NAD H-dependent Oxidoreductases Research Articles

Design of a cofactor self-sufficient whole-cell biocatalyst for enzymatic asymmetric reduction via engineered metabolic pathways and multi-enzyme cascade.

NAD(P)H-dependent oxidoreductases are crucial biocatalysts for synthesizing chiral compounds. Yet, the industrial implementation of enzymatic redox reactions is often hampered by an insufficient supply of expensive nicotinamide cofactors. Here, a cofactor self-sufficient whole-cell biocatalyst was developed for the enzymatic asymmetric reduction of 2-oxo-4-[(hydroxy)(-methyl)phosphinyl] butyric acid (PPO) to L-phosphinothricin (L-PPT). The endogenous NADP+ pool was significantly enhanced by regulating Preiss-Handler pathway toward NAD(H) synthesis and, in the meantime, introducing NAD kinase to phosphorylate NAD(H) toward NADP+. The intracellular NADP(H) concentration displayed a 2.97-fold increase with the strategy compared with the wild-type strain. Furthermore, a recombinant multi-enzyme cascade biocatalytic system was constructed based on the Escherichia coli chassis. In order to balance multi-enzyme co-expression levels, the strategy of modulating rate-limiting enzyme PmGluDH by RBS strengths regulation successfully increased the catalytic efficiency of PPO conversion. Finally, the cofactor self-sufficient whole-cell biocatalyst effectively converted 300mM PPO to L-PPT in 2h without the need to add exogenous cofactors, resulting in a 2.3-fold increase in PPO conversion (%) from 43% to 100%, with a high space-time yield of 706.2gL-1d-1 and 99.9% ee. Overall, this work demonstrates a technological example for constructing a cofactor self-sufficient system for NADPH-dependent redox biocatalysis.

Identification and functional characterization of a novel aldo-keto reductase from Aloe vera.

The present investigation profoundly asserted the catalytic potential of plant-based aldo-ketoreductase, postulating its role in polyketide biosynthesis and providing new insights for tailored biosynthesis of vital plant polyketides for therapeutics. Plants hold great potential as a future source of innovative biocatalysts, expanding the possibilities within chemical reactions and generating a variety of benefits. The aldo-keto reductase (AKR) superfamily includes a huge collection of NAD(P)H-dependent oxidoreductases that carry out a variety of redox reactions essential for biosynthesis, detoxification, and intermediary metabolism. The present study involved the isolation, cloning, and purification of a novel aldo-ketoreductase (AvAKR) from the leaves of Aloe vera (Aloe barbadensis Miller) by heterologous gene expression in Escherichia coli based on the unigene sequences of putative ketoreductase and cDNA library screening by oligonucleotide hybridization. The in-silico structural analysis, phylogenetic relationship, and molecular modeling were outranged to approach the novelty of the sequence. Additionally, agroinfiltration of the candidate gene tagged with a green fluorescent protein (GFP) was employed for transient expression in the Nicotiana benthamiana to evaluate the sub-cellular localization of the candidate gene. The AvAKR preferred cytoplasmic localization and shared similarities with the known plant AKRs, keeping the majority of the conserved active-site residues in the AKR superfamily enzymes. The enzyme facilitated the NADPH-dependent reduction of various carbonyl substrates, including benzaldehyde and sugars, proclaiming a broad spectrum range. Our study successfully isolated and characterized a novel aldo-ketoreductase (AvAKR) from Aloe vera, highlighting its versatile NADPH-dependent carbonyl reduction proficiency therewith showcasing its potential as a versatile biocatalyst in diverse redox reactions.

