)H cofactors. The electrocatalytically mediated oxidation of NAD(P)H by 1 led to amperometric responses in the system. Similarly, an electrically contacted bilirubin oxidase (BOD)-SWCNT electrode was prepared by the deposition of BOD onto the SWCNTs and the subsequent crosslinking of the BOD units using glutaric dialdehyde. The BOD-SWCNT electrode revealed bioelectrocatalytic functions for the reduction of O(2) to H(2)O. The different electrically contacted SWCNT-based enzyme electrodes were used to construct biofuel cell elements. The electrically contacted GDH-SWCNT electrode was used as the anode for the oxidation of the glucose fuel in conjunction with the BOD-SWCNT electrode in the presence of O(2), which acted as an oxidizer in the system. The power output of the cell was 23 muW cm(-2). Similarly, the AlcDH-SWCNT electrode was used as the anode for the oxidation of ethanol, which was acting as the fuel, with the BOD-SWCNT electrode as the cathode for the reduction of O(2). The power output of the system was 48 microW cm(-2).     

Recognizing five molecular ligand-binding sites with similar chemical structure

Accurate identification of ligand-binding sites and discovering the protein–ligand interaction mechanism are important for understanding proteins' functions and designing new drugs. Meanwhile, accurate computational prediction and mechanism research are two grand challenges in proteomics. In this article, ligand-binding residues of five ligands (ATP, ADP, GTP, GDP, and NAD) are predicted as a group, due to their similar chemical structures and close biological function relations. The data set of binding sites by five ligands (ATP, ADP, GTP, GDP, and NAD) are collated from Biolip database. Then, five features, containing increment of diversity value, matrix scoring value, auto-covariance, secondary structure information, and surface accessibility information are used in binding site predictions. The support vector machine (SVM) model is used with the five features to predict ligand-binding sites. Finally, prediction results are tested by fivefold cross validation. Accuracy (Acc) of five ligands (ATP, ADP, GTP, GDP, and NAD) achieves 77.4%, 71.2%, 82.1%, 82.9%, and 85.3%, respectively; and Matthew correlation coefficient (MCC) of the above five ligands achieves 0.549, 0.424, 0.643, 0.659, and 0.702, respectively. The research result shows that for ligands with similar chemical structures, microenvironment of their binding sites and their sensitivities to features are similar, while, differences of their ligand-binding properties exist at the same time. © 2019 Wiley Periodicals, Inc.     

Including tightly-bound water molecules in de novo drug design. Exemplification through the in silico generation of poly(ADP-ribose)polymerase ligands

Different strategies for the in silico generation of ligand molecules in the binding site of poly(ADP-ribose)polymerase (PARP) were studied in order to observe the effect of the targeting and displacement of tightly bound water molecules. Several molecular scaffolds were identified as having better interactions in the binding site when targeting one or two tightly bound water molecules in the NAD binding site. Energy calculations were conducted in order to assess the ligand-protein and ligand-water-protein interactions of different functional groups of the generated ligands. These calculations were used to evaluate the energetic consequences of the presence of tightly bound water molecules and to identify those that contribute favorably to the binding of ligands.     

烟酰型辅酶NAD(P)+和NAD(P)H再生的研究进展

大部分氧化还原酶的催化反应需要烟酰型辅酶NAD(P) 和NAD(P)H作为氧化剂或还原剂参与,由于氧化还原酶应用广泛而辅酶价格昂贵,使得辅酶再生逐渐成为研究热点.综述了近年来NAD(P) 和NAD(P)H酶法再生、电化学法及光化学法再生的研究进展,并介绍了各再生技术的应用和开发状况.     

Reactivity of the B-H Bond in tris(pyrazolyl)hydroborato zinc complexes: unexpected example of zinc hydride formation in a protic solvent and its relevance towards hydrogen transfer to NAD(+) mimics by tris(pyrazolyl)hydroborato zinc complexes in alcoholic media

Solutions of the zinc hydroxide complex [Tp(Bu(t),Me)]ZnOH in alcohols (ROH; R = Me, Et, Pr(i)) achieve hydride transfer to the NAD(+) model, 10-methylacridinium perchlorate. Deuterium labeling studies, however, demonstrate that the source of the hydride is not the alcohol but, rather, the B [bond] H group of the [Tp(Bu(t),Me)] ligand. A further example in which a [Tp(Bu(t),Me)] ligand acts as a hydride donor is provided by the reaction of the aqua complex [[Tp(Bu(t),Me)]Zn(OH(2))][HOB(C(6)F(5))(3)] with MeOH to generate the zinc hydride complex [Tp(Bu(t),Me)]ZnH. The present study therefore provides a caveat for the often assumed inertness of the B [bond] H group in tris(pyrazolyl)hydroborato ligands, especially in the presence of reactive cationic species.     

