- Oxygen Activation and Electron Transfer in Flavocytochrome P450 BM3
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In flavocytochrome P450 BM3, there is a conserved phenylalanine residue at position 393 (Phe393), close to Cys400, the thiolate ligand to the heme. Substitution of Phe393 by Ala, His, Tyr, and Trp has allowed us to modulate the reduction potential of the heme, while retaining the structural integrity of the enzyme's active site. Substrate binding triggers electron transfer in P450 BM3 by inducing a shift from a low- to high-spin ferric heme and a 140 mV increase in the heme reduction potential. Kinetic analysis of the mutants indicated that the spin-state shift alone accelerates the rate of heme reduction (the rate determining step for overall catalysis) by 200-fold and that the concomitant shift in reduction potential is only responsible for a modest 2-fold rate enhancement. The second step in the P450 catalytic cycle involves binding of dioxygen to the ferrous heme. The stabilities of the oxy-ferrous complexes in the mutant enzymes were also analyzed using stopped-flow kinetics. These were found to be surprisingly stable, decaying to superoxide and ferric heme at rates of 0.01-0.5 s-1. The stability of the oxy-ferrous complexes was greater for mutants with higher reduction potentials, which had lower catalytic turnover rates but faster heme reduction rates. The catalytic rate-determining step of these enzymes can no longer be the initial heme reduction event but is likely to be either reduction of the stabilized oxy-ferrous complex, i.e., the second flavin to heme electron transfer or a subsequent protonation event. Modulating the reduction potential of P450 BM3 appears to tune the two steps in opposite directions; the potential of the wild-type enzyme appears to be optimized to maximize the overall rate of turnover. The dependence of the visible absorption spectrum of the oxy-ferrous complex on the heme reduction potential is also discussed.
- Ost, Tobias W. B.,Clark, Jonathan,Mowat, Christopher G.,Miles, Caroline S.,Walkinshaw, Malcolm D.,Reid, Graeme A.,Chapman, Stephen K.,Daff, Simon
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- The Role of Adsorption in the Initial One-Electron Electrochemical Reduction of Nicotinamide Adenine Dinucleotide (NAD+)
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The title reduction was investigated at pH 9.1 in several base electrolytes of varying surface activity in order to elucidate the role of adsorption of NAD+, its free radical, and the resulting dimer at the aqueous solution/mercury electrode interface.In the presence of electrolytes of low activity, NAD+ is adsorbed at potentials positive to its 1-e reduction (ca. -0.9 V vs.SCE); the electrochemically generated dimer, (NAD)2, is adsorbed positive of -1.20 and -1.32 V in 0.06 and 0.4 M KCl solutions, respectively.NAD+ undergoes both diffusion- and adsorption-controlled reduction; the former predominates on slow time scale experiments and the latter on fast time scales.From low concentration surfactant (0.06 and 0.1 M tetraethylammonium (Tea+) chloride) solutions, NAD+ is only adsorbed positive of -0.65 V and an adsorption-controlled prewave appears, indicating that an adsorbed layer of NAD. and/or (NAD)2 is formed on reduction of dissolved NAD+.From a high concentration (0.4 M) Tea+ solution, NAD+ is adsorbed positive of -0.66 V, but the adsorption-controlled prewave is suppressed and the reduction is entirely diffusion controlled.Under diffusion control, the heterogeneous rate constant for the title reduction is ca. 0.1 cm s-1 and the rate constant for dimerization of NAD. is ca. 3E6 M-1 s-1.
- Bresnahan, William T.,Elving, Philip J.
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- The Peroxidase-NADH Biochemical Oscillator. 1. Examination of Oxygen Mass Transport, the Effect of Light, and the Role of Methylene Blue
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The peroxidase-NADH oscillator examined here initially consists of four chemical components.The well-mixed aqueous solution includes native horseradish peroxidase, reduced β-nicotinamide adenine dinucleotide (NADH), methylene blue (MB+), and dissolved oxygen combined in a semi-batch reactor under a set of standard conditions.In this system, the macroscopic appearance of the process of oxygen dissolution from the gas phase is dependent on k-m, the mass transport constant of oxygen out of solution.Additional details of oxygen mass transport are derived.The amplitude of oxygen oscillations is decreased by continuous illumination by the deuterium source of a diode array spectrophotometer.This attenuation effect of light is dependent on wavelengths =+ allows several damped oscillations of small amplitude.Subsequent addition of MB+ to the oscillator results in oscillations of much larger amplitude.MB+ is seen to either directly or indirectly enhance the conversion of peroxidase compound III to the native enzyme and then inhibit oxygen consumption, allowing the initiation of relatively large, prolonged oscillations.MB+ is seen to function either as a system catalyst, or as a peroxidase inhibitor in the oxidation of NADH by oxygen.
