cas 58-68-4, dihydronicotinamide-adenine dinucleotide

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Mechanistic Aspects of the Electrochemical Oxidation of Dihydronicotinamide Adenine Dinucleotide (NADH)
The apparently single stage anodic oxidation of NADH involving removal of two electrons and a proton to form NAD+ has been examined with particular attention to the deprotonation step and its relationship to the initial potential-determining electron-transfer step, primarily at glassy carbon electrodes (GCE) in aqueous media with supplementary studies at pyrolytic graphite and platinum electrodes in aqueous media and at GCE in Me2SO; the carbon electrodes were generally first covered with an adsorbed NAD+ layer in order to eliminate adsorption-controlled faradaic processes.The initial step is an irreversible heterogeneous electron transfer (transfer coefficient β = 0.37 at carbon electrodes and 0.43 at platinum).The resulting cation radical NAD.H+ loses a proton (first-order reaction; rate constant k) to form the neutral radical NAD. which may participate in a second heterogeneous electron transfer (ECE mechanism) or in a homogeneous electron transfer with NAD.H+ (disproportionation mechanism DISP 1 or half-regeneration mechanism), yielding NAD+.The near identities of current functions, viscosity-corrected diffusion coefficients D and β values, point to essentially similar solute species and charge-transfer paths being involved in different media and at different electrodes.D is ca. 2 x 10-6 cm2 s-1 in aqueous solution; k is ca. 60 s-1 at the GCE covered with adsorbed NAD+.
Moiroux, Jacques,Elving, Philip J.
p. 6533 - 6538
(1980)
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Engineering Olefin-Linked Covalent Organic Frameworks for Photoenzymatic Reduction of CO2
It is of profound significance concerning the global energy and environmental crisis to develop new techniques that can reduce and convert CO2. To address this challenge, we built a new type of artificial photoenzymatic system for CO2 reduction, using a rationally designed mesoporous olefin-linked covalent organic framework (COF) as the porous solid carrier for co-immobilizing formate dehydrogenase (FDH) and Rh-based electron mediator. By adjusting the incorporating content of the Rh electronic mediator, which facilitates the regeneration of nicotinamide cofactor (NADH) from NAD+, the apparent quantum yield can reach as high as 9.17±0.44 %, surpassing all reported NADH-regenerated photocatalysts constructed by crystalline framework materials. Finally, the assembled photocatalyst–enzyme coupled system can selectively convert CO2 to formic acid with high efficiency and good reusability. This work demonstrates the first example using COFs to immobilize enzymes for artificial photosynthesis systems that utilize solar energy to produce value-added chemicals.
Chen, Yao,Guo, Menglei,Yu, Jiangyue,Zhang, Sainan,Zhang, Zhenjie,Zhao, Zhengfeng,Zheng, Dong
supporting information

(2022/02/10)

A highly active Cp*Ir complex with an anionic N,N-donor chelate ligand catalyzes the robust regeneration of NADH under physiological conditions
A highly active [N^N?] iridium complex [Cp*Ir(pba)Cl] (3, Cp* = pentamethylcyclopentadiene, pba = 4-(picolinamido)benzoic acid) has been obtained with an anionic ligand, which exhibited the most robust performance for cofactor NADH regeneration in physiological conditions with HCOONa as the hydrogen source. The structure of complex3was revealed by X-ray single-crystal structure analysis. The turnover frequency (TOF) of complex3in the regeneration of NADH is 7825 h?1, which is about 22.7 times and 178 times higher than that of the C?^N type complex2(345 h?1) and N^N complex1(44 h?1) at 37 °C, respectively. The high activity of complex3seems to be critically affected by the negatively charged N?of the amide chelating ligand, which could promote the reaction rate of Ir-Cl conversion to Ir-H2O. Furthermore, complex3shows good biocompatibility for various biomolecules except SH-compounds (such as reduced glutathione (GSH)). When combined with NADH-dependent enzymes (KRED-101), the complex3-based NADH-regeneration catalytic system shows stable chemoenzymatical coordinate catalytic activity for reducing acetophenone to the corresponding alcohol with high enantioselectivity.
Qi, Caixia,Shi, Yusheng,Su, Huijuan,Sun, Libo,Sun, Wen,Sun, Xun,Xia, Linyan,Yin, Zequn,Zhang, Weiling,Zhao, Li-Jun
p. 7982 - 7991
(2021/12/27)

Highly Efficient S-g-CN/Mo-368 Catalyst for Synergistically NADH Regeneration Under Solar Light
Sulfur-doped graphitic carbon nitride (S-g-CN) has gained significant attention in recent years. Sulfur-doped graphitic carbon nitride (S-g-CN) is a promising metal-free photocatalyst because of its band orientation, natural abundance and groundwork. Improved photocatalytic activity of S-g-CN material for solar chemical production persists a hot yet challenging problem. Herein, we provide an adaptable method for the synthesis of S-g-CN nanocomposite decorated with the moiety of giant polyoxometalate (S-g-CN/Mo-368) that subsequently showed highly efficient photocatalytic activity. The as-synthesized S-g-CN/Mo-368 as a recyclable artificial photocatalyst revealed excellent activity for solar chemical production, that is nicotinamide adenine dinucleotide (NADH) regeneration under visible light. The immobilized Mo-368 on the S-g-CN surface increased the visible light adsorption capacity of the S-g-CN/Mo-368 photocatalyst. The visible light absorption activity, morphology, element compositions, particle size and zeta potential of S-g-CN powder and S-g-CN/Mo-368 were thoroughly investigated. From the application point of view, S-g-CN/Mo-368 was applied to determine the solar chemical production (i.e. NADH regeneration) under visible light with a higher yield% of about ~ 94.85%.
Gupta, Abhishek Kumar,Gupta, Sarvesh Kumar,Singh, Ajeet,Yadav, Bal Chandra,Yadav, Rajesh Kumar
(2021/07/31)

Chemo-bio catalysis using carbon supports: application in H2-driven cofactor recycling
Heterogeneous biocatalytic hydrogenation is an attractive strategy for clean, enantioselective CX reduction. This approach relies on enzymes powered by H2-driven NADH recycling. Commercially available carbon-supported metal (metal/C) catalysts are investigated here for direct H2-driven NAD+reduction. Selected metal/C catalysts are then used for H2oxidation with electrons transferredviathe conductive carbon support material to an adsorbed enzyme for NAD+reduction. These chemo-bio catalysts show improved activity and selectivity for generating bioactive NADH under ambient reaction conditions compared to metal/C catalysts. The metal/C catalysts and carbon support materials (all activated carbon or carbon black) are characterised to probe which properties potentially influence catalyst activity. The optimised chemo-bio catalysts are then used to supply NADH to an alcohol dehydrogenase for enantioselective (>99% ee) ketone reductions, leading to high cofactor turnover numbers and Pd and NAD+reductase activities of 441 h?1and 2347 h?1, respectively. This method demonstrates a new way of combining chemo- and biocatalysis on carbon supports, highlighted here for selective hydrogenation reactions.
Cleary, Sarah E.,Grobert, Nicole,Reeve, Holly A.,Vincent, Kylie A.,Zhao, Xu,Zor, Ceren
p. 8105 - 8114
(2021/06/22)

Visible light driven selective NADH regeneration using a system of water-soluble zinc porphyrin and homogeneous polymer-dispersed rhodium nanoparticles
We discovered the catalytic activity of Rh nanoparticles dispersed by polyvinylpyrrolidone (Rh-PVP) for NADH regeneration. The selective reduction of NAD+to enzyme active NADH has successfully been achieved in the presence of an electron donor, ZnTPPS as a photosensitizer, and Rh-PVP. Only 1,4-NADH was produced as the reduction product of NAD+, which was validated by an enzymatic assay and HPLC.
Amao, Yutaka,Katagiri, Takayuki
p. 15748 - 15752
(2021/09/22)