Analysis of the mechanism of aldo-keto reductase dependent cis-platin resistance in HepG2 cells based on transcriptomic and NADH metabolic state

Background: Aldo-keto oxidoreductase (AKR) inhibitors could reverse the resistance of several cancer cells to cis-platin, but their role in resistance remains unclear.Methods: We verified the difference of AKR1Cs expression by Western blot, RNA sequencing and qRT-PCR. The differences of AKR1Cs expression were analyzed and inferred. Use Assay of NADH and NAD+ content to verify the inference. The Docking experience was used to verify the affinity between MPA, MCFLA, MLS and AKR1C3.Results: Our RNA-seq results showed de novo NAD biosynthesis-related genes and NAD(P)H-dependent oxidoreductases were significantly upregulated in cis-platin-resistant HepG2 hepatic cancer cells (HepG2-RC cells) compared with HepG2 cells. At least 63 NAD(P)H-dependent reductase/oxidases were upregulated in HepG2-RC cells at least twofold. Knockdown of AKR1Cs could increase cis-platin sensitivity in HepG2-RC cells about two-fold. Interestingly, the AKR1C inhibitor meclofenamic acid could increase the cis-platin sensitivity of HepG2-RC cells about eight-fold, indicating that the knockdown of AKR1Cs only partially reversed the resistance. Meanwhile, the amount of total NAD and the ratio of NADH/NAD+ were increased in HepG2-RC cells compared with HepG2 cells. The ratio of NADH/NAD+ in HepG2-RC cells was almost seven-fold higher than in HepG2 or HL-7702 cells. Increased NADH expression could be explained as a directly operating antioxidant to scavenge cis-platin-induced radicals.Conclusion: We report here that NADH, which is produced by NAD(P)H-dependent oxidoreductases, plays a key role in the AKR-associated cis-platin resistance of HepG2 hepatic cancer cells.

Redox Biocatalysis: Quantitative Comparisons of Nicotinamide Cofactor Regeneration Methods.

Enzymatic processes, particularly those capable of performing redox reactions, have recently been of growing research interest. Substrate specificity, optimal activity at mild temperatures, high selectivity, and yield are among the desirable characteristics of these oxidoreductase catalyzed reactions. Nicotinamide adenine dinucleotide (phosphate) or NAD(P)H-dependent oxidoreductases have been extensively studied for their potential applications like biosynthesis of chiral organic compounds, construction of biosensors, and pollutant degradation. One of the main challenges associated with making these processes commercially viable is the regeneration of the expensive cofactors required by the enzymes. Numerous efforts have pursued enzymatic regeneration of NAD(P)H by coupling a substrate reduction with a complementary enzyme catalyzed oxidation of a co-substrate. While offering excellent selectivity and high total turnover numbers, such processes involve complicated downstream product separation of a primary product from the coproducts and impurities. Alternative methods comprising chemical, electrochemical, and photochemical regeneration have been developed with the goal of enhanced efficiency and operational simplicity compared to enzymatic regeneration. Despite the goal, however, the literature rarely offers a meaningful comparison of the total turnover numbers for various regeneration methodologies. This comprehensive Review systematically discusses various methods of NAD(P)H cofactor regeneration and quantitatively compares performance across the numerous methods. Further, fundamental barriers to enhanced cofactor regeneration in the various methods are identified, and future opportunities are highlighted for improving the efficiency and sustainability of commercially viable oxidoreductase processes for practical implementation.

Metalloproteinases and NAD(P)H-dependent oxidoreductase within of Bay nettle (Chrysaora chesapeakei) venom

The venom of jellyfish has been a source of biologically active substances, so it is interesting to study the components of the venom of Chrysaora chesapeakei from the estuaries of the Gulf of Mexico. Proteomic techniques in this study identified the most abundant venom proteins (100, 50, and 37 kDa). Homology searches suggest that the main toxins in this venom correspond to metalloproteinase-zinc disintegrin, astacin-type metalloprotease, and NAD (P) H-dependent oxidoreductases, which could be responsible for the main effects of this jellyfish venom; The tentacles were also identified as containing nonpoisonous metalloproteinases in connective tissue.

