NADPH Recycling System Research Articles

Stable and efficient immobilization of bi-enzymatic NADPH cofactor recycling system under consecutive microwave irradiation.

One of the challenges in biocatalysis is the development of stable and efficient bi-enzymatic cascades for bio-redox reactions coupled to the recycling of soluble cofactors. Aldo-keto reductase (LEK) and glucose dehydrogenase (GDH) can be utilized as the NADPH recycling system for economic and efficient biocatalysis of (R)-4-chloro-3-hydroxybutanoate ((R)-CHBE), an important chiral pharmaceutical intermediate. The LEK and GDH was efficiently co-immobilized in mesocellular siliceous foams (MCFs) under microwave irradiation (CoLG-MIA). while they were also co-immobilized by entrapment in calcium alginate without MIA as control (CoLG-CA). The relative activity of CoLG-MIA was increased to 140% compared with that of free LEK. The CoLG-MIA exhibited a wider range of pH and temperature stabilities compared with other preparations. The thermal, storage and batch operational stabilities of microwave-assisted immobilized LEK-GDH were also improved. The NADPH recycling system exhibited the potential as the stable and efficient catalyst for the industrial preparation of (R)-CHBE.

Highly selective synthesis of d-amino acids from readily available l-amino acids by a one-pot biocatalytic stereoinversion cascade.

d-Amino acids are key intermediates required for the synthesis of important pharmaceuticals. However, establishing a universal enzymatic method for the general synthesis of d-amino acids from cheap and readily available precursors with few by-products is challenging. In this study, we constructed and optimized a cascade enzymatic route involving l-amino acid deaminase and d-amino acid dehydrogenase for the biocatalytic stereoinversions of l-amino acids into d-amino acids. Using l-phenylalanine (l-Phe) as a model substrate, this artificial biocatalytic cascade stereoinversion route first deaminates l-Phe to phenylpyruvic acid (PPA) through catalysis involving recombinant Escherichia coli cells that express l-amino acid deaminase from Proteus mirabilis (PmLAAD), followed by stereoselective reductive amination with recombinant meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum (StDAPDH) to produce d-phenylalanine (d-Phe). By incorporating a formate dehydrogenase-based NADPH-recycling system, d-Phe was obtained in quantitative yield with an enantiomeric excess greater than 99%. In addition, the cascade reaction system was also used to stereoinvert a variety of aromatic and aliphatic l-amino acids to the corresponding d-amino acids by combining the PmLAAD whole-cell biocatalyst with the StDAPDH variant. Hence, this method represents a concise and efficient route for the asymmetric synthesis of d-amino acids from the corresponding l-amino acids.

Peroxisomal plant metabolism – an update on nitric oxide, Ca2+ and the NADPH recycling network

Plant peroxisomes are recognized organelles that - with their capacity to generate greater amounts of H2O2 than other subcellular compartments - have a remarkable oxidative metabolism. However, over the last 15 years, new information has shown that plant peroxisomes contain other important molecules and enzymes, including nitric oxide (NO), peroxynitrite, a NADPH-recycling system, Ca2+ and lipid-derived signals, such as jasmonic acid (JA) and nitro-fatty acid (NO2-FA). This highlights the potential for complex interactions within the peroxisomal nitro-oxidative metabolism, which also affects the status of the cell and consequently its physiological processes. In this review, we provide an update on the peroxisomal interactions between all these molecules. Particular emphasis will be placed on the generation of the free-radical NO, which requires the presence of Ca2+, calmodulin and NADPH redox power. Peroxisomes possess several NADPH regeneration mechanisms, such as those mediated by glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) proteins, which are involved in the oxidative phase of the pentose phosphate pathway, as well as that mediated by NADP-isocitrate dehydrogenase (ICDH). The generated NADPH is also an essential cofactor across other peroxisomal pathways, including the antioxidant ascorbate-glutathione cycle and unsaturated fatty acid β-oxidation, the latter being a source of powerful signaling molecules such as JA and NO2-FA.

In Silico Analysis of Arabidopsis thaliana Peroxisomal 6-Phosphogluconate Dehydrogenase.