Design of artificial metalloenzymes for the reduction of nicotinamide cofactors
Artificial metalloenzymes result from the insertion of a catalytically active metal complex into a biological scaffold, generally a protein devoid of other catalytic functionalities. As such, their design requires efforts to engineer substrate binding, in addition to accommodating the artificial catalyst. Here we constructed and characterised artificial metalloenzymes using alcohol dehydrogenase as starting point, an enzyme which has both a cofactor and a substrate binding pocket. A docking approach was used to determine suitable positions for catalyst anchoring to single cysteine mutants, leading to an artificial metalloenzyme capable to reduce both natural cofactors and the hydrophobic 1-benzylnicotinamide mimic. Kinetic studies revealed that the new construct displayed a Michaelis-Menten behaviour with the native nicotinamide cofactors, which were suggested by docking to bind at a surface exposed site, different compared to their native binding position. On the other hand, the kinetic and docking data suggested that a typical enzyme behaviour was not observed with the hydrophobic 1-benzylnicotinamide mimic, with which binding events were plausible both inside and outside the protein. This work demonstrates an extended substrate scope of the artificial metalloenzymes and provides information about the binding sites of the nicotinamide substrates, which can be exploited to further engineer artificial metalloenzymes for cofactor regeneration. Synopsis about graphical abstract: The manuscript provides information on the design of artificial metalloenzymes based on the bioconjugation of rhodium complexes to alcohol dehydrogenase, to improve their ability to reduce hydrophobic substrates. The graphical abstract presents different binding modes and results observed with native cofactors as substrates, compared to the hydrophobic benzylnicotinamide.
Basle, Mattias,Padley, Henry A.W.,Martins, Floriane L.,Winkler, Gerlof Sebastiaan,J?ger, Christof M.,Pordea, Anca
(2021/04/19)

Implantable Biosupercapacitor Inspired by the Cellular Redox System
The carbon nanotube (CNT) yarn supercapacitor has high potential for in vivo energy storage because it can be used in aqueous environments and stitched to inner parts of the body, such as blood vessels. The biocompatibility issue for frequently used pseudocapacitive materials, such as metal oxides, is controversial in the human body. Here, we report an implantable CNT yarn supercapacitor inspired by the cellular redox system. In all living cells, nicotinamide adenine dinucleotide (NAD) is a key redox biomolecule responsible for cellular energy transduction to produce adenosine triphosphate (ATP). Based on this redox system, CNT yarn electrodes were fabricated by inserting a twist in CNT sheets with electrochemically deposited NAD and benzoquinone for redox shuttling. Consequently, the NAD/BQ/CNT yarn electrodes exhibited the maximum area capacitance (55.73 mF cm?2) under physiological conditions, such as phosphate-buffered saline and serum. In addition, the yarn electrodes showed a negligible loss of capacitance after 10 000 repeated charge/discharge cycles and deformation tests (bending/knotting). More importantly, NAD/BQ/CNT yarn electrodes implanted into the abdominal cavity of a rat's skin exhibited the stable in vivo electrical performance of a supercapacitor. Therefore, these findings demonstrate a redox biomolecule-applied platform for implantable energy storage devices.
Jang, Yongwoo,Park, Taegyu,Kim, Eunyoung,Park, Jong Woo,Lee, Dong Yeop,Kim, Seon Jeong
p. 10563 - 10567
(2021/04/05)

Fluorescent and Biocompatible Ruthenium-Coordinated Oligo(p-phenylenevinylene) Nanocatalysts for Transfer Hydrogenation in the Mitochondria of Living Cells
It is challenging to design metal catalysts for in situ transformation of endogenous biomolecules with good performance inside living cells. Herein, we report a multifunctional metal catalyst, ruthenium-coordinated oligo(p-phenylenevinylene) (OPV-Ru), for intracellular catalysis of transfer hydrogenation of nicotinamide adenine dinucleotide (NAD+) to its reduced format (NADH). Owing to its amphiphilic characteristic, OPV-Ru possesses good self-assembly capability in water to form nanoparticles through hydrophobic interaction and π–π stacking, and numerous positive charges on the surface of nanoparticles displayed a strong electrostatic interaction with negatively charged substrate molecules, creating a local microenvironment for enhancing the catalysis efficiency in comparison to dispersed catalytic center molecule (TOF value was enhanced by about 15 fold). OPV-Ru could selectively accumulate in the mitochondria of living cells. Benefiting from its inherent fluorescence, the dynamic distribution in cells and uptake behavior of OPV-Ru could be visualized under fluorescence microscopy. This work represents the first demonstration of a multifunctional organometallic complex catalyzing natural hydrogenation transformation in specific subcellular compartments of living cells with excellent performance, fluorescent imaging ability, specific mitochondria targeting and good chemoselectivity with high catalysis efficiency.
Chen, Yanyan,Dai, Nan,Liu, Libing,Lv, Fengting,Qi, Ruilian,Wang, Shu,Zhao, Hao
(2020/03/23)

Construction of Fully Conjugated Covalent Organic Frameworks via Facile Linkage Conversion for Efficient Photoenzymatic Catalysis
Covalent organic frameworks (COFs) with improved stability and extended ?-conjugation structure are highly desirable. Here, two imine-linked COFs were converted into ultrastable and ?-conjugated fused-aromatic thieno[3,2-c]pyridine-linked COFs (B-COF-2 and T-COF-2). The successful conversion was confirmed by infrared and solid-state 13C NMR spectroscopies. Furthermore, the structures of thieno[3,2-c]pyridine-linked COFs were evaluated by TEM and PXRD. It is noted that a slight difference in the structure leads to totally different photoactivity. The fully ?-conjugated T-COF-2 containing triazine as the core exhibited an excellent photocatalytic NADH regeneration yield of 74% in 10 min.
Chen, Renzeng,Hao, Wenbo,Li, Zhibo,Liu, Hui,Liu, Jian,Pan, Qingyan,Wang, Yuancheng,Wu, Chenyu,Xu, Jie,Zhao, Yingjie
supporting information
p. 5958 - 5963
(2020/04/27)

Can a Nonorganometallic Ruthenium(II) Polypyridylamine Complex Catalyze Hydride Transfer? Mechanistic Insight from Solution Kinetics on the Reduction of Coenzyme NAD+by Formate
Application of organometallic ruthenium(II) arene complexes has been successful for the modulation of cellular redox processes via their interaction with species such as formate to control the NAD+/NADH balance in cells. Here we present the first evidence that similar effects can be reached with the application of a nonorganometallic ruthenium(II) polypyridyl complex. Kinetic studies performed demonstrate the ability of [RuII(terpy)(en)(H2O/EtOH)]2+ in water/ethanol (1:9, v/v) solution, where terpy = 2,2′:6′,2″-terpyridine and en = ethylenediamine, to catalyze the reduction of the NAD+ coenzyme to NADH in the presence of formate as hydride transfer source. In this case, terpy instead of arene is responsible for the labilization of coordinated solvent. The suggested catalytic cycle begins with the fast anation of the [RuII(terpy)(en)(H2O/EtOH)]2+ complex by formate. This is followed by the rate-determining formate-catalyzed decarboxylation of the generated ruthenium(II) formato complex to form [RuII(terpy)(en)H]+. Rapid hydride transfer to NAD+ from [RuII(terpy)(en)H]+ to form NADH and to regenerate the starting ruthenium(II) solvato complex, closes the overall catalytic cycle.
Chrzanowska, Marta,Katafias, Anna,Van Eldik, Rudi
supporting information
p. 14944 - 14953
(2020/11/02)