The evolution and new exploration of the catalytic mechanism of oxidoreductase

<p indent="0mm">Oxidoreductases play an important role in the metabolism of life so that revealing their catalytic mechanism is momentous for further reveal the influence of electron and proton transfer on the growth of living organisms. More importantly, oxidoreductase accounts for about one-third among all enzymes newly registered on the BRENDA website, of which half using NAD(P)H/NAD(P)⁺ as a coenzyme. Hugo Theorell is awarded a Nobel Prize in 1955 for achievements that discovered the nature and mode of action of oxidation enzymes in 1951. Cooperate with Britton Chance, they propose the famous Theorell-Chance mechanism based on the research of the catalysis kinetics mechanism of liver alcohol dehydrogenase, which has always been a hot topic since then. Thus, in the past <sc>70 years,</sc> the study of the mechanism and its modification has always been a focus of researches since the classic oxidoreductase’s catalysis mechanism is put forward in 1951. Based on the previous work, we review the evolution of the catalytic mechanism of oxidoreductases systematically and thus put forward two valuable scientific problems. (1) Do intracellular coenzymes bound to oxidoreductases enzymes have to dissociate after oxidation or reduction? (2) Do intracellular NAD(P)H-dependent oxidoreductases enzyme and its corresponding coenzyme have self-assembly? Then, we also detailed introduce some discoveries of oxidoreductases catalytic mechanism based on our recent work. Based on the whole-cell catalytic process of 1,3-propanediol oxidoreductase and glycerol dehydrogenase, we find that the whole-cell can also catalyze the extracellularly NAD⁺ to NADH. After then, technology development brings us advanced precise instruments, which give us a deeper insight into the catalyzation process at a single-molecular level. Among them, the STM-BJ (scanning tunneling microscope break junction) technique provides excellent performance in spatial resolution and electrical detection sensitivity, which has been one of the most frequently used techniques in single-molecule electrical detection in recent years. Thus, we provide the first demonstration of the STM-BJ technique for investigating charge transport through a single active enzyme junction, in which the binding of NAD⁺ with proteins boosts the charge transport by over 2100% than neutral FDH. Combine with site-specific mutagenesis, we demonstrate the conductance of FDH-NAD⁺ highly correlate with their bioactivities. And we will also employ it to study the real-time conductivity of single NAD(P)H-depending formate dehydrogenase in the catalytic process to reveal the new catalytic mechanism at the single-molecule level in future work. Studying the combination of single NAD(P)H-depending oxidoreductase and its co-enzyme and the reaction of substrate catalyzing is an original innovative research, which will help us to understand the essence of enzyme catalyzation in life activity and to improve technologies in directed enzyme evolution and development of biomedical drugs. The proposed catalytic mechanism of the NAD(P)H-depending oxidoreductase is expected not only to revolutionize the theory that has been followed for <sc>70 years</sc> but also to improve a wide range of traditional technologies, such as whole-cell fermentation, multiple enzymes coupling catalyzation, enzyme activity detection, bio-sensor and so on.

NAD(P)H-dependent oxidoreductases for synthesis of chiral amines by asymmetric reductive amination of ketones

Asymmetric reductive amination is one of the most important reactions in the preparation of chiral amine pharmaceutical intermediates. Different kinds of enzymes have been applied to the synthesis of chiral amines Among them, the development of reductive amination reactions catalyzed by NAD(P)H-dependent oxidoreductases is attractive, as it would permit a one-step transformation of prochiral ketones into chiral amines with high optical purity. In this review, the recent advances in enzyme structure, catalytic mechanism, protein engineering and the application in reductive amination reactions of NAD(P)H-dependent oxidoreductases, including imine reductase, amino acid dehydrogenase, opine dehydrogenase and reductive aminase, are summarized.

The characterization of a short chain dehydrogenase/reductase (SDRx) in Comamonas testosteroni