NADPH, whose regeneration is critical for reductive biosynthesis and detoxification pathways, is an essential component in cell redox homeostasis. Peroxisomes are subcellular organelles with a complex biochemical machinery involved in signaling and stress processes by molecules such as hydrogen peroxide (H2O2) and nitric oxide (NO). NADPH is required by several peroxisomal enzymes involved in β-oxidation, NO, and glutathione (GSH) generation. Plants have various NADPH-generating dehydrogenases, one of which is 6-phosphogluconate dehydrogenase (6PGDH). Arabidopsis contains three 6PGDH genes that probably are encoded for cytosolic, chloroplastic/mitochondrial, and peroxisomal isozymes, although their specific functions remain largely unknown. This study focuses on the in silico analysis of the biochemical characteristics and gene expression of peroxisomal 6PGDH (p6PGDH) with the aim of understanding its potential function in the peroxisomal NADPH-recycling system. The data show that a group of plant 6PGDHs contains an archetypal type 1 peroxisomal targeting signal (PTS), while in silico gene expression analysis using affymetrix microarray data suggests that Arabidopsis p6PGDH appears to be mainly involved in xenobiotic response, growth, and developmental processes.

Simple Method for Measuring Endothelial Nitric Oxide Synthase Activity in Clinical Researches

Antiatherogenic Effect of Almond Oil in Streptozotocin Induced Diabetic RatsMona Anwar, Wafaa Ghoneim Shousha, Hatem A. El-mezayen, Raafat A wadallah, Maha El-Wassef, Naglaa M. Nazif and Mona A. El-bana

Biocatalytic reduction of prochiral aromatic ketones to optically pure alcohols by a coupled enzyme system for cofactor regeneration

A simple, highly efficient, and economical biphasic cell-free system was developed for biocatalytic reduction of prochiral aromatic ketones to furnish enantiopure alcohols. This system is characterized by using endogenous enzymes of the cell-free extract to form enzyme-coupled NADPH recycling system. Besides, it offered much higher productivity than whole cells and greatly simplified the preparation process of biocatalysts in comparison with isolated enzymes. Various prochiral aromatic ketones, especially α-substituted acetophenone derivatives, were reduced to chiral alcohols with excellent enantiomeric excess (ee) and moderate to good yield by this cell-free system.

Asymmetric synthesis of ( S)-ethyl-4-chloro-3-hydroxy butanoate using a Saccharomyces cerevisiae reductase: Enantioselectivity and enzyme–substrate docking studies

Ethyl ( S)-4-chloro-3-hydroxy butanoate (ECHB) is a building block for the synthesis of hypercholesterolemia drugs. In this study, various microbial reductases have been cloned and expressed in Escherichia coli. Their reductase activities toward ethyl-4-chloro oxobutanoate (ECOB) have been assayed. Amidst them, Baker's yeast YDL124W, YOR120W, and YOL151W reductases showed high activities. YDL124W produced ( S)-ECHB exclusively, whereas YOR120W and YOL151W made ( R)-form alcohol. The homology models and docking models with ECOB and NADPH elucidated their substrate specificities and enantioselectivities. A glucose dehydrogenase-coupling reaction was used as NADPH recycling system to perform continuously the reduction reaction. Recombinant E. coli cell co-expressing YDL124W and Bacillus subtilis glucose dehydrogenase produced ( S)-ECHB exclusively.

Purification and Characterization of a (R)-1-Phenyl-1,3-propanediol-producing Enzyme fromTrichosporon fermentansAJ-5152 and Enzymatic (R)-1-Phenyl-1,3-propanediol Production

An (R)-1-phenyl-1,3-propanediol-producing enzyme was purified from Trichosporon fermentans AJ-5152. It was NADPH-dependent and converted 3-hydroxy-1-phenylpropane-1-one (HPPO) to (R)-1-phenyl-1,3-propanediol [(R)-PPD] with anti-Prelog's specificity. It showed maximum activity at pH 7.0 and 40 degrees C. Its K(m) and V(max) values toward HPPO were 20.1 mM and 3.4 mumol min(-1) mg protein(-1) respectively. The relative molecular weight of the enzyme was estimated to be 68,000 on gel filtration and 32,000 on SDS-polyacrylamide gel electrophoresis. An (R)-PPD-producing reaction using the (R)-PPD-producing enzyme and an NADPH recycling system was carried out by successive feeding of HPPO. A total (R)-PPD yield of 8.9 g/l was produced in 16 h. The molar yield was 76%, and the optical purity of the (R)-PPD produced was over 99% e.e.