Emissive Synthetic Cofactors: A Highly Responsive NAD+ Analogue Reveals Biomolecular Recognition Features
Apart from its vital function as a redox cofactor, nicotinamide adenine dinucleotide (NAD+) has emerged as a crucial substrate for NAD+-consuming enzymes, including poly(ADP-ribosyl)transferase 1 (PARP1) and CD38/CD157. Their association with severe diseases, such as cancer, Alzheimer's disease, and depressions, necessitates the development of new analytical tools based on traceable NAD+ surrogates. Here, the synthesis, photophysics and biochemical utilization of an emissive, thieno[3,4-d]pyrimidine-based NAD+ surrogate, termed NthAD+, are described. Its preparation was accomplished by enzymatic conversion of synthetic thATP by nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1). The new NAD+ analogue possesses useful photophysical features including redshifted absorption and emission maxima as well as a relatively high quantum yield. Serving as a versatile substrate, NthAD+ was reduced by alcohol dehydrogenase (ADH) to NthADH and afforded thADP-ribose (thADPr) upon hydrolysis by NAD+-nucleosidase (NADase). Furthermore, NthAD+ was engaged in cholera toxin A (CTA)-catalyzed mono(thADP-ribosyl)ation, but was found incapable in promoting PARP1-mediated poly(thADP-ribosyl)ation. Due to its high photophysical responsiveness, NthAD+ is suited for spectroscopic real-time monitoring. Intriguingly, and as an N7-lacking NAD+ surrogate, the thieno-based cofactor showed reduced compatibility (i.e., functional similarity compared to native NAD+) relative to its isothiazolo-based analogue. The distinct tolerance, displayed by diverse NAD+ producing and consuming enzymes, suggests unique biological recognition features and dependency on the purine N7 moiety, which is found to be of importance, if not essential, for PARP1-mediated reactions.
Feldmann, Jonas,Li, Yao,Tor, Yitzhak
p. 4379 - 4389
(2019/03/07)

N, O -Chelating quinoline-based half-sandwich organorhodium and -iridium complexes: Synthesis, antiplasmodial activity and preliminary evaluation as transfer hydrogenation catalysts for the reduction of NAD+
Two Rh(iii) and Ir(iii) half-sandwich quinoline-based complexes were synthesised and evaluated for their in vitro antiplasmodial activity against the chloroquine-sensitive NF54 and multi-drug resistant K1 strains of the human malaria parasite, Plasmodium falciparum. These half-sandwich organometallic complexes can also facilitate transfer hydrogenation, by converting β-nicotinamide adenine dinucleotide (NAD+) to its reduced form (NADH) in the presence of sodium formate. Co-administration of the iridium(iii) complex with sodium formate enhances the antiplasmodial activity in the chloroquine-resistant (K1) strain of Plasmodium falciparum, intimating that metal-mediated transfer hydrogenations can be achieved in malarial parasitic cells.
Stringer, Tameryn,Melis, Diana R.,Smith, Gregory S.
supporting information
p. 13143 - 13148
(2019/09/18)

WS2/g-C3N4 composite as an efficient heterojunction photocatalyst for biocatalyzed artificial photosynthesis
A heterogeneous WS2/g-C3N4 composite photocatalyst was prepared by a facile ultrasound-assisted hydrothermal method. The WS2/g-C3N4 composite was used for photocatalytic regeneration of NAD+ to NADH, which were coupled with dehydrogenases for sustainable bioconversion of CO2 to methanol under visible light irradiation. Compared with pristine g-C3N4 and the physical mixture of WS2 and g-C3N4, the fabricated WS2/g-C3N4 composite catalyst with 5 wt% of WS2 showed the highest activity for methanol synthesis. The methanol productivity reached 372.1 μmol h-1 gcat-1, which is approximately 7.5 times higher than that obtained using pure g-C3N4. For further application demonstration, the activity of the WS2/g-C3N4 composite catalyst toward photodegradation of Rhodamine B (RhB) was evaluated. RhB removal ratio approaching 100% was achieved in 1 hour by using the WS2/g-C3N4 composite catalyst with 5 wt% of WS2, at an apparent degradation rate approximately 2.6 times higher than that of pure g-C3N4. Based on detailed investigations on physiochemical properties of the photocatalysts, the significantly enhanced reaction efficiency of the WS2/g-C3N4 composite was considered to be mainly benefiting from the formation of a heterojunction interface between WS2 and g-C3N4. Upon visible-light irradiation, the photo-induced electrons can transfer from the conduction band of g-C3N4 to WS2, thus recombination of electrons and holes was decreased and the photo-harvesting efficiency was enhanced.
Zeng, Peng,Ji, Xiaoyuan,Su, Zhiguo,Zhang, Songping
p. 20557 - 20567
(2018/06/14)

Emissive Synthetic Cofactors: Enzymatic Interconversions of tzA Analogues of ATP, NAD+, NADH, NADP+, and NADPH
A series of enzymatic transformations, which generate visibly emissive isofunctional cofactors based on an isothiazolo[4,3-d]pyrimidine analogue of adenosine (tzA), was developed. Nicotinamide adenylyl transferase condenses nicotinamide mononucleotide and tzATP to yield NtzAD+, which can be enzymatically phosphorylated by NAD+ kinase and ATP or tzATP to the corresponding NtzADP+. The latter can be engaged in NADP-specific coupled enzymatic transformations involving conversion to NtzADPH by glucose-6-phosphate dehydrogenase and reoxidation to NtzADP+ by glutathione reductase. The NtzADP+/NtzADPH cycle can be monitored in real time by fluorescence spectroscopy.
Hallé, Fran?ois,Fin, Andrea,Rovira, Alexander R.,Tor, Yitzhak
supporting information
p. 1087 - 1090
(2017/12/28)

Transfer Hydrogenation and Antiproliferative Activity of Tethered Half-Sandwich Organoruthenium Catalysts
We report the synthesis and characterization of four neutral organometallic tethered complexes, [Ru(η6-Ph(CH2)3-ethylenediamine-N-R)Cl], where R = methanesulfonyl (Ms, 1), toluenesulfonyl (Ts, 2), 4-trifluoromethylbenzenesulfonyl (Tf, 3), and 4-nitrobenzenesulfonyl (Nb, 4), including their X-ray crystal structures. These complexes exhibit moderate antiproliferative activity toward human ovarian, lung, hepatocellular, and breast cancer cell lines. Complex 2 in particular exhibits a low cross-resistance with cisplatin. The complexes show potent catalytic activity in the transfer hydrogenation of NAD+ to NADH with formate as hydride donor in aqueous solution (310 K, pH 7). Substituents on the chelated ligand decreased the turnover frequency in the order Nb > Tf > Ts > Ms. An enhancement of antiproliferative activity (up to 22%) was observed on coadministration with nontoxic concentrations of sodium formate (0.5-2 mM). Complex 2 binds to nucleobase guanine (9-EtG), but DNA appears not to be the target, as little binding to calf thymus DNA or bacterial plasmid DNA was observed. In addition, complex 2 reacts rapidly with glutathione (GSH), which might hamper transfer hydrogenation reactions in cells. Complex 2 induced a dose-dependent G1 cell cycle arrest after 24 h exposure in A2780 human ovarian cancer cells while promoting an increase in reactive oxygen species (ROS), which is likely to contribute to its antiproliferative activity.
Chen, Feng,Romero-Canelón, Isolda,Soldevila-Barreda, Joan J.,Song, Ji-Inn,Coverdale, James P. C.,Clarkson, Guy J.,Kasparkova, Jana,Habtemariam, Abraha,Wills, Martin,Brabec, Viktor,Sadler, Peter J.
p. 1555 - 1566
(2018/06/04)

Construction of PQQ-enzyme multi-immobilized electrodes for electrocataltyic reduction of carbonyl compounds
A thin poly(arylamine) (PAA) and poly(acrylic acid) (PAAc) layers-coated graphite felt (GF) electrode immobilizing all mediation components of pyrroloquinolinequinone (PQQ), diaphorase (Dp), oxidized nicotinamide adenine dinucleotide (NAD+) and alcohol dehydrogenase (ADH) to construct a complete bioelectrochemical reactor was prepared and applied to electrocatalytic reduction of carbonyl compounds in a phosphate buffer at constant potential of -0.65 V vs. Ag/AgCl. The carbonyl compounds were reduced to the corresponding alcohols with high current efficiency (97.2 - 100%) and high yield (97.4 - 100%), respectively.
Kashiwagi, Yoshitomo,Ono, Tetsuya,Yoshida, Kentaro,Ito, Toshinori,Sakurai, Nobuki
p. 1216 - 1225
(2018/08/06)