- Olson, Dean L.,Scheeline, Alexander
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- NADH oxidase activity of Bacillus subtilis nitroreductase NfrA1: Insight into its biological role
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NfrA1 nitroreductase from the Gram-positive bacterium Bacillus subtilis is a member of the NAD(P)H/FMN oxidoreductase family. Here, we investigated the reactivity, the structure and kinetics of NfrA1, which could provide insight into the unclear biological role of this enzyme. We could show that NfrA1 possesses an NADH oxidase activity that leads to high concentrations of oxygen peroxide and an NAD+ degrading activity leading to free nicotinamide. Finally, we showed that NfrA1 is able to rapidly scavenge H2O2 produced during the oxidative process or added exogenously. Structured summary: MINT- 7990140: nfrA1 (uniprotkb:. P39605) and nfrA1 (uniprotkb:. P39605) bind (MI:. 0407) by X-ray crystallography (MI:. 0114).
- Cortial, Sylvie,Chaignon, Philippe,Iorga, Bogdan I.,Aymerich, Stephane,Truan, Gilles,Gueguen-Chaignon, Virginie,Meyer, Philippe,Morera, Solange,Ouazzani, Jamal
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- Rate-limiting one-electron transfer in the oxidation of NADH by polyoxometalates
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The kinetics of NADH oxidation by 3 Dawson-type mixed heteropolyanions were studied in buffered aqueous pH = 7 medium, by the stopped flow technique and UV-visible spectroscopy.The log of k was a linear function of the E0 of the first redox systems of the heteropolyanions with a slope of 16.5 V-1.The results indicate that, in the present case, the oxidation of NADH proceeds by a multistep mechanism involving initial rate-limiting one-electron transfer.An estimate of the E0 value for the one-electron NADH/NADHcation radical couple has been obtained.
- Keita, Bineta,Essaadi, Khalid,Nadjo, Louis,Desmadril, Michel
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- The Peroxidase-NADH Biochemical Oscillator. 2. Examination of the Roles of Hydrogen Peroxide and Superoxide
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The peroxidase-NADH oscillator examined here initially consists of a well-mixed aqueous solution of native horseradish peroxidase, reduced β-nicotinamide adenine dinucleotide (NADH), methylene blue (MB+), and dissolved oxygen combined in a semi-batch reactor under a set of standard conditions.Hydrogen peroxide and superoxide have been implicated as important chemical intermediates.A comprehensive model which includes such intermediates and all initial chemical species has appeared elsewhere.To experimentally explore the role of hydrogen peroxide in the oscillator, H2O2 was substituted for MB+ as an initial ingredient.This substitution allows relatively small, quasi-sinusoidal oscillations sensitive to the oxygen mass transport constant, and predicted earlier in a theoretical model.The oscillations become much larger when MB+ is added, suggesting that MB+ might serve as a chemical mediator between the small oscillations seen when H2O2 is substituted for MB+, and the relatively large oscillations observed when MB+ is present.Catalase and superoxide dismutase are used as enzymatic scavengers for H2O2 and O2.-, respectively.The enzymes are added individually to a working oscillator at oxygen minima and maxima to examine the roles and approximate the concentrations of H2O2 and O2.-.For the enzyme addition experiments, a perturbation model for oxygen behavior is proposed and applied to the interpretation of experimental data.Two methods of analysis for the addition of the enzyme probes indicate a higher concentration of H2O2 and O2.- at oxygen maxima than at minima.Comparison of experimental and simulated data indicate that the relatively simple model presented here is a resonable, yet apparently incomplete, representation of oxygen dynamics for the addition of scavenger enzymes to this oscillator.
- Olson, Dean L.,Scheeline, Alexander
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- Formation of pyridine nucleotides under symbiotic and non-symbiotic conditions between soybean nodules and free-living rhizobia
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Enzymatic regulation of pyricline nucleotide formation, under symbiotic and non-symbiotic conditions, was analyzed using soybeans (Glycine max L, cv. 'Akisengoku') and rhizobia (Bradyrhizobia japonicum strain A1017), respectively. It was found that levels
- Tezuka, Takafumi,Murayama, Yuko
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- Poly(aniline)-poly(acrylate) composite films as modified electrodes for the oxidation of NADH
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Poly(aniline), electrochemically deposited on an electrode surface in the presence of poly(acrylic acid), forms a film which remains protonated, and conducting, at pH 7. The resulting modified electrode is an electrocatalytic surface for NADH oxidation at +0.05 V vs. SCE in 0.1 M citrate-phosphate buffer at pH 7. The amperometric responses of these composite poly(aniline) films for NADH oxidation were studied in detail and fitted to a kinetic model in which the NADH diffuses into the polymer film and then binds to catalytic sites within the film where it undergoes reduction to NAD+. The rate determining process depends on the concentration of NADH present and the polymer film thickness. A comparison of the results presented here for the poly(aniline)-poly(acrylate) films with earlier work on poly(aniline)-poly(vinylsulfonate) films shows that the currents obtained for NADH at these poly(aniline)-poly(acrylate) films are approximately one third of those obtained for the poly(aniline)-poly(vinylsulfonate) films under similar conditions, that the currents saturate at lower NADH concentration and that the response is less stable towards repeated measurements. The poly(aniline)-poly(acrylate) films are, however, less readily inhibited by NAD+ and possess the potential advantage that the carboxylate groups can be used as sites for chemical attachment of enzymes or NADH derivatives by using simple coupling reactions.
