53-57-6Relevant academic research and scientific papers
Kinetic studies of the inhibition of a human liver 3α-hydroxysteroid/dihydrodiol dehydrogenase isozyme by bile acids and anti-inflammatory drugs
Miyabe,Amano,Deyashiki,Hara,Tsukada
, p. 9 - 12 (1995)
We have investigated the steady-state kinetics for a cytosolic 3α-hydroxysteroid/dihydrodiol dehydrogenase isozyme of human liver and its inhibition by several bile acids and anti-inflammatory drugs such as indomethacin, flufemanic acid and naproxen. Initial velocity and product inhibition studies performed in the NADP+-linked (S)-1-indanol oxidation at pH 7.4 were consistent with a sequential ordered mechanism in which NADP+ binds first and leaves last. The bile acids and drugs, competitive inhibitors with respect to the alcohol substrate, exhibited uncompetitive inhibition with respect to the coenzyme, with K(i) values less than 1 μM, whereas indomethacin exhibited noncompetitive inhibition (K(i) 24 μM). The kinetics of the inhibition by a mixture of the two inhibitors suggests that bile acids and drugs, except indomethacin, bind to overlapping sites at the active center of the enzyme-coenzyme binary complex.
Photochemical reduction of NADP+ by zinc protoporphyrin reconstituted myoglobin as a simple model of photosystem I
Nishiyama, Katsuhiko,Uchiyama, Miho,Mie, Yasuhiro,Taniguchi, Isao
, p. 357 - 358 (1999)
Photoinduced electron transfer between zinc protoporphyrin reconstituted myoglobin (Zn-Mb) and NADP+ functions as a model of photosystem I by forming NADPH. The reduction efficiency of NADP+ depended strongly on the solution pH, which was explained by the difference in the redox potential and/or the static interaction between Zn-Mb and a sacrificial donor triethanolamine (TEA).
16-Bromoepiandrosterone, an activator of the mammalian immune system, inhibits glucose 6-phosphate dehydrogenase from Trypanosoma cruzi and is toxic to these parasites grown in culture
Cordeiro, Artur T.,Thiemann, Otavio H.
, p. 4762 - 4768 (2010)
Glucose 6-phosphate dehydrogenase (G6PDH) catalyzes the first step of the pentose-phosphate pathway which supplies cells with ribose 5-phosphate (R5P) and NADPH. R5P is the precursor for the biosynthesis of nucleotides while NADPH is the cofactor of several dehydrogenases acting in a broad range of biosynthetic processes and in the maintenance of the cellular redox state. RNA interference-mediated reduction of G6PDH levels in bloodstream-form Trypanosoma brucei validated this enzyme as a drug target against Human African Trypanosomiasis. Dehydroepiandrosterone (DHEA), a human steroidal pro-hormone and its derivative 16α-bromoepiandrosterone (16BrEA) are uncompetitive inhibitors of mammalian G6PDH. Such steroids are also known to enhance the immune response in a broad range of animal infection models. It is noteworthy that the administration of DHEA to rats infected by Trypanosoma cruzi, the causative agent of Human American Trypanosomiasis (also known as Chagas' disease), reduces blood parasite levels at both acute and chronic infection stages. In the present work, we investigated the in vitro effect of DHEA derivatives on the proliferation of T. cruzi epimastigotes and their inhibitory effect on a recombinant form of the parasite's G6PDH (TcG6PDH). Our results show that DHEA and its derivative epiandrosterone (EA) are uncompetitive inhibitors of TcG6PDH, with Ki values of 21.5 ± 0.5 and 4.8 ± 0.3 μM, respectively. Results from quantitative inhibition assays indicate 16BrEA as a potent inhibitor of TcG6PDH with an IC50 of 86 ± 8 nM and those from in vitro cell viability assays confirm its toxicity for T. cruzi epimastigotes, with a LD50 of 12 ± 8 μM. In summary, we demonstrated that, in addition to host immune response enhancement, 16BrEA has a direct effect on parasite viability, most likely as a consequence of TcG6PDH inhibition.
Fabrication of novel electrochemical reduction systems using alcohol dehydrogenase as a bifunctional electrocatalyst
Yuan, Ruo,Kuwabata, Susumu,Yoneyama, Hiroshi
, p. 137 - 138 (1996)
Electrochemical reduction of NADP+ to NADPH and of NAD+ to NADH with current efficiencies of more than 97% has been achieved at alcohol dehydrogenase (ALDH) in the presence of acctophenone as an electron mediator. Addition of acetone or acctaldehyde as a substrate to the above electrolytic system allowed reduction of the substrate to the corresponding alcohol at ALDH accompanied by oxidation of the resulting NAD(P)H.