18O Kinetic Isotope Effects Reveal an Associative Transition State for Phosphite Dehydrogenase Catalyzed Phosphoryl Transfer
Phosphite dehydrogenase (PTDH) catalyzes an unusual phosphoryl transfer reaction in which water displaces a hydride leaving group. Despite extensive effort, it remains unclear whether PTDH catalysis proceeds via an associative or dissociative mechanism. Here, primary 2H and secondary 18O kinetic isotope effects (KIEs) were determined and used together with computation to characterize the transition state (TS) catalyzed by a thermostable PTDH (17X-PTDH). The large, normal 18O KIEs suggest an associative mechanism. Various transition state structures were computed within a model of the enzyme active site and 2H and 18O KIEs were predicted to evaluate the accuracy of each TS. This analysis suggests that 17X-PTDH catalyzes an associative process with little leaving group displacement and extensive nucleophilic participation. This tight TS is likely a consequence of the extremely poor leaving group requiring significant P-O bond formation to expel the hydride. This finding contrasts with the dissociative TSs in most phosphoryl transfer reactions from phosphate mono- and diesters.
Howe, Graeme W.,Van Der Donk, Wilfred A.
supporting information
p. 17820 - 17824
(2019/01/09)

G-C3N4α-Fe2O3/C Photocatalysts: Synergistically Intensified Charge Generation and Charge Transfer for NADH Regeneration
Graphitic carbon nitride (g-C3N4) is an emergent metal-free photocatalyst because of its band position, natural abundance, and facile preparation. Synergetic intensification of charge generation and charge transfer of g-C3N4 to increase solar-to-chemical efficiency remains a hot yet challenging issue. Herein, a nanoshell with two moieties of α-Fe2O3 and carbon (C) is in situ formed on the surface of a g-C3N4 core through calcination of Fe3+/polyphenol-coated melamine, thus acquiring g-C3N4α-Fe2O3/C coreshell photocatalysts. The α-Fe2O3 moiety acts as an additional photosensitizer, offering more photogenerated electrons, whereas the C moiety bridges a "highway" to facilitate the electron transfer either from α-Fe2O3 moiety to g-C3N4 or from g-C3N4 to C moiety. By tuning the proportion of these two moieties in the nanoshell, a photocurrent density of 3.26 times higher than pristine g-C3N4 is obtained. When utilized for photocatalytic regeneration of reduced nicotinamide adenine dinucleotide (NADH, a dominant cofactor in biohydrogenation reaction), g-C3N4α-Fe2O3/C exhibits an equilibrium NADH yield of 76.3% with an initial reaction rate (r) of 7.7 mmol h-1 g-1, among the highest r for photocatalytic NADH regeneration ever reported. Manipulating the coupling between charge generation and charge transfer may offer a facile, generic strategy to improve the catalytic efficiency of a broad range of photocatalysts other than g-C3N4.
Wu, Yizhou,Ward-Bond, Jesse,Li, Donglin,Zhang, Shaohua,Shi, Jiafu,Jiang, Zhongyi
p. 5664 - 5674
(2018/05/29)

Selective Usage of Isozymes for Stress Response
Isozymes are enzymes with similar sequences that catalyze the same reaction in a given species. In Saccharomyces cerevisiae, most isozymes have major isoforms with high expression levels and minor isoforms with little expression under normal growth conditions. In a proteomic study aimed at identifying yeast protein regulated by rapamycin, we found an interesting phenomenon, that, for several metabolic enzymes, the major isozymes are downregulated while the minor isozymes are upregulated. Through enzymological and biochemical studies, we demonstrate that a rapamycin-upregulated enolase isozyme (ENO1) favors gluconeogenesis and a rapamycin-upregulated alcohol dehydrogenase isozyme (ALD4) promotes the reduction of NAD+ to NADH (instead of NADP+ to NADPH). Gene deletion study in yeast showed that the ENO1 and ALD4 are important for yeast survival under less-favorable growth conditions. Therefore, our study highlights the different metabolic needs of cells under different conditions and how nature chooses different isozymes to fit the metabolic needs.
Zhang, Yugang,Lin, Zhewang,Wang, Miao,Lin, Hening
p. 3059 - 3064
(2018/11/23)

Effect of sulfonamidoethylenediamine substituents in RuII arene anticancer catalysts on transfer hydrogenation of coenzyme NAD+ by formate
A series of neutral pseudo-octahedral RuII sulfonamidoethylenediamine complexes [(η6-p-cym)Ru(N,N′)Cl] where N,N′ is N-(2-(R1,R2-amino)ethyl)-4-toluenesulfonamide (TsEn(R1,R2)) R1,R2 = Me,H (1); Me,Me (2); Et,H (3); benzyl,H (Bz, 4); 4-fluorobenzyl,H (4-F-Bz, 5) or naphthalen-2-ylmethyl,H (Naph, 6), were synthesised and characterised including the X-ray crystal structure of 3. These complexes catalyse the reduction of NAD+ regioselectively to 1,4-NADH by using formate as the hydride source. The catalytic efficiency depends markedly on the steric and electronic effects of the N-substitutent, with turnover frequencies (TOFs) increasing in the order: 1 -1 for 4 with a 95% yield of 1,4-NADH. The reduction rate was highest between pH? (deuterated solvent) 6 and 7.5 and improved with an increase in formate concentration (TOF of 18.8 h-1, 140 mM formate). The calculations suggested initial substitution of an aqua ligand by formate, followed by hydride transfer to RuII and then to NAD+, and indicated specific interactions between the aqua complex and both NAD+ and NADH, the former allowing a preorganisation involving interaction between the aqua ligand, formate anion and the pyridine ring of NAD+. The complexes exhibited antiproliferative activity towards A2780 human ovarian cancer cells with IC50 values ranging from 1 to 31 μM, the most potent complex, [(η6-p-cym)Ru(TsEn(Bz,H))Cl] (4, IC50 = 1.0 ± 0.1 μM), having a potency similar to the anticancer drug cisplatin. Co-administration with sodium formate (2 mM), increased the potency of all complexes towards A2780 cells by 20-36%, with the greatest effect seen for complex 6.
Chen, Feng,Soldevila-Barreda, Joan J.,Romero-Canelón, Isolda,Coverdale, James P. C.,Song, Ji-Inn,Clarkson, Guy J.,Kasparkova, Jana,Habtemariam, Abraha,Brabec, Viktor,Wolny, Juliusz A.,Schünemann, Volker,Sadler, Peter J.
supporting information
p. 7178 - 7189
(2018/06/04)

Elucidation of a Self-Sustaining Cycle in Escherichia coli l -Serine Biosynthesis That Results in the Conservation of the Coenzyme, NAD+
The equilibrium of the reaction catalyzed by d-3-phosphoglycerate dehydrogenase (PGDH), the first enzyme in the l-serine biosynthetic pathway, is far in the direction away from serine synthesis. As such, the enzyme is usually assayed in this direction. To
Grant, Gregory A.
p. 1798 - 1806
(2018/03/26)

S-allyl-L-cysteine sulfoxide, a garlic odor precursor, suppresses elevation in blood ethanol concentration by accelerating ethanol metabolism and preventing ethanol absorption from gut
Alcoholic beverages are enjoyed together with meals worldwide, but their excessive intake is associated with an increased risk of various diseases. We investigated whether S-allyl-L-cysteine sulfoxide (ACSO), a sulfuric odor precursor of garlic, suppresses elevation in plasma ethanol concentration by accelerating ethanol metabolism and preventing ethanol absorption from the gut in rats. ACSO and garlic extract with a high ACSO content (Garlic-H) suppressed elevation in concentrations of ethanol and acetaldehyde in plasma and promoted the activities of alcohol dehydrogenase and aldehyde dehydrogenase. However, ACSO and Garlic-H did not affect plasma acetate so much. Furthermore, we examined the change in plasma ethanol concentration by injecting ACSO or Garlic-H into the ligated stomach or jejunum together with ethanol solution. ACSO and Garlic-H suppressed the absorption of ethanol from the stomach and jejunum, but suppression in the jejunum was less than in the stomach. In conclusion, ACSO inhibits ethanol absorption and accelerates ethanol metabolism.
Uto-Kondo, Harumi,Hase, Ayumu,Yamaguchi, Yusuke,Sakurai, Ayaka,Akao, Makoto,Saito, Takeshi,Kumagai, Hitomi
p. 724 - 731
(2018/04/12)