Simultaneous measurement of NAD metabolome in aged mice tissue using liquid chromatography tandem‐mass spectrometry

Nicotinamide adenine dinucleotide (NAD) is a major co‐factor that mediates multiple biological processes including redox reaction and gene expression. Recently, NAD metabolism has received considerable attention because administration of NAD precursors exhibited beneficial effects against aging‐related metabolic disorders in animals. Although numerous studies have reported that NAD levels decline with aging in multiple animal tissues, the pathway and kinetics of NAD metabolism in aged organs are not completely understood. To determine the NAD metabolism upon aging, we developed targeted metabolomics based on an LC/MS/MS system. Our method is simple and applicable to crude biological samples, including culture cells and animal tissues. Unlike a conventional enzymatic cycling assay, our approach can determine NAD and NADH (reduced form of NAD) by performing a single sample preparation. Further, we validated our method using biological samples and investigated the alteration of the NAD metabolome during aging. Consistent with previous reports, the NAD levels in the liver and skeletal muscle decreased with aging. Further, we detected a significant increase in nicotinamide mononucleotide and nicotinamide riboside in the kidney upon aging. The LC/MS/MS‐based NAD metabolomics that we have developed is extensively applicable to biomedical studies, and the results will present innovative ideas for the aging studies, especially for that of NAD metabolism.     

Coenzyme analogs: excellent substitutes (not poor imitations) for electrochemical regeneration

For the first time, employment of nicotinamide coenzyme NAD analogs has overcome the limitations of NAD in electrochemical regeneration. It has been shown that NAD analogs, APAD and PAAD, were electrochemically reduced more efficiently than original NAD and that the stability of their reduced products was also much higher than NADH.     

Artificial electron carriers for photoenzymatic synthesis under visible light

NAD analogues can be employed as artificial electron carriers for photoenzymatic synthesis under visible light. Four different NAD analogues that have a 3-substituted pyridine ring have been investigated. 3-Acetylpyridine adenine dinucleotide and 3-pyridinealdehyde adenine dinucleotide were photochemically reduced much more efficiently than NAD, while their reduced products showed coenzyme activity comparable to natural NAD.     

烟酰胺腺嘌呤二核苷酸在纳米α-氧化铝上的吸附作用

用紫外-可见吸收光谱、X射线衍射、荧光光谱和衰减全反射傅里叶变换红外(ATR-FTIR）光谱等方法,研究了烟酰胺腺嘌呤二核苷酸(NAD+)在纳米α-Al2O3粒子上的吸附行为。 实验结果显示,NAD+的吸附量受pH值和离子强度影响较大,说明NAD+主要通过静电作用吸附在纳米α-Al2O3粒子上。 采用ATR-FTIR光谱分析了不同pH值溶液中及被吸附的NAD+,发现吸附后的NAD+与溶液中NAD+ 的ATR-FTIR光谱相似,但磷酸根的吸收峰向高波数位移,说明磷酸根参与了表面静电作用。 吸附过程符合Langmuir和Freundlich等温式。 荧光实验结果显示,随着吸附剂α-Al2O3用量的变化,NAD+构象也发生变化。     

Loss of NAD(H) from swollen yeast mitochondria

Background

The mitochondrial electron transport chain oxidizes matrix space NADH as part of the process of oxidative phosphorylation. Mitochondria contain shuttles for the transport of cytoplasmic NADH reducing equivalents into the mitochondrial matrix. Therefore for a long time it was believed that NAD(H) itself was not transported into mitochondria. However evidence has been obtained for the transport of NAD(H) into and out of plant and mammalian mitochondria. Since Saccharomyces cerevisiae mitochondria can directly oxidize cytoplasmic NADH, it remained questionable if mitochondrial NAD(H) transport occurs in this organism.

Results

NAD(H) was lost more extensively from the matrix space of swollen than normal, condensed isolated yeast mitochondria from Saccharomyces cerevisiae. The loss of NAD(H) in swollen organelles caused a greatly decreased respiratory rate when ethanol or other matrix space NAD-linked substrates were oxidized. Adding NAD back to the medium, even in the presence of a membrane-impermeant NADH dehydrogenase inhibitor, restored the respiratory rate of swollen mitochondria oxidizing ethanol, suggesting that NAD is transported into the matrix space. NAD addition did not restore the decreased respiratory rate of swollen mitochondria oxidizing the combination of malate, glutamate, and pyruvate. Therefore the loss of matrix space metabolites is not entirely specific for NAD(H). However, during NAD(H) loss the mitochondrial levels of most other nucleotides were maintained. Either hypotonic swelling or colloid-osmotic swelling due to opening of the yeast mitochondrial unspecific channel (YMUC) in a mannitol medium resulted in decreased NAD-linked respiration. However, the loss of NAD(H) from the matrix space was not mediated by the YMUC, because YMUC inhibitors did not prevent decreased NAD-linked respiration during swelling and YMUC opening without swelling did not cause decreased NAD-linked respiration.

Conclusion

Loss of endogenous NAD(H) from isolated yeast mitochondria is greatly stimulated by matrix space expansion. NAD(H) loss greatly limits NAD-linked respiration in swollen mitochondria without decreasing the NAD-linked respiratory rate in normal, condensed organelles. NAD addition can totally restore the decreased respiration in swollen mitochondria. In live yeast cells mitochondrial swelling has been observed prior to mitochondrial degradation and cell death. Therefore mitochondrial swelling may stimulate NAD(H) transport to regulate metabolism during these conditions.     