C. testosteroni is a research topic that can degrade steroid hormones into water and carbon dioxide through a series of enzymes in the body. Short-chain dehydrogenase (SDR) are a class of NAD (P) H-dependent oxidoreductases in C. testosteroni. Its main function is catalyzing the redox of the hydroxyl/ketone group of the hormone. In this paper, a SDR gene(SDRx) is cloned from C. testosteroni ATCC11996 and expressed. The polyclonal antibody was prepared and the SDRx gene knocked out by homologous recombination. Wild type and mutant C. testosteroni induced by testosterone, estradiol, estrone and estriol. The growth curves of the bacteria were measured by spectrophotometer. ELISA established the expression of SDRx protein, and high-performance liquid chromatography(HPLC) detected the contents of various hormones. The results show that the growth of wild type was faster than mutant type induced by testosterone. The concentration of SDRx is 0.318 mg/ml under testosterone induction. It has a great change in steroid hormones residue in culture medium measured by HPLC: Testosterone residue in the mutant type group was 42.4 % more than the wild type in culture medium. The same thing happens with induced by estrone. In summary, this SDRx gene involved in the degradation of testosterone and estradiol, and effects the growth of C. testosteroni.

208 The Aryl hydrocarbon receptor (AhR) induces the expression of aldo-keto reductase (AKR) 1C3 in keratinocytes in a ligand-specific manner

The aldo-keto reductase 1C3 (AKR1C3) belongs to a superfamily of NAD(P)H-dependent oxidoreductases, which metabolize prostaglandins and steroid hormones. It is involved in the pathogenesis of various skin malignancies, such as atopic dermatitis (AD) and squamous cell carcinoma, which are triggered by polycyclic aromatic hydrocarbons and related environmental pollutants. These chemicals are able to bind to the aryl hydrocarbon receptor (AhR), a cytosolic transcription factor that, upon activation, rapidly translocates to the nucleus to induce the expression of target genes, for instance encoding for cytochrome P450 (CYP) 1A monooxygenases. In this study, we identified AKR1C3 as a target gene of the AhR in immortalized keratinocytes. Interestingly, activation of AhR by benzo(a)pyrene (BaP), but not by PCB126, a dioxin-like polychlorinated biphenyl, induced AKR1C3 expression. Chemical and genetic inhibition of AhR reversed the BaP-mediated expression of AKR1C3 and CYP1A1, providing evidence for an AhR-dependent effect. As the expression of CYP1A1 was induced by both ligands, we hypothesize that AKR1C3 is regulated by a non-canonical AhR signaling pathway. Upon ligand-binding, AhR undergoes conformational changes and c-Src, as an integral component of the cytosolic multiprotein complex, is released. This tyrosine kinase, in turn, activates EGFR-MEK-ERK signal transduction. Accordingly, chemical inhibition of either c-Src, EGFR and MEK abrogated the BaP-triggered induction of AKR1C3. SDS-PAGE/western blot analyses confirmed the AhR-dependent activation of this cascade in response to BaP treatment. Taken together, these data provide a new mechanism through which pollutants may regulate AKR1C3 expression and thereby contribute to the progression of inflammatory and malignant skin diseases.

3-Bromopyruvate induces expression of antioxidant genes

An alkylating compound, 3-bromopyruvic acid (3-3-bromopyruvic acid (BP)) is a promising anti-cancer agent, potentially able to act on multidrug-resistant cells. Its action has been attributed mainly to inhibition of glycolysis. This compound induces also oxidative stress at a cellular level. The effects of 3-BP on gene expression have not been studied although they may determine the survival of cells exposed to 3-BP. The aim of this paper was to examine the effect 3-BP on gene expression pattern in breast MCF-7 cancer cells. Detection of the differences in gene expression was performed using microarrays and dysregulated genes were validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Exposure of cells to 100 µM 3-BP for 6, 12 and 24 increased expression and diminished expression of 39 and 6 genes, respectively. Among the induced genes, 22 belong to general cellular stress response genes, maintenance genes involved in redox homeostasis, responding to oxidative stress (among them metallothioneins, low-molecular-weight thiol homeostasis enzymes and genes coding for NAD(P)H-dependent oxidoreductases operating on complex organic substrates, including aldo-keto reductases). These results demonstrate that transient oxidative stress in cells exposed to 3-BP is followed by antioxidant response.