Emissive Synthetic Cofactors: An Isomorphic, Isofunctional, and Responsive NAD+ Analogue
The synthesis, photophysics, and biochemical utility of a fluorescent NAD+ analogue based on an isothiazolo[4,3-d]pyrimidine core (NtzAD+) 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+.
Rovira, Alexander R.,Fin, Andrea,Tor, Yitzhak
supporting information
p. 15556 - 15559
(2017/11/14)

Plasmonic substrates comprising gold nanostars efficiently regenerate cofactor molecules
The light harvesting capacity of plasmonic nanoparticles is a fundamental feature for catalysing chemical reactions close to their surface. The efficiency of the photochemical processes depends not only on the geometrical aspects on a single particle level but also on the complexity of the multiparticle architectures. Although, the effect of the particle geometry is progressively understood in the relevant photochemical processes (water splitting and hydrogen evolution), there are experimental and theoretical needs for understanding the role of the shape in the multiparticle systems in the photocatalytic processes. Here we have shown that macroscopic plasmonic substrates comprising gold nanostars exhibit better efficiencies than nanorods or cubes in the photoregeneration of cofactor molecules. We performed photochemical and photoelectrochemical measurements, supported by theoretical simulations, showing that the unique geometry of nanostars-radially distributed spikes-contributes to stronger light absorption by the plasmonic film containing that type of nanoparticles.
Sánchez-Iglesias, Ana,Barroso, Javier,Solís, Diego M.,Taboada, José M.,Obelleiro, Fernando,Pavlov, Valeri,Chuvilin, Andrey,Grzelczak, Marek
p. 7045 - 7052
(2016/05/24)

A highly efficient covalent organic framework film photocatalyst for selective solar fuel production from CO2
Two-dimensional covalent organic frameworks (2D COFs) are a class of crystalline polymers with a design controllable platform that may be developed into a new type of metal-free photocatalyst. The exploration of new frameworks is, however, critical for further progress in this emerging field. To realize their full potential in practical light harvesting applications, the fabrication of a film type photocatalyst is equally essential. Here, we report the successful development of a triazine based covalent organic framework (2D CTF) as an inexpensive and highly efficient visible light active flexible film photocatalyst for solar fuel production from CO2. For this research work, the condensation polymerization between cyanuric chloride and perylene diimide has been exploited for the first time as a new synthetic approach to the construction of 2D CTFs. The visible light-harvesting capacity, suitable band gap and highly ordered π electron channels contribute to the excellent performance of the film photocatalyst. The current study is a benchmark example of COF based photocatalysts for solar fuel production from CO2 and is expected to trigger further interest in potential solar energy conversion applications such as wearable devices.
Yadav, Rajesh K.,Kumar, Abhishek,Park, No-Joong,Kong, Ki-Jeong,Baeg, Jin-Ook
supporting information
p. 9413 - 9418
(2016/07/06)

Upregulation of an Artificial Zymogen by Proteolysis
Regulation of enzymatic activity is vital to living organisms. Here, we report the development and the genetic optimization of an artificial zymogen requiring the action of a natural protease to upregulate its latent asymmetric transfer hydrogenase activity.
Liu, Zhe,Lebrun, Vincent,Kitanosono, Taku,Mallin, Hendrik,K?hler, Valentin,H?ussinger, Daniel,Hilvert, Donald,Kobayashi, Shu,Ward, Thomas R.
supporting information
p. 11587 - 11590
(2016/10/24)

Highly selective solar-driven methanol from CO2 by a photocatalyst/biocatalyst integrated system
The successful development of a photocatalyst/biocatalyst integrated system that carries out selective methanol production from CO2 is reported herein. The fine-tuned system was derived from a judicious combination of graphene-based visible light active photocatalyst (CCG-IP) and sequentially coupled enzymes. The covalent attachment of isatin-porphyrin (IP) chromophore to chemically converted graphene (CCG) afforded newly developed CCG-IP photocatalyst for this research endeavor. The current work represents a new benchmark for carrying out highly selective methanol formation from CO2 in an environmentally benign manner.
Yadav, Rajesh K.,Oh, Gyu Hwan,Park, No-Joong,Kumar, Abhishek,Kong, Ki-Jeong,Baeg, Jin-Ook
supporting information
p. 16728 - 16731
(2015/02/19)

Mussel-inspired plasmonic nanohybrids for light harvesting
Core-shell plasmonic nanohybrids are synthesized through a simple solutionbased process utilizing mussel-inspired polydopamine (PDA). The multi-purpose PDA not only facilitates plasmonic metal formation, but also serves as a scaffold to incorporate photosensitizers around the metal cores, as well as an adhesive between the nanohybrids and the substrate. The resulting plasmonic assembly exhibits highly enhanced light absorption in photo catalytic systems to augment artificial photosynthesis.
Lee, Minah,Kim, Jong Uk,Lee, Joon Seok,Lee, Byung Il,Shin, Jonghwa,Park, Chan Beum
p. 4463 - 4468
(2014/07/22)

A genetically encodable ligand for transfer hydrogenation
Simple tripeptides are shown here to be versatile ligands for iridium-catalyzed transfer hydrogenations affording large acceleration effects. A water-soluble iridium complex with Gly-Gly-Phe, for example, catalyzes the reduction of diverse ketones, aldehydes, and imines by formate with turnover frequencies rivaling or outperforming those of established ligand systems. Regioselective reduction of coenzyme NAD+ to NADH illustrates the potential utility of this system for biotechnological applications. Because peptides are genetically encodable, they represent an attractive class of foldamer ligands for creating artificial metalloenzymes.
Mayer, Clemens,Hilvert, Donald
p. 3427 - 3431
(2013/07/19)

Production and characterization of Escherichia coli glycerol dehydrogenase as a tool for glycerol recycling
NAD+-dependent glycerol (Gro) dehydrogenase (GroDHase) catalyzes the conversion of Gro into dihydroxyacetone (DHA), the first step for fermentative Gro metabolism in Escherichia coli. In this work, we cloned the gldA gene that codes for the E. coli GroDHase and homologously expressed, purified, and kinetically characterized the recombinant protein. To achieve this, the enzyme was over-produced using Gro supplemented growth medium and lactose as the inducer. The enzyme was highly purified using either pseudo-affinity chromatography or a simple heat-shock treatment, which is potentially valuable for industrial production of GroDHase. We detected efficient oxidation of Gro derived from biodiesel production to DHA by gas chromatography. The results presented in this work support recombinant GroDHase production in a biorefinery setting as a relevant tool for converting Gro into DHA for future biotechnological applications.
Piattoni, Claudia Vanesa,Figueroa, Carlos María,Asención Diez, Matías Damián,Parcerisa, Ivana Lorna,Antu?a, Sebastián,Comelli, Raúl Alberto,Guerrero, Sergio Adrián,Beccaria, Alejandro José,Iglesias, Alberto álvaro
p. 406 - 412
(2013/06/27)

Functionalized bipyridyl rhodium complex capable of electrode attachment for regeneration of NADH
A Rh(III) complex having a functionalized bpy-OH ligand that is potentially linkable to electrode surfaces was synthesized and fully characterized. The hydrido complex, which could be generated either electrochemically by cathodic reduction of the [η5-Cp*Rh(bpy-OH)Cl]Cl complex at -771 mV (versus Ag/AgCl) or chemically with formate, transformed NAD + efficiently into NADH with a TOF = 710 at 60 C.
Sivanesan, Dharmalingam,Yoon, Sungho
p. 52 - 56
(2013/07/19)