辅酶NAD(P)H模型分子的研究进展

辅酶NAD(P)H在生物体内起着重要的调节作用, 已引起了有机化学工作者极大的兴趣, 尤其是在还原反应的立体选择性上, 人们已经开展了大量的研究工作. 讨论了NAD(P)H模型分子进行立体专一性还原反应的影响因素, 并对NAD(P)H模型分子的研究工作做了总结.     

Simultaneous spectrofluorimetric determination of 1-naphthylacetic acid and 1-naphthalenacetamide in commercial formulations and soil samples

A spectrofluorimetric method for the simultaneous determination of 1-naphthylacetic acid (NAA) and 1-naphthalenacetamide (NAD) was developed. The sample solution containing both analytes was equilibrated with Sephadex QAE A-25 gel by agitation and then only NAA was fixed on gel, while the remaining NAD stayed in the solution. The relative fluorescence intensity of NAA fixed on Sephadex QAE A-25 gel was measured directly after packing the gel beads in a 1-mm silica cell, using a solid-phase attachment. NAD was determined spectrofluorimetrically in the solution. The wavelengths of excitation and emission chosen for the determination of NAA were 280 and 336 nm, respectively, and for NAD determination 222 and 337 nm, respectively. The applicable concentration range was 12-60 ng ml(-1) for NAA and 6-120 ng ml(-1) for NAD. The detection limit was 3 ng ml(-1) for NAA and 2 ng ml(-1) for NAD. The method was applied satisfactorily to the determination of NAA and NAD in commercial formulations of phytohormones and soil samples.     

辅酶Ⅰ在苯并咪唑修饰银电极上的还原反应

辅酶Ⅰ在苯并咪唑修饰银电极上的还原反应李根喜,陈洪渊,朱德煦（南京大学生物化学系,生物医药技术国家重点实验室,化学系,南京,２１００９３）关键词ＮＡＤ~＋,苯并咪唑,化学修饰电极还原态烟酰胺辅酶Ⅰ（ＮＡＤＨ）的氧化反应已进行了大量研究。但有关氧化态辅?..     

脱氢酶电化学生物传感器的研究进展

自然界中超过400种脱氢酶使用辅酶-烟酰胺腺嘌呤二核苷酸(NAD+)或烟酰胺腺嘌呤二核苷酸磷酸(NADP+)作为生物催化反应中氢和电子的传递体,因此烟酰胺型辅酶的电化学氧化对构筑此类脱氢酶电化学生物传感器具有重要的意义.本文介绍了还原型辅酶在人工电子媒介体存在下的电化学氧化,以及脱氢酶电化学生物传感器的设计和应用.     

A label-free fluorescence DNA probe based on ligation reaction with quadruplex formation for highly sensitive and selective detection of nicotinamide adenine dinucleotide

A simple label-free fluorescent sensing scheme for sensitive and selective detection of nicotinamide adenine dinucleotide (NAD(+)) has been developed based on DNA ligation reaction with ligand-responsive quadruplex formation. This approach can detect 0.5 nM NAD(+) with high selectivity against other NAD(+) analogs.     

Polymer-supported 1,4-dihydronicotinamide:Synthesis and Application in the Reduction of the Activated Olefins

Three new polymer-supported NAD(P)H models (Ⅰ, Ⅱ, Ⅲ) were designed and synthesized, which can efficiently reduce many activated olefins under mild conditions.1 The most advantageous featureof the three NAD(P)H models is (i) easy work-up and separation of the reaction products and (ii) good potential for recycle use of the NAD(P)H models, which makes the three new polymer-supported NAD(P)H models a promising alternative both in research laboratories and in industrial processes.     

Ultrasensitive and selective detection of nicotinamide adenine dinucleotide by target-triggered ligation-rolling circle amplification

An ultrasensitive fluorescence assay for nicotinamide adenine dinucleotide (NAD(+)) was developed by target-triggered ligation-rolling circle amplification (L-RCA). This novel approach can detect as low as 1 pM NAD(+), much lower than those of previously reported biosensors, and exhibits high discrimination ability even against 200 times excess of NAD(+) analogs.     

Electrochemical Studies of the Interaction of 2‐Nitroacridone with DNA and Determination of DNA

A new acridone derivate 2‐nitroacridone (NAD) was synthesized and a new method of electrochemical probe has been proposed for the determination of salmon sperm DNA based on its interaction with NAD. The electrochemical behavior of interaction of NAD with DNA was investigated on glassy carbon electrode (GCE). In the presence of DNA, the peak current of NAD decreases and the peak potential shifts to a more positive potential without appearance of a new peak. The binding ratio between NAD and salmon sperm DNA was calculated to be 2 : 1 and the binding constant was 3.19×10⁵ L/mol. The decrease of the peak current (ΔI_p) of NAD was proportional to the concentration of DNA in the range from 1.55×10^?7 M to 2.02×10^?6 M with the detection limit of 3.10×10^?8 M, and DNA of synthetic sample was determined satisfactorily. Additionally, the binding mechanism was preliminarily discussed. The mode of interaction between NAD and DNA was found to be intercalation binding.