- Bartlett, Philip N.,Simon, Evelyne
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- Altering the substrate specificity of glutamate dehydrogenase from Bacillus subtilis by site-directed mutagenesis
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The Lys80, Gly82 and Met101 residues of glutamate dehydrogenase from Bacillus subtilis were mutated into a series of single mutants. The wild-type enzyme was highly specific for 2-oxoglutarate, whereas G82K and M101S dramatically switched to increased specificity for oxaloacetate with k cat values 3.45 and 5.68s-1, which were 265-fold and 473-fold higher respectively than those for 2-oxoglutarate.
- Khan, Md. Iqbal Hassan,Kim, Hyeung,Ashida, Hiroyuki,Ishikawa, Takahiro,Shibata, Hitoshi,Sawa, Yoshihiro
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- Switching the Mechanism of NADH Photooxidation by Supramolecular Interactions
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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.
- Mengele, Alexander K.,Weixler, Dominik,Chettri, Avinash,Maurer, Maite,Huber, Fabian Lukas,Seibold, Gerd M.,Dietzek, Benjamin,Eikmanns, Bernhard J.,Rau, Sven
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supporting information
p. 16840 - 16845
(2021/10/25)
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- Emissive Synthetic Cofactors: A Highly Responsive NAD+ Analogue Reveals Biomolecular Recognition Features
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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
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p. 4379 - 4389
(2019/03/07)
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- Fluorescent half-sandwich phosphine-sulfonate iridium(III) and ruthenium(II) complexes as potential lysosome-targeted anticancer agents
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The synthesis, characterization and biological activity of neutral fluorescent Ir(III) and Ru(II) half-sandwich organometallic complexes containing phosphine-sulfonate ligands are reported. X-ray crystal structure of complexes 1–3, 10 and 11 exhibits the expected half-sandwich “three-legged piano-stool” pseudo-octahedral geometry. Spectroscopic properties study displays that these complexes show rich fluorescence properties. With the exception of 9, 10 and 11 toward A549 human lung cancer cells and 10 towards HeLa human cervical cancer cells, each complex shows promising cytotoxicity toward HeLa and A549 cells line with IC50 values in the range of 3.6–53.1 μM, and 6.5–34.5 μM, respectively. Hydrolysis, DNA cleavage and depolarization of the mitochondrial membrane potential (MMP) appear not to be the main mechanism of action. However, these complexes are able to covert NADH to NAD+ via the transfer hydrogenation. Mechanism studies by flow cytometry display that the complexes exert their anticancer efficacy by inducing apoptosis, perturbing the cell cycle and increasing the intracellular ROS level. Furthermore, fluorescence property of these complexes provides a tool to investigate the microscopic mechanism by confocal microscopy. Notably, the typical Ir(III) complex 3 can specially localize to lysosome and damage it. In addition, complex 3 enters into HeLa cells mainly through energy-dependent pathway.
- Du, Qing,Yang, Yuliang,Guo, Lihua,Tian, Meng,Ge, Xingxing,Tian, Zhenzhen,Zhao, Liping,Xu, Zhishan,Li, Juanjuan,Liu, Zhe
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p. 821 - 830
(2018/11/23)
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- Organometallic ruthenium and iridium phosphorus complexes: Synthesis, cellular imaging, organelle targeting and anticancer applications
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The use of metal complexes containing phosphorus ligands as anticancer agents has not been well studied. In this work, eight novel half-sandwich IrIII and RuII compounds with P^P-chelating ligands have been synthesized and fully characterized, and alongside two crystal structures were reported. All eight complexes displayed highly potent antiproliferative activity, up to nine times more potent than the clinical anticancer drug cisplatin towards A549 lung cancer cells. Complex Ir1, which has a simpler structure and highly potent antiproliferative activity, was selected to investigate in further mechanistic studies. No hydrolysis and nucleobase binding occurred for complex Ir1. In order to elucidate subcellular localization, the self-luminescence of the complex Ir1 was utilized. Ir1 can specifically target lysosomes and facilitate excessive production of reactive oxygen species, resulting in lysosomal membrane permeabilization in A549 cells. Release of cathepsin B and changes in the mitochondria membrane potential also contributed to the observed cytotoxicity of Ir1, which demonstrated an anticancer action mechanism that was different from that of cisplatin. The favorable results from biological and chemical research demonstrated that these types of complexes hold significant theranostic potential.