Identification of a long-range protein network that modulates active site dynamics in extremophilic alcohol dehydrogenases
A tetrameric thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (ht-ADH) has been mutated at an aromatic side chain in the active site (Trp-87). The ht-W87A mutation results in a loss of the Arrhenius break seen at 30 °C for the wild-type enzyme and an increase in cold lability that is attributed to destabilization of the active tetrameric form. Kinetic isotope effects (KIEs) are nearly temperature-independent over the experimental temperature range, and similar in magnitude to those measured above 30 °C for the wild-type enzyme. This suggests that the rigidification in the wild-type enzyme below 30 °C does not occur for ht-W87A. A mutation at the dimer-dimer interface in a thermolabile psychrophilic homologue of ht-ADH, ps-A25Y, leads to a more thermostable enzyme and a change in the rate-determining step at low temperature. The reciprocal mutation in ht-ADH, ht-Y25A, results in kinetic behavior similar to that of W87A. Collectively, the results indicate that flexibility at the active site is intimately connected to a subunit interaction 20 A away. The convex Arrhenius curves previously reported for ht-ADH (Kohen, A., Cannio, R., Bartolucci, S., and Klinman, J. P. (1999) Nature 399, 496-499) are proposed to arise, at least in part, from a change in subunit interactions that rigidifies the substrate-binding domain below 30 °C, and impedes the ability of the enzyme to sample the catalytically relevant conformational landscape. These results implicate an evolutionarily conserved, long-range network of dynamical communication that controls C-H activation in the prokaryotic alcohol dehydrogenases.
Nagel, Zachary D.,Cun, Shujian,Klinman, Judith P.
p. 14087 - 14097
(2013/07/28)

Application of reduced graphene oxide and carbon nanotube modified electrodes for measuring the enzymatic activity of alcohol dehydrogenase
An electrochemical method was developed to measure the enzymatic activity of alcohol dehydrogenase (ADH) by monitoring the amount of reduced nicotinamide adenine dinucleotide (NADH) generated in the catalysed oxidation of ethanol by ADH. The concentration
Wang, Xianlong,Li, Li,Wang, Yanping,Xu, Chongzheng,Zhao, Bo,Yang, Xiaodi
p. 2195 - 2200
(2013/06/26)

The first nonradioactive fluorescence assay for phosphatidylglycerol: prolipoprotein diacylglyceryl transferase that initiates bacterial lipoprotein biosynthesis
The unique and physiologically vital bacterial enzyme, prolipoprotein diacylglyceryl transferase (Lgt), which catalyzes the committed first step in the posttranslational transfer of diacylglyceryl group from phosphatidylglycerol to the prospective N-terminal cysteine of prolipoproteins, remains to be characterized for want of a simpler but equally sensitive nonradioactive assay. We, for the first time, report a coupled enzymatic fluorescence assay for Lgt using the de novo synthetic peptide substrate MKATKSAVGSTLAGCSSHHHHHH. The assay is based on the conversion of the by-product, glycerol-1-phosphate, to dihydroxyacetone using an alkaline phosphatase-glycerol dehydrogenase combination and estimating the fluorescence of the coupled reduction of resazurin to resorufin. The minimum amount of glycerol-1-phosphate, and hence the modified peptide, detected by this method is approximately 20 pmol, thereby making this assay a promising alternative to the radioactive assays. The assay is rapid, more convenient, less laborious, and suitable for purification and characterization of Lgt.
Sundaram, Srividhya,Banerjee, Sanchari,Sankaran, Krishnan
experimental part
p. 163 - 170
(2012/07/28)

Efficient catalytic interconversion between NADH and NAD+ accompanied by generation and consumption of hydrogen with a water-soluble iridium complex at ambient pressure and temperature
Regioselective hydrogenation of the oxidized form of β-nicotinamide adenine dinucleotide (NAD+) to the reduced form (NADH) with hydrogen (H2) has successfully been achieved in the presence of a catalytic amount of a [C,N] cyclometalated organoiridium complex [IrIII(Cp)(4- (1H-pyrazol-1-yl-κN2)benzoic acid-κC3)(H 2O)]2 SO4 [1]2·SO4 under an atmospheric pressure of H2 at room temperature in weakly basic water. The structure of the corresponding benzoate complex Ir III(Cp)(4-(1H-pyrazol-1-yl-κN2)-benzoate- κC3)(H2O) 2 has been revealed by X-ray single-crystal structure analysis. The corresponding iridium hydride complex formed under an atmospheric pressure of H2 undergoes the 1,4-selective hydrogenation of NAD+ to form 1,4-NADH. On the other hand, in weakly acidic water the complex 1 was found to catalyze the hydrogen evolution from NADH to produce NAD+ without photoirradiation at room temperature. NAD+ exhibited an inhibitory behavior in both catalytic hydrogenation of NAD+ with H2 and H2 evolution from NADH due to the binding of NAD+ to the catalyst. The overall catalytic mechanism of interconversion between NADH and NAD+ accompanied by generation and consumption of H2 was revealed on the basis of the kinetic analysis and detection of the catalytic intermediates.
Maenaka, Yuta,Suenobu, Tomoyoshi,Fukuzumi, Shunichi
experimental part
p. 367 - 374
(2012/03/07)

Enhanced reactivity of Lys182 explains the limited efficacy of biogenic amines in preventing the inactivation of glucose-6-phosphate dehydrogenase by methylglyoxal
This study examines the inactivation of the enzyme glucose 6-phosphate dehydrogenase (G6PD) by methylglyoxal (MG) and the eventual protection exerted by endogenous amines. To determine the protective effect of amines, the rate constant of the reaction of MG with the amino group of N-α-acetyl-lysine, carnosine, spermine and spermidine was measured at pH 7.4, and the behavior of endogenous amines was analyzed on the basis of quantum chemical reactivity descriptors. A 63% reduction in the enzyme activity was found upon incubation of G6PD with MG at pH 7.4. The inactivation of G6PD was even larger when the pH was increased to 9.4, revealing a weak protective effect by the amines. The results suggest that some basic residues of G6PD exhibit an anomalous reactivity, which likely reflects a shift in the standard pKa value due to the local environment in the enzyme. Under the experimental conditions used in the assays, this hypothesis was corroborated by mass spectrometry analysis, which points out that modification of Lys182 in the binding site is responsible for the inactivation of G6PD by MG. These results emphasize the need to search for more effective antiglycating agents, which can compete with basic amino acid residues possessing enhanced reactivity in proteins.
Flores-Morales, Patricio,Diema, Claudio,Vilaseca, Marta,Estelrich, Joan,Luque, F. Javier,Gutierrez-Oliva, Soledad,Toro-Labbe, Alejandro,Silva, Eduardo
experimental part
p. 1613 - 1622
(2011/04/16)

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.
Lee, Hye Jung,Lee, Sahng Ha,Park, Chan Beum,Won, Keehoon
body text
p. 12538 - 12540
(2012/02/02)

Biomimetic artificial photosynthesis by light-harvesting synthetic wood
Tree of a kind: An integrated artificial photosynthetic system is developed by reassembling raw materials from plants as support matrix for the encapsulation of porphyrins. The hybrids allow visible-light-driven regeneration of NADH and production of fine chemicals. The synthetic wood not only provides a microenvironment for porphyrin encapsulation but also makes the photosynthesis more effective due to the redox-active lignin component.
Lee, Minah,Kim, Jae Hong,Lee, Sang Hyun,Lee, Sahng Ha,Park, Chan Beum
experimental part
p. 581 - 586
(2012/03/22)

Zn-containing porphyrin as a biomimetic light-harvesting molecule for biocatalyzed artificial photosynthesis
Among the porphyrin molecules with different metal insertion sites and functional ligands, Zn-porphyrin most efficiently regenerates NADH through photo-induced electron transfer in the presence of [Cp*Rh(bpy)H 2O]2+, a rhodium-based electron mediator. Photochemical regeneration of NADH by Zn-porphyrin is successfully coupled with redox enzymatic synthesis under dark state conditions.
Kim, Jae Hong,Lee, Sahng Ha,Lee, Joon Seok,Lee, Minah,Park, Chan Beum
supporting information; experimental part
p. 10227 - 10229
(2011/10/19)