Enzymatic recycling of NADPH at high temperature utilizing a thermostable glucose-6-phosphate dehydrogenase from Bacillus stearothermophilus

A high temperature NADPH recycling system was designed utilizing a thermostable glucose-6-phosphate dehydrogenase (tG6PDH) from Bacillus stearothermophilus with glucose-6-sulfate (G6S) as the substrate. The thermostable alcohol dehydrogenase (tADH) from Thermoanaerobacter (formerly Thermoanaerobium) brockii was employed to catalyze the reduction of 2-butanone to 2-butanol at 55 °C. It was established that the B. stearothermophilus G6PDH is capable of utilizing G6S as the substrate ( K M=50 mM at 55 °C). NADPH recycling based on the G6S/tG6PDH system performed optimally with a 3:1 ratio of tG6PDH:tADH, and with 1 mM NADP. The system produced 75 mM 2-butanol and retained 60% of NADPH after 5 h at 55 °C. Under the same conditions, the system using glucose-6-phosphate (G6P), the natural substrate for tG6PDH, produced only 25 mM 2-butanol and retained negligible amount of the co-factor.

Sources of interference in the use of 2,3-diaminonaphthalene for the fluorimetric determination of nitric oxide synthase activity in biological samples

The use of 2,3-diaminonaphthalene (DAN) for the fluorimetric determination of nitric oxide synthase (NOS) activity in rat brain extracts has been re-examined. Two types of interference were observed, due either to components of the reaction mixture or to the enzymatic sample itself. One of the substrates (NADPH) and some cofactors (FADH 2, FMNH 2) required for the enzyme activity interfere in the assay by quenching the fluorescence produced. Interference was minimized by using lower FADH 2, FMNH 2 and NADPH concentrations (1 μmol/l) and a NADPH recycling system in the reaction mixture. The addition of bovine serum albumin or hemoglobin to the sample quenched fluorescence intensity, but these protein interferences could be reduced by filtering the samples after reaction. We conclude that the DAN fluorimetric assay as originally described is not suitable for the determination of NOS activity in crude extracts such as rat brain cytosolic fraction, due to the presence of interfering substances. Nevertheless, DAN could be used for the determination of enzyme activity after reducing protein interference by filtering, or in less complex samples such as cell cultures (e.g. activated macrophages), or in chromatographic fractions obtained during the purification of the enzyme. A careful use of the commercial kits based on the use of DAN for the determination of NOS activity is recommended.

Large-scale chemoenzymic synthesis of calcium (6S)-5-formyl-5,6,7,8-tetrahydrofolate [(–)-leucovorin] using the NADPH recycling method

Chemoenzymic large-scale synthesis of the calcium salt of (6S)-5-formyltetrahydrofolic acid [(–)-leucovorin, (6S)-5] was achieved from folic acid 1via(6S)-tetrahydrofolic acid [(6S)-3] by using dihydrofolate reductase (DHFR) produced by Escherichia coil, harbouring a high-expression plasmid, pTP64–1. On the other hand, for the diastereoselective reduction of 7,8-dihydrofolic acid 2 to tetrahydrofolate (6S)-3, a new NADPH recycling system was constructed by coupling with glucose dehydrogenase from Gluconobacter scieroides. Having these enzymic systems to hand, compound 1 was reduced by zinc powder in alkaline solution to give compound 2 which, without isolation, was reduced enzymatically to afford tetrahydrofolate (6S)-3(94% de). The pH adjustment of the reaction mixture containing dihydrofolate 2 was done with phosphoric acid in order to remove zinc ion which inhibited the following enzymic reduction. The formed tetrahydrofolate (6S)-3 was converted into entirely optically pure N-formyl compound (6S)-5 on a large scale. The specific rotation value of (–)-leucovorin was [α]D20–13.3 (c 1, water). For the comparison of pharmacological effects, a completely optically pure form of (+)-leucovorin [(6R)-5] was also prepared on a preparative scale. Compound (6S)-5 was 300-fold more active compared with the (6R)-diastereoisomer.