- Li, JuanJuan,Tian, Zhenzhen,Zhang, Shumiao,Xu, Zhishan,Mao, Xudong,Zhou, Yumin,Liu, Zhe
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- Toward Automated Enzymatic Glycan Synthesis in a Compartmented Flow Microreactor System
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Immobilized microfluidic enzyme reactors (IMER) are of particular interest for automation of enzyme cascade reactions. Within an IMER, substrates are converted by paralleled immobilized enzyme modules and intermediate products are transported for further conversion by subsequent enzyme modules. By optimizing substrate conversion in the spatially separated enzyme modules purification of intermediate products is not necessary, thus shortening process time and increasing space-time yields. The IMER enables the development of efficient enzyme cascades by combining compatible enzymatic reactions in different arrangements under optimal conditions and the possibility of a cost-benefit analysis prior to scale-up. These features are of special interest for automation of enzymatic glycan synthesis. We here demonstrate a compartmented flow microreactor system using six magnetic enzyme beads (MEBs) for the synthesis of the non-sulfated human natural killer cell-1 (HNK-1) glycan epitope. MEBs are assembled to build compartmented enzyme modules, consisting of enzyme cascades for the synthesis of uridine 5′- diphospho-α- d-galactose (UDP-Gal) and uridine 5′-diphospho-α-d-glucuronic acid (UDP-GlcA), the donor substrates for the Leloir glycosyltransferases β4-galactosyltransferase and β3-glucuronosyltransferase, respectively. Glycan synthesis was realized in an automated microreactor system by a cascade of individual enzyme module compartments each performing under optimal conditions. The products were analyzed inline by an MS-system connected to the microreactor. The high synthesis yield of 96% for the non-sulfated HNK-1 glycan epitope indicates the excellent performance of the automated enzyme module cascade. Furthermore, combinations of other MEBs for nucleotide sugars synthesis with MEBs of glycosyltransferases have the potential for a fully automated and programmed glycan synthesis in a compartmented flow microreactor system. (Figure presented.).
- Heinzler, Raphael,Fisch?der, Thomas,Elling, Lothar,Franzreb, Matthias
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supporting information
p. 4506 - 4516
(2019/08/20)
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- Ferrocene-appended iridium(III) Complexes: Configuration regulation, anticancer application, and mechanism research
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A series of ferrocene-appended half-sandwiched iridium(III) phenylpyridine complexes have been designed and synthesized. These complexes show better anticancer activity than cisplatin widely used in clinic under the same conditions. Meanwhile, complexes could effectively inhibit cell migration and colony formation. Complexes could interact with protein and transport through serum protein, effectively catalyzing the oxidation of nicotinamide-adenine dinucleotid and inducing the accumulation of reactive oxygen species (ROS, 1O2), which confirmed the anticancer mechanism of oxidation. Furthermore, laser scanning confocal detection indicates that these complexes can enter cells followed by a non-energy-dependent cellular uptake mechanism, effectively accumulating in the lysosome (Pearson's colocalization coefficient: ～0.90), leading to lysosome damage, and reducing the mitochondrial membrane potential (MMP). Taken together, ferrocene-appended iridium(III) complexes possess the prospect of becoming a new multifunctional therapeutic platform, including lysosome-targeted imaging and anticancer drugs.
- Ge, Xingxing,Chen, Shujiao,Liu, Xicheng,Wang, Qinghui,Gao, Lijun,Zhao, Chengfeng,Zhang, Lei,Shao, Mingxiao,Yuan, Xiang-Ai,Tian, Laijin,Liu, Zhe
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supporting information
p. 14175 - 14184
(2019/10/16)
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- MftD Catalyzes the Formation of a Biologically Active Redox Center in the Biosynthesis of the Ribosomally Synthesized and Post-translationally Modified Redox Cofactor Mycofactocin
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Mycofactocin (MFT) is a putative ribosomally synthesized and post-translationally modified (RiPP) redox cofactor. The biosynthesis of MFT is encoded by the gene cluster mftABCDEF. While processing of the precursor peptide by MftB, MftC, and MftE has been shown to result in the formation of the small molecule 3-amino-5-[(p-hydroxyphenyl)methyl]-4,4-dimethyl-2-pyrrolidinone (AHDP), no activity has been shown for the putative dehydrogenase MftD and the putative glycosyltransferase MftF. In addition, evidence demonstrating that MFT is a redox cofactor has only been limited to the requirement of mft genes for ethanol assimilation in Mycobacterium smegmatis mc2155. Here, we demonstrate that MftD catalyzes the oxidative deamination of AHDP, forming an α-keto moiety on the resulting molecule, which we call pre-mycofactocin (PMFT). We characterize PMFT by 1D and 2D NMR spectroscopy techniques and by high-resolution mass spectrometry data to solve its structure. We further characterized PMFT by cyclic voltammetry and found its midpoint potential to be ～255 mV. Lastly, we demonstrate that PMFT is a biologically active redox cofactor that oxidizes NADH bound by M. smegmatis carveol dehydrogenase (MsCDH) and can be used by MsCDH in the oxidation of carveol. These data demonstrate for the first time that PMFT functions as a biologically active redox mediator and provides the most direct evidence to date that MFT is a RiPP-derived redox cofactor.
- Ayikpoe, Richard S.,Latham, John A.