Purification and characterization of two isoforms of glucose 6-phosphate dehydrogenase (G6PDH) from Chlorella vulgaris C-27
Honjoh, Ken-Ichi,Mimura, Ayano,Kuroiwa, Eiko,Hagisako, Takahiro,Suga, Koushirou,Shimizu, Hideyuki,Dubey, Rama Shanker,Miyamoto, Takahisa,Hatano, Shoji,Iio, Masayoshi
, p. 1888 - 1896 (2003)
Two kinds of isoforms of glucose 6-phosphate dehydrogenase (G6PDH) were purified from cells of a freezing-tolerant strain, Chlorella vulgaris C-27, by sequential steps of chromatography on five kinds of columns, including a HiTrap Blue column which showed excellent separation of the isoforms from each other. The two isoforms (G6PDH1 and G6PDH2) were purified up to 109-fold and 197-fold with specific activity of 14.4 and 26.0 U/mg-protein, respectively. G6PDH1 showed an apparent Mr of 200,000 with a subunit Mr of about 58,000, whereas G6PDH2 showed an apparent Mr of 450,000 with a subunit Mr of about 52,000. The kinetic parameters were measured and several enzymatic features of the isoforms, such as effects of metal ions on the enzyme activity, were clarified, which showed that the two isoforms were different from each other in many respects. Among the effective ions, Cd2+ showed marked stimulating effects on both isoforms. G6PDH1 and G6PDH2 seem to be a cytosolic and a chloroplastic type, respectively, as judged by their sensitivity to DTT, and also from the results of sequence similarity searches using their N-terminal and internal amino acid sequences.
Design of artificial metalloenzymes for the reduction of nicotinamide cofactors
Basle, Mattias,Padley, Henry A.W.,Martins, Floriane L.,Winkler, Gerlof Sebastiaan,J?ger, Christof M.,Pordea, Anca
, (2021/04/19)
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.
Selective Usage of Isozymes for Stress Response
Zhang, Yugang,Lin, Zhewang,Wang, Miao,Lin, Hening
, p. 3059 - 3064 (2018/11/23)
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.
Biocatalyst-artificial metalloenzyme cascade based on alcohol dehydrogenase
Morra, Simone,Pordea, Anca
, p. 7447 - 7454 (2018/10/21)
Chemo-enzymatic cascades of enzymes with transition metal catalysts can offer efficient synthetic strategies, but their development is challenging due to the incompatibility between proteins and transition metal complexes. Rhodium catalysts can be combined with alcohol dehydrogenases to regenerate nicotinamide cofactors using formate as the hydride donor. However, their use is limited, due to binding of the metals to residues on the enzyme surface, leading to mutual enzyme and catalyst inactivation. In this work, we replaced the zinc from Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) with Rh(iii) catalysts possessing nitrogen donor ligands, by covalent conjugation to the active site cysteine, to create artificial metalloenzymes for NADP+ reduction. TbADH was used as protein scaffold for both alcohol synthesis and the recycling of the cofactor, by combination of the chemically modified species with the non-modified recombinant enzyme. Stability studies revealed that the incorporation of the catalysts into the TbADH pocket provided a shielding environment for the metal catalyst, resulting in increased stability of both the recycling catalyst and the ADH. The reduction of a representative ketone using this novel alcohol dehydrogenase-artificial formate dehydrogenase cascade yielded better conversions than in the presence of free metal catalyst.
Emissive Synthetic Cofactors: Enzymatic Interconversions of tzA Analogues of ATP, NAD+, NADH, NADP+, and NADPH
Hallé, Fran?ois,Fin, Andrea,Rovira, Alexander R.,Tor, Yitzhak
, p. 1087 - 1090 (2017/12/28)
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.
A solar light-driven, eco-friendly protocol for highly enantioselective synthesis of chiral alcohols via photocatalytic/biocatalytic cascades
Choudhury, Sumit,Baeg, Jin-Ook,Park, No-Joong,Yadav, Rajesh K.
, p. 4389 - 4400 (2014/09/29)
The judicious utilization of solar light for the asymmetric synthesis of optically active compounds by imitating natural photosynthesis introduces a new concept that harnesses this renewable energy in vitro for ultimate transformation into chiral chemical bonds. Herein, we present a comprehensive description of such a biomimetic endeavor towards the design and construction of an asymmetric artificial photosynthesis system that comprises an efficient method of nicotinamide cofactor (NADPH) regeneration under visible light employing a graphene-based light harvesting photocatalyst and its subsequent utilization in an enzyme-catalyzed asymmetric reduction of prochiral ketones to expediently furnish the corresponding chiral secondary alcohols. A detailed optimization study revealed a major dependency of the reaction outcome on the amount of cofactor, photocatalyst and enzyme used, as well as the mode of their addition. A series of structurally diverse ketones bearing an array of (hetero)aryl/alkyl substituents proved to be highly suitable to our photocatalytic-biocatalytic cascade approach, providing (R/S)-1-(hetero)aryl/ alkylethanols in excellent enantioselectivities (ee ～ 95->99.9%) under mild and environmentally benign conditions. To the best of our knowledge, the synthesis of these enantiopure alcohols employing a visible-light-driven nicotinamide cofactor regeneration strategy has been reported for the first time. Such enantioenriched alcohols act as versatile chiral building blocks for the synthesis of compounds having industrial and pharmaceutical relevance. In addition, this solar-to-chiral chemicals prototype appears advantageous from ecological and economical perspectives. We describe mechanistic pathways to demonstrate how the present catalytic synthesis protocol functions through perfect orchestration between visible-light-driven photocatalysis and biocatalysis to be successively applied in inducing asymmetry in an achiral molecule for the ultimate goal of solar energy utilization in the synthesis of valuable chiral fine chemicals. This work highlights the potential advantages of a bioinspired system to the pertinence of solar energy in asymmetric transformations leading to enantioenriched alcohol precursors, and thus opens up a new field of research that might emerge as an important breakthrough with promising implications towards generating a sustainable and non-fossil/non- nuclear energy future. the Partner Organisations 2014.