Substitutions of coenzyme-binding, nonpolar residues improve the low-temperature activity of thermophilic dehydrogenases
Although enzymes of thermophilic organisms are often very resistant to thermal denaturation, they are usually less active than their mesophilic or psychrophilic homologues at moderate or low temperatures. To explore the structural features that would improve the activity of a thermophilic enzyme at less than optimal temperatures, we randomly mutated the DNA of single-site mutants of the thermostable Thermus thermophilus 3-isopropylmalate dehydrogenase that already had improved low-temperature activity and selected for additional improved low-temperature activity. A mutant (Ile279 → Val) with improved low-temperature activity contained a residue that directly interacts with the adenine of the coenzyme NAD+, suggesting that modulation of the coenzyme-binding pocket's volume can enhance low-temperature activity. This idea was further supported by a saturation mutagenesis study of the two codons of two other residues that interact with the adenine. Furthermore, a similar type of amino acid substitution also improved the catalytic efficiency of another thermophilic dehydrogenase, T. thermophilus lactate dehydrogenase. Steady-state kinetic experiments showed that the mutations all favorably affected the catalytic turnover numbers. Thermal stability measurements demonstrated that the mutants remain very resistant to heat. Calculation of the energetic contributions to catalysis indicated that the increased turnover numbers are the result of destabilized enzyme-substrate-coenzyme complexes. Therefore, small changes in the side chain volumes of coenzyme-binding residues improved the catalytic efficiencies of two thermophilic dehydrogenases while preserving their high thermal stabilities and may be a way to improve low-temperature activities of dehydrogenases in general.
Hayashi, Sayaka,Akanuma, Satoshi,Onuki, Wakana,Tokunaga, Chihiro,Yamagishi, Akihiko
experimental part
p. 8583 - 8593
(2012/07/27)

On the nature of mutual inactivation between [Cp*Rh(bpy)(H2O)]2+ and enzymes - analysis and potential remedies
Pentamethylcyclopentadienyl rhodium bipyridine ([Cp*Rh(bpy)(H2O)]2+) is a versatile catalyst to promote biocatalytic redox reactions. However, its major drawback lies in the mutual inactivation of [Cp*Rh(bpy)(H2O)]2+ and the biocatalyst. This interaction was investigated using the alcohol dehydrogenase from Thermus sp. ATN1 (TADH) as model enzyme. TADH binds 4equiv. of [Cp*Rh(bpy)(H2O)]2+ without detectable decrease in catalytic activity and stability. Higher molar ratios lead to time-, temperature-, and concentration-dependent inactivation of the enzyme suggesting [Cp*Rh(bpy)(H2O)]2+ to function as an 'unfolding catalyst'. This detrimental activity can be circumvented using strongly coordinating buffers (e.g. (NH4)2SO4) while preserving its activity as NAD(P)H regeneration catalyst under electrochemical reaction conditions.
Poizat, Mael,Arends, Isabel W.C.E.,Hollmann, Frank
body text
p. 149 - 156
(2010/10/19)

The functions of key residues in the inhibitor, substrate and cofactor sites of human 3β-hydroxysteroid dehydrogenase type 1 are validated by mutagenesis
In postmenopausal women, human 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD1) is a critical enzyme in the conversion of DHEA to estradiol in breast tumors, while 3β-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland. The goals of this project are to determine if Arg195 in 3β-HSD1 vs. Pro195 in 3β-HSD2 in the substrate/inhibitor binding site is a critical structural difference responsible for the higher affinity of 3β-HSD1 for inhibitor and substrate steroids compared to 3β-HSD2 and whether Asp61, Glu192 and Thr8 are fingerprint residues for cofactor and substrate binding using site-directed mutagenesis. The R195P-1 mutant of 3β-HSD1 and the P195R-2 mutant of 3β-HSD2 have been created, expressed, purified and characterized kinetically. Dixon analyses of the inhibition of the R195P-1 mutant, P195R-2 mutant, wild-type 3β-HSD1 and wild-type 3β-HSD2 by trilostane has produced kinetic profiles that show inhibition of 3β-HSD1 by trilostane (Ki=0.10μM, competitive) with a 16-fold lower Ki and different mode than measured for 3β-HSD2 (Ki=1.60μM, noncompetitive). The R195P-1 mutation shifts the high-affinity, competitive inhibition profile of 3β-HSD1 to a low-affinity (trilostane Ki=2.56μM), noncompetitive inhibition profile similar to that of 3β-HSD2 containing Pro195. The P195R-2 mutation shifts the low-affinity, noncompetitive inhibition profile of 3β-HSD2 to a high-affinity (trilostane Ki=0.19μM), competitive inhibition profile similar to that of 3β-HSD1 containing Arg195. Michaelis-Menten kinetics for DHEA, 16β-hydroxy-DHEA and 16α-hydroxy-DHEA substrate utilization by the R195P-1 and P195R-2 enzymes provide further validation for higher affinity binding due to Arg195 in 3β-HSD1. Comparisons of the Michaelis-Menten values of cofactor and substrate for the targeted mutants of 3β-HSD1 (D61N, D61V, E192A, T8A) clarify the functions of these residues as well.
Thomas, James L.,Mack, Vance L.,Sun, Jingping,Terrell, J. Ross,Bucholtz, Kevin M.
scheme or table
p. 192 - 199
(2011/10/30)

Aldehyde dehydrogenase 1B1: Molecular cloning and characterization of a novel mitochondrial acetaldehyde-metabolizing enzyme
Ethanol-induced damage is largely attributed to its toxic metabolite, acetaldehyde. Clearance of acetaldehyde is achieved by its oxidation, primarily catalyzed by the mitochondrial class II aldehyde dehydrogenase (ALDH2). ALDH1B1 is another mitochondrial aldehyde dehydrogenase (ALDH) that shares 75% peptide sequence homology with ALDH2. Recent population studies in whites suggest a role for ALDH1B1 in ethanol metabolism. However, to date, no formal documentation of the biochemical properties of ALDH1B1 has been forthcoming. In this current study, we cloned and expressed human recombinant ALDH1B1 in Sf9 insect cells. The resultant enzyme was purified by affinity chromatography to homogeneity. The kinetic properties of purified human ALDH1B1 were assessed using a wide range of aldehyde substrates. Human ALDH1B1 had an exclusive preference for NAD + as the cofactor and was catalytically active toward short- and medium-chain aliphatic aldehydes, aromatic aldehydes, and the products of lipid peroxidation, 4-hydroxynonenal and malondialdehyde. Most importantly, human ALDH1B1 exhibited an apparent Km of 55 μM for acetaldehyde, making it the second low Km ALDH for metabolism of this substrate. The dehydrogenase activity of ALDH1B1 was sensitive to disulfiram inhibition, a feature also shared with ALDH2. The tissue distribution of ALDH1B1 in C57BL/6J mice and humans was examined by quantitative polymerase chain reaction, Western blotting, and immunohistochemical analysis. The highest expression occurred in the liver, followed by the intestinal tract, implying a potential physiological role for ALDH1B1 in these tissues. The current study is the first report on the expression, purification, and biochemical characterization of human ALDH1B1 protein. Copyright
Stagos, Dimitrios,Chen, Ying,Brocker, Chad,Donald, Elizabeth,Jackson, Brian C.,Orlicky, David J.,Thompson, David C.,Vasiliou, Vasilis
experimental part
p. 1679 - 1687
(2011/10/31)