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- Emissive Synthetic Cofactors: Enzymatic Interconversions of tzA Analogues of ATP, NAD+, NADH, NADP+, and NADPH
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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
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supporting information
p. 1087 - 1090
(2017/12/28)
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- Half-sandwich Iridium(III) Benzimidazole-Appended Imidazolium-Based N-heterocyclic Carbene Complexes and Antitumor Application
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A series of half-sandwich iridium(III) benzimidazole-appended imidazolium-based N-heterocyclic carbene (NHC) antitumor complexes [(η5-Cpx)Ir(C^N)Cl]Cl, where Cpx is pentamethylcyclopentadienyl (Cp*) or its biphenyl derivative (Cpxbiph) and C^N is a NHC chelating ligand, were successfully synthesized and characterized. The IrIII complexes showed potential antitumor activity against A549 cells, at most three times more potent than cis-platin under the same conditions. Complexes could bind to BSA by a static quenching mode, catalyzing the change of NADH to NAD+ and inducing the production of reactive oxygen species (maximum turnover number, 9.8), which play an important role in regulating cell apoptosis. Confocal microscopy showed that the complexes could specifically target lysosomes in cells with a Pearson's co-localization coefficient 0.76 and 0.72 after 1 h and 6 h, respectively, followed an energy-dependent cellular uptake mechanism and damaged the integrity of lysosomes. At the same time, complexes caused a marked loss of mitochondrial membrane potential.
- Han, Yali,Liu, Xicheng,Tian, Zhenzhen,Ge, Xingxing,Li, Juanjuan,Gao, Min,Li, Yanru,Liu, Yi,Liu, Zhe
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supporting information
p. 3697 - 3705
(2018/11/03)
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- Photobiocatalytic alcohol oxidation using LED light sources
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The photocatalytic oxidation of NADH using a flavin photocatalyst and a simple blue LED light source is reported. This in situ NAD+ regeneration system can be used to promote biocatalytic, enantioselective oxidation reactions. Compared to the traditional use of white light bulbs this method enables very significant reductions in energy consumption and CO2 emission.
- Rauch,Schmidt,Arends,Oppelt,Kara,Hollmann
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supporting information
p. 376 - 379
(2017/08/14)
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- Novel half-sandwich iridium(iii) imino-pyridyl complexes showing remarkable: In vitro anticancer activity
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Seven novel half-sandwich IrIII cyclopentadienyl complexes, [(η5-Cpx)Ir(N^N)Cl]PF6, have been prepared and characterized, where Cpx is Cp? or the biphenyl derivative Cpxbiph (C5Me4C6H4C6H5), and the N^N-chelating ligands are imino-pyridyl Schiff-bases. The X-ray crystal structures of complexes 2A, 2B, and 3A have been determined. Excitingly, most of the complexes show potent antiproliferative activity towards A549 and HeLa cancer cells, except for Cp? complex 1A towards HeLa cells. Cpxbiph complex 2B displayed the highest potency, about 19 and 6 times more active than the clinically used drug cisplatin toward A549 and HeLa cells, respectively. These complexes undergo hydrolysis, and the kinetics data have been calculated. DNA binding has been studied by interaction with nucleobases 9-ethylguanine and 9-methyladenine, cleavage of plasmid DNA, and interaction with ctDNA. Interaction with DNA does not appear to be the major mechanism of action. Protein binding (bovine serum albumin, BSA) has been established by UV-Vis, fluorescence and synchronous spectroscopic studies. The stability of complex 2B in the presence of GSH was evaluated. The complexes catalytically convert coenzyme NADH to NAD+via hydride transfer. Cpxbiph complexes 2B and 4B induce cell apoptosis and arrest cell cycles at the S and G2/M phases towards A549 cancer cells and increase the reactive oxygen species dramatically, which appear to contribute to the remarkable anticancer activity.
- Li, Juanjuan,Guo, Lihua,Tian, Zhenzhen,Tian, Meng,Zhang, Shumiao,Xu, Ke,Qian, Yuchuan,Liu, Zhe
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supporting information
p. 15520 - 15534
(2017/11/22)
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- Contrasting Anticancer Activity of Half-Sandwich Iridium(III) Complexes Bearing Functionally Diverse 2-Phenylpyridine Ligands
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We report the synthesis, characterization, and antiproliferative activity of 15 iridium(III) half-sandwich complexes of the type [(η5-Cp?)Ir(2-(R′-phenyl)-R-pyridine)Cl] bearing either an electron-donating (-OH, -CH2OH, -CH3) or electron-withdrawing (-F, -CHO, -NO2) group at various positions on the 2-phenylpyridine (2-PhPy) chelating ligand giving rise to six sets of structural isomers. The X-ray crystal structures of [(η5-Cp?)Ir(2-(2′-fluorophenyl)pyridine)Cl] (1) and [(η5-Cp?)Ir(2-(4′-fluorophenyl)pyridine)Cl] (2) exhibit the expected "piano-stool" configuration. DFT calculations showed that substituents caused only localized effects on the electrostatic potential surface of the chelating 2-PhPy ligand of the complexes. Hydrolysis of all complexes is rapid, but readily reversed by addition of NaCl. The complexes show preferential binding to 9-ethylguanine over 9-methyladenine and are active catalysts for the oxidation of NADH to NAD+. Antiproliferative activity experiments in A2780 ovarian, MCF-7 breast, A549 lung, and HCT116 colon cancer cell lines showed IC50 values ranging from 1 to 89 μM, with the most potent complex, [(η5-Cp?)Ir(2-(2′-methylphenyl)pyridine)Cl] (13) (A2780 IC50 = 1.18 μM), being 10× more active than the parent, [(η5-Cp?)Ir(2-phenylpyridine)Cl], and 2× more active than [(η5-CpxPh)Ir(2-phenylpyridine)Cl]. Intriguingly, contrasting biological activities are observed between structural isomers despite exhibiting similar chemical reactivity. For pairs of structural isomers both the nature and position of the functional group can affect the hydrophobicity of the complex. An increase in hydrophobicity resulted in enhanced cellular-iridium accumulation in A2780 ovarian cells, which generally gave rise to an increase in potency. The structural isomers [(η5-Cp?)Ir(2-(4′-fluorophenyl)pyridine)Cl] (2) and [(η5-Cp?)Ir(2-phenyl-5-fluoropyridine)Cl] (4) preferentially localized in the cytosol > membrane and particulate > nucleus > cytoskeleton. This work highlights the strong dependence of biological behavior on the nature and position of the substituent on the chelating ligand and shows how this class of organometallic anticancer complexes can be fine-tuned to increase their potency without using extended cyclopentadienyl systems. (Chemical Equation Presented).