P-220 - 3-Bromopyruvate induces expression of antioxidant genes

3-bromopyruvic acid (3-BP) is a promising anticancer agent, potentially able to act on multidrug-resistant cells. Its action has been attributed mainly to inhibition of glycolysis. This compound induces also oxidative stress at a cellular level. The effects of 3-BP on gene expression have not been studied, although they may determine the survival of cells exposed to 3-BP. The aim of this paper was to examine the effect 3-BP on gene expression pattern in breast MCF-7 cancer cells. Detection of the differences in gene expression was performed using microarrays and dysregulated genes were validated by reverse transcription quantitative polymerase chain reaction. Exposure of cells to 100 µM 3-BP for 6, 12 and 24 increased expression and diminished expression of 39 and 6 genes, respectively. Among the induced genes, 22 belong to general cellular stress response genes, maintenance genes involved in redox homeostasis, responding to oxidative stress (among them metallothioneins, low-molecular-weight thiol homeostasis enzymes and genes coding for NAD(P)H-dependent oxidoreductases operating on complex organic substrates, including aldo-keto reductases). These results demonstrate that transient oxidative stress in cells exposed to 3-BP is followed by antioxidant response.

Discovery of a new metal and NAD+-dependent formate dehydrogenase from Clostridium ljungdahlii

ABSTRACTOver the next decades, with the growing concern of rising atmospheric carbon dioxide (CO2) levels, the importance of investigating new approaches for its reduction becomes crucial. Reclamation of CO2 for conversion into biofuels represents an alternative and attractive production method that has been studied in recent years, now with enzymatic methods gaining more attention. Formate dehydrogenases (FDHs) are NAD(P)H-dependent oxidoreductases that catalyze the conversion of formate into CO2 and have been extensively used for cofactor recycling in chemoenzymatic processes. A new FDH from Clostridium ljungdahlii (ClFDH) has been recently shown to possess activity in the reverse reaction: the mineralization of CO2 into formate. In this study, we show the successful homologous expression of ClFDH in Escherichia coli. Biochemical and kinetic characterization of the enzyme revealed that this homologue also demonstrates activity toward CO2 reduction. Structural analysis of the enzyme through homology modeling is also presented.

Ochrobactrum sp. MPV1 from a dump of roasted pyrites can be exploited as bacterial catalyst for the biogenesis of selenium and tellurium nanoparticles

BackgroundBacteria have developed different mechanisms for the transformation of metalloid oxyanions to non-toxic chemical forms. A number of bacterial isolates so far obtained in axenic culture has shown the ability to bioreduce selenite and tellurite to the elemental state in different conditions along with the formation of nanoparticles—both inside and outside the cells—characterized by a variety of morphological features. This reductive process can be considered of major importance for two reasons: firstly, toxic and soluble (i.e. bioavailable) compounds such as selenite and tellurite are converted to a less toxic chemical forms (i.e. zero valent state); secondly, chalcogen nanoparticles have attracted great interest due to their photoelectric and semiconducting properties. In addition, their exploitation as antimicrobial agents is currently becoming an area of intensive research in medical sciences.ResultsIn the present study, the bacterial strain Ochrobactrum sp. MPV1, isolated from a dump of roasted arsenopyrites as residues of a formerly sulfuric acid production near Scarlino (Tuscany, Italy) was analyzed for its capability of efficaciously bioreducing the chalcogen oxyanions selenite (SeO32−) and tellurite (TeO32−) to their respective elemental forms (Se0 and Te0) in aerobic conditions, with generation of Se- and Te-nanoparticles (Se- and TeNPs). The isolate could bioconvert 2 mM SeO32− and 0.5 mM TeO32− to the corresponding Se0 and Te0 in 48 and 120 h, respectively. The intracellular accumulation of nanomaterials was demonstrated through electron microscopy. Moreover, several analyses were performed to shed light on the mechanisms involved in SeO32− and TeO32− bioreduction to their elemental states. Results obtained suggested that these oxyanions are bioconverted through two different mechanisms in Ochrobactrum sp. MPV1. Glutathione (GSH) seemed to play a key role in SeO32− bioreduction, while TeO32− bioconversion could be ascribed to the catalytic activity of intracellular NADH-dependent oxidoreductases. The organic coating surrounding biogenic Se- and TeNPs