NAD-malic enzymes of Arabidopsis thaliana display distinct kinetic mechanisms that support differences in physiological control
The Arabidopsis thaliana genome contains two genes encoding NAD-MEs [NAD-dependent malic enzymes; NAD-ME1 (TAIR accession number At4G13560) and NAD-ME2 (TAIR accession number At4G00570)]. The encoded proteins are localized to mitochondria and assemble as homo- and hetero- dimers in vitro and in vivo. In the present work, the kinetic mechanisms of NAD-ME1 and -ME2 homodimers and NAD-MEH (NAD-ME heterodimer) were studied as an approach to understand the contribution of these enzymes to plant physiology. Product-inhibition and substrate-analogue analyses indicated that NAD-ME2 follows a sequential ordered Bi-Ter mechanism, NAD being the leading substrate followed by L-malate. On the other hand, NAD-ME1 and NAD-MEH can bind both substrates randomly. However, NAD-ME1 shows a preferred route that involves the addition ofNADfirst.As a consequence of the kinetic mechanism, NAD-ME1 showed a partial inhibition by L-malate at low NAD concentrations. The analysis of a protein chimaeric forNAD-ME1 and -ME2 indicated that the first 176 amino acids are associated with the differences observed in the kinetic mechanisms of the enzymes. Furthermore, NAD-ME1, -ME2 and -MEH catalyse the reverse reaction (pyruvate reductive carboxylation) with very low catalytic activity, supporting the notion that these isoforms act only in L-malate oxidation in plant mitochondria. The different kinetic mechanism of each NAD-ME entity suggests that, for a metabolic condition in which the mitochondrial NAD level is low and the L-malate level is high, the activity of NAD-ME2 and/or -MEH would be preferred over that of NAD-ME1. The Authors.
Tronconi, Marcos A.,Gerrard Wheeler, Mariel C.,Maurino, Veronica G.,Drincovich, Maria F.,Andreo, Carlos S.
experimental part
p. 295 - 303
(2012/05/20)

Factors influencing the operational stability of NADPH-dependent alcohol dehydrogenase and an NADH-dependent variant thereof in gas/solid reactors
The continuous enzymatic gas/solid bio-reactor serves both for the production of volatile fine chemicals and flavors on an industrial scale and for thermodynamically controlled investigation of substrate and water effects on enzyme preparations for research purposes. Here, we comparatively investigated the molecular effects on the operational stability of NADPH-dependent Lactobacillus brevis alcohol dehydrogenase and an NADH-dependent variant thereof, LbADH G37D, in the gas/solid bioreactor. The reference reaction is the reduction of acetophenone to (R)-1-phenylethanol with concomitant oxidation of 2-propanol to acetone for the purpose of regeneration of the redox cofactor. It could be clearly shown that not the thermostability of the cofactor, but the thermostability of the proteins in the solid dry state govern the order of magnitude of the operational stability of both purified enzymes in the gas/solid reactor at low thermodynamic activity of water and substrate. However, at higher thermodynamic activity the operational stability in the gas/solid reactor is overlaid by stabilizing and destabilizing effects of the substrates that require further investigation. We demonstrated first evidence that the substrate affinity of the two variants in the gas/solid reactor is similar to the affinity in aqueous medium. We could also show that partial unfolding of the proteins with subsequent aggregation are the factors governing protein thermo-in-stability both in the dissolved and in the dry state. Thus, stability investigations of enzymes in the dry state are suggested to predict their basal level of operational stability in gas/solid reactions.
Kulishova, Liliya,Dimoula, Kerasina,Jordan, Max,Wirtz, Astrid,Hofmann, Diana,Santiago-Schuebel, Beatrix,Fitter, Joerg,Pohl, Martina,Spiess, Antje C.
experimental part
p. 271 - 283
(2011/02/22)

Discrimination of aliphatic substrates by a single Amino acid substitution in bacillus and bacillus shaericus phenylalanine dehydrogenases
Replacement of glycine by serine at positions 123 and 124 of phenylalanine dehydrogenases from Bacillus badius and Bacillus sphaericus respectively strikingly decreased enzyme activity toward aromatic amino acids and resulted in an elevation of relative activity toward aliphatic amino acids. The mutant from B. badius preferentially dehydrogenated branched-chain amino acids, while that from B. sphaericus acted on amino acids with straight-chain amino acids.
Tachibana, Shinjirao,Kuwamori, Yuko,Asano, Yasuhisa
body text
p. 729 - 732
(2010/04/26)

A combined experimental and theoretical study of the pH-dependent binding mode of NAD+ by water-soluble molecular clips
The highly selective recognition process of NAD+ and NADH (as important cofactors of many redox enzymes) by molecular clips in aqueous solution is studied systematically by a combined experimental and quantum-chemical approach. The strongly pH-
Polkowska, Jolanta,Bastkowski, Frank,Schrader, Thomas,Klaerner, Frank-Gerrit,Zienau, Jan,Koziol, Felix,Ochsenfeld, Christian
supporting information; scheme or table
p. 779 - 790
(2010/06/13)

Characterization of a dihydrolipoyl dehydrogenase having diaphorase activity of Clostridium kluyveri
The Clostridium kluyveri bfmBC gene encoding a putative dihydrolipoyl dehydrogenase (DLD; EC 1.8.1.4) was expressed in Escherichia coli, and the recombinant enzyme rBfmBC was characterized. UV-visible absorption spectrum and thin layer chromatography analysis of rBfmBC indicated that the enzyme contained a noncovalently but tightly attached FAD molecule. rBfmBC catalyzed the oxidation of dihydrolipoamide (DLA) with NAD+ as a specific electron acceptor, and the apparent Km values for DLA and NAD+ were 0.3 and 0.5 mM respectively. In the reverse reaction, the apparent K m values for lipoamide and NADH were 0.42 and 0.038 mM respectively. Like other DLDs, this enzyme showed NADH dehydrogenase (diaphorase) activity with some synthetic dyes, such as 2,6-dichlorophenolindophenol and nitro blue tetrazolium. rBfmBC was optimally active at 40°C at pH 7.0, and the enzyme maintained some activity after a 30-min incubation at 60°C.
Chakraborty, Saikat,Sakka, Makiko,Kimura, Tetsuya,Sakka, Kazuo
p. 982 - 988
(2008/09/21)

Inhibitors of inosine monophosphate dehydrogenase: Probes for antiviral drug discovery
The role of inosine monophosphate dehydrogenase (IMPDH) at the metabolic branch point of de novo purine nucleotide biosynthesis makes this enzyme an attractive probe for the discovery of antiviral compounds. Introduction of unsaturation at the 2-position of IMP, the natural substrate for IMPDH, produces Michael acceptors at that position, which results in these compounds being inhibitors of IMPDH. Consistent with this mechanism-based molecular design, some of the parent nucleosides exhibited antiviral activity. Copyright Taylor & Francis, Inc.
Story, Sherry,Gupta, Mukta,Bonsu, Eric,Nair, Vasu
p. 717 - 720
(2007/10/03)

Purification, characterization, and overexpression of psychrophilic and thermolabile malate dehydrogenase of a novel antarctic psychrotolerant, Flavobacterium frigidimaris KUC-1
We purified the psychrophilic and thermolabile malate dehydrogenase to homogeneity from a novel psychrotolerant, Flavobacterium frigidimaris KUC-1, isolated from Antarctic seawater. The enzyme was a homotetramer with a molecular weight of about 123 k and that of the subunit was about 32 k. The enzyme required NAD(P)+ as a coenzyme and catalyzed the oxidation of L-malate and the reduction of oxalacetate specifically. The reaction proceeded through an ordered bi-bi mechanism. The enzyme was highly susceptible to heat treatment, and the half-life time at 40°C was estimated to be 3.0 min. The kcat/Km (μM-1·s-1) values for L-malate and NAD+ at 30°C were 289 and 2,790, respectively. The enzyme showed pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of the coenzyme. The enzyme contained 311 amino acid residues and much lower numbers of proline and arginine residues than other malate dehydrogenases.
Oikawa, Tadao,Yamamoto, Noriko,Shimoke, Koji,Uesato, Shinichi,Ikeuchi, Toshihiko,Fujioka, Toru
p. 2146 - 2154
(2008/02/03)