- Millett, Adam J.,Habtemariam, Abraha,Romero-Canelón, Isolda,Clarkson, Guy J.,Sadler, Peter J.
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p. 2683 - 2694
(2015/06/23)
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- Light-accelerated biocatalytic oxidation reactions
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The influence of (visible) light on the performance of laccase mediator systems (LMS) was investigated. As a model reaction, LMS-mediated nicotinamide adenine dinucleotide (NAD+) regeneration to promote alcohol dehydrogenase catalyzed oxidation reactions was examined.
- Kochius, Svenja,Ni, Yan,Kara, Selin,Gargiulo, Serena,Schrader, Jens,Holtmann, Dirk,Hollmann, Frank
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p. 1554 - 1557
(2015/02/05)
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- Expanding the scope of laccase-mediator systems
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The laccase-mediator system (LMS) for the regeneration of oxidised nicotinamide co-factors was revisited to broaden the mediator scope. Among the 18 mediators screened, acetosyringone, syringaldehyde and caffeic acid excelled with respect to activity and stability under process conditions. The LMS based on the laccase from Myceliophthora thermophila and acetosyringone was further investigated and applied to promote the nicotinamide adenine dinucleotide (NAD+)-dependent oxidation of glucose as well as the oxidative lactonisation of 1,4-butanediol to the corresponding γ-butyrolactone. Laccase gets a look-in: The laccase-mediator system (LMS) for the regeneration of oxidized nicotinamide co-factors is revisited to broaden the mediator scope. The LMS based on the laccase from Myceliophthora thermophila and acetosyringone is applied to promote the oxidation of glucose and the oxidative lactonization of 1,4-butanediol to the corresponding γ-butyrolactone.
- Koenst, Paul,Kara, Selin,Kochius, Svenja,Holtmann, Dirk,Arends, Isabel W. C. E.,Ludwig, Roland,Hollmann, Frank
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p. 3027 - 3032
(2013/10/21)
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- MONONUCLEAR METAL COMPLEX, HYDROGENATION REDUCTION CATALYST, DEHYDROGENATION CATALYST, METHOD FOR PRODUCING HYDROGENATION REDUCTION PRODUCT, METHOD FOR PRODUCING HYDROGEN (H2), AND METHOD FOR PRODUCING DEHYDROGENATION REACTION
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The present invention provides: a mononuclear metal complex that has high catalytic activity and can be used as a hydrogenation reduction catalyst that allows efficient hydrogenation reduction of a substance to be reduced; a tautomer or stereoisomer thereof; or a salt thereof. Provided is the mononuclear metal complex represented by the following formula (1), a tautomer or stereoisomer thereof; or a salt thereof. In the formula (1), Ar1 is an aromatic anionic ligand or an aromatic ligand, or is not present, Ar2 is a ligand having aromaticity and may or may not be substituted, and when Ar2 is substituted, the number of substituents may be one or more, M is an atom or ion of a transition metal, A1 and A2 are both carbon atoms, or one of A1 and A2 is a carbon atom and the other is a nitrogen atom, Y is an anionic group or a cationic group, or is not present, L is any ligand or is not present, and m is a positive integer, 0, or a negative integer.
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Page/Page column 37-38
(2013/02/27)
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- Substitutions of coenzyme-binding, nonpolar residues improve the low-temperature activity of thermophilic dehydrogenases
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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
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experimental part
p. 8583 - 8593
(2012/07/27)
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- The functions of key residues in the inhibitor, substrate and cofactor sites of human 3β-hydroxysteroid dehydrogenase type 1 are validated by mutagenesis
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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.
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scheme or table
p. 192 - 199
(2011/10/30)
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- Characterization of a dihydrolipoyl dehydrogenase having diaphorase activity of Clostridium kluyveri
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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
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p. 982 - 988
(2008/09/21)
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- Molecular properties and enhancement of thermostability by random mutagenesis of glutamate dehydrogenase from Bacillus subtilis
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The rocG gene encoding glutamate dehydrogenase from Bacillus subtilis (Bs-GluDH) was cloned, and expressed at considerable magnitude in Escherichia coli. The recombinant Bs-GluDH was purified to homogeneity and has been determined to have a hexameric structure (Mr 270 kDa) with strict specificity for 2-oxoglutarate and L-glutamate, requiring NADH and NAD + as cofactors respectively. The enzyme showed low thermostability with Tm = 41°C due to dissociation of the hexamer. To improve the thermostability of this enzyme, we performed error-prone PCR, introducing random mutagenesis on cloned GluDH. Two single mutant enzymes, Q144R and E27F, were isolated from the final mutant library. Their Tm values were 61°C and 49°C respectively. Furthermore, Q144R had a remarkably high kcat value (435 s-1) for animation reaction at 37°C, 1.3 times higher than that of the wild-type. Thus, Q144R can be used as a template gene to modify the substrate specificity of Bs-GluDH for industrial use.
- Khan, Md. Iqbal Hassan,Ito, Kousuke,Kim, Hyeung,Ashida, Hiroyuki,Ishikawa, Takahiro,Shibata, Hitoshi,Sawa, Yoshihiro
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p. 1861 - 1870
(2008/02/04)
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- Borate-nucleotide complex formation depends on charge and phosphorylation state
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Flow injection analysis with electrospray ionization mass spectrometry was used to investigate borate-nucleotide complex formation. Solutions containing 100 μM nucleotide and 500 μM boric acid in water-acetonitrile-triethylamine (50:50:0.2, v/v/v; pH 10.3
- Kim, Danny H.,Faull, Kym F.,Norris, Andrew J.,Eckhert, Curtis D.
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p. 743 - 751
(2007/10/03)
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- Measurement of the kinetic isotope effect for the oxidation of NADH at a poly(aniline)-modified electrode
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Kinetic isotope measurements using [4,4-2H2]NADH and [4-1H, 4-2H]NADH have been used to investigate the mechanism of the electrochemical oxidation of NADH at poly(aniline)-poly(vinyl sulfonate)-modified electrodes. The experiments show a primary kinetic isotope effect for the reaction of 4.2. This is consistent with literature values for the corresponding isotope effect for the oxidation of NADH by two-electron oxidants in homogeneous solution. The result demonstrates that transfer of H from NADH to the modified electrode occurs in the rate-limiting step within the reaction complex. Copyright
- Bartlett, Philip N.,Simon, Evelyne
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p. 4014 - 4015
(2007/10/03)
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- Reaction of cyclic nitroxides with nitrogen dioxide: The intermediacy of the oxoammonium cations
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Piperidine and pyrrolidine nitroxides, such as 2,2,6,6-tetramethylpiperidinoxyl (TPO) and 3-carbamoylproxyl (3-CP), respectively, are cell-permeable stable radicals, which effectively protect cells, tissues, isolated organs, and laboratory animals from radical-induced damage. The kinetics and mechanism of their reactions with .OH, superoxide, and carbon-centered radicals have been extensively studied, but not with .NO2, although the latter is a key intermediate in cellular nitrosative stress. In this research, .NO2 was generated by pulse radiolysis, and its reactions with TPO, 4-OH-TPO, 4-oxo-TPO, and 3-CP were studied by fast kinetic spectroscopy, either directly or by using ferrocyanide or 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate), which effectively scavenge the product of this reaction, the oxoammonium cation. The rate constants for the reactions of .NO2 with these nitroxides were determined to be (7-8) × 108 M-1 s-1, independent of the pH over the range 3.9-10.2. These are among the highest rate constants measured for .NO2 and are close to that of the reaction of .NO2 with .NO, that is, 1.1 × 109 M-1 s-1. The hydroxylamines TPO-H and 4-OH-TPO-H are less reactive toward .NO2, and an upper limit for the rate constant for these reactions was estimated to be 1 × 105 M-1 s-1. The kinetics results demonstrate that the reaction of nitroxides with .NO2 proceeds via an inner-sphere electron-transfer mechanism to form the respective oxoammonium cation, which is reduced back to the nitroxide through the oxidation of nitrite to .NO2. Hence, the nitroxide slows down the decomposition of .NO2 into nitrite and nitrate and could serve as a reservoir of .NO2 unless the respective oxoammonium is rapidly scavenged by other reductant. This mechanism can contribute toward the protective effect of nitroxides against reactive nitrogen-derived species, although the oxoammonium cations themselves might oxidize essential cellular targets if they are not scavenged by common biological reductants, such as thiols.
- Goldstein, Sara,Samuni, Amram,Russo, Angelo
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p. 8364 - 8370
(2007/10/03)
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- Cofactor Regeneration of NAD+ from NADH: Novel Water-Forming NADH Oxidases
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Dehydrogenases with their superb enantioselectivity can be employed advantageously to prepare enantiomerically pure alcohols, hydroxy acids, and amino acids. For economic syntheses, however, the co-substrate of dehydrogenases, the NAD(P)(H) cofactor, has to be regenerated. Whereas the problem of regenerating NADH from NAD+ can be considered solved, the inverse problem of regenerating NAD+ from NADH still awaits a definitive and practical solution. A possible solution is the oxidation of NADH to NAD+ with concomitant reduction of oxygen catalyzed by NADH oxidase (E.C. 1.6.-.-) which can reduce O2 either to undesirable H2O2 or to innocuous H2O. We have found and cloned two novel genes from Borrelia burgdorferi and Lactobacillus sanfranciscensis with hitherto only machine-annotated NADH oxidase function. We have overexpressed the corresponding proteins and could prove the annotated function to be correct. As demonstrated with a more sensitive assay than employed previously, the two novel NADH oxidases reduce O2 to H2O.
- Riebel, Bettina R.,Gibbs, Phillip R.,Wellborn, William B.,Bommarius, Andreas S.
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p. 1156 - 1168
(2007/10/03)
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- A chemical synthesis of nicotinamide adenine dinucleotide (NAD+)
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A practical synthesis of nicotinamide mononucleotide (β-NMN) and a high yield coupling with AMP-morpholidate that also provides NAD+ in an efficient manner are reported.
- Lee, Jaemoon,Churchil, Hywyn,Choi, Woo-Baeg,Lynch, Joseph E.,Roberts,Volante,Reider, Paul J.
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p. 729 - 730
(2007/10/03)
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- Oxidative activity of organometallic compounds in relation to the coenzymes NADH and NADPH
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Kinetic investigations of the interaction of Hg and Sn-containing organometallic compounds with NADH and NADPH using spectra-photometric methods have shown that these compounds may act as oxidizing agents in relation to the coenzyme. Their oxidative activity depends on the nature and number of organic groups in the molecule. Comparison of the kinetic data for the activity of Hg and Sn compounds with those for Fe porphyrins imitating the active centers of redox enzymes indicates the competitiveness of the organometallic compounds and models of natural electron acceptors. 1999 KluwerAcademic/Plenum Publishers.
- Medvedev,Tyurin,Rozhkova,Milaeva
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p. 907 - 911
(2007/10/03)
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- Artificial Redox Enzymes. Part 3. Structure and Properties
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The structural and catalytic properties of models of flavoenzymes in which flavin is covalently attached to the catalytically important secondary side of 2--α-cyclodextrin and 2--β-cyclodextrin as well as the primary side 6-(10-N-isoalloxazinylmethyl)-β-cyclodextrin are reported.
- Ye, Hongping,Rong, Ding,Tong, Weida,D'Souza, Valerian T.
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p. 2071 - 2076
(2007/10/02)
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- Rose Bengal Radicals and their Reactivity
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The one-electron oxidised (radical cation) and one-electron reduced (radical anion) forms of the photosensitiser rose bengal have absorption maxima at 470 nm (molar absorption coefficient 21100 dm3 mol-1cm-1) and 420 nm (molar absorption coefficient 37600 dm3mol-1cm1-), respectively.The radical anion (RB.-) undergoes electron-transfer reactions both with oxygen (k=1.5E8 dm3mol-1s-1) and iron(III) complexed with ethylenediamineteteraacetic acid (EDTA) (k=4.5E8 dm3mol1-s1-) and diethylenetriaminepentaacetic acid (DTPA) (k=8.6E8 dm3mol-1s-1).Rose bengal reacts both with solvated electron and semireduced nicotinamide adenine dinucleotide radical (NAD.) to give RB.- with the second-order rate constants of 3.1E10 and 9.6E8 dm3mol1-s1-.The one electron-reduction potential E21 for rose bengal, measured vis pluse radiolysis, was found to be -511 mV.
- Lambert, C.,Sarna, T.,Truscott, T. G.
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p. 3879 - 3882
(2007/10/02)
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- Efficient NAD+-Regeneration System with Heterocyclic o-Quinones and Molecular Oxygen Catalyzed by Diaphorase
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Addition of catalase and diaphorase drastically improved efficiency of the NAD+-regeneration system with heterocyclic o-quinones and molecular oxygen.
- Itoh, Shinobu,Mita, Naruyoshi,Ohshiro, Yoshiki
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p. 1949 - 1952
(2007/10/02)
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- Mechanism of the Oxidation of NADH by Quinones. Energetics of One-Electron and Hydride Routes
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The kinetics of NADH oxidation by 7 o-benzoquinones and 14 p-benzoquinones were studied by using buffered aqueous solutions and UV/vis spectroscopy.For each quinone the rate law was first order in NADH and first order in quinone.The rate constants varied from 0.0745 to 9220 M-1s-1.Variation of the pH from 6 to 8 gave no change in rate.The use of 4-D and 4,4-D2NADH revealed kinetic isotope effects.The dideutero data gave kH/kD in the range 1.6-3.1 for p-quinones and 4.2 for 3,5-di-tert-butyl-o-quinone.When p-quinones were used, the log k was a linear function of Eo for the quinone/hydroquinone monoanion (Q/QH(1-)) couple with a slope of 16.9 V-1. o-Quinones reacted about 100 times more rapidly, but the same linear relationship with a slope of 16.4 V-1 was observed.Comparisons to data for one-electron-transfer reactions indicate that such mechanisms are not involved.A hydride-transfer mechanism accommodates all the data, and rate-limiting hydrogen atom transfer followed by electron transfer cannot be ruled out.
- Carlson, Brian W.,Miller, Larry L.
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p. 479 - 485
(2007/10/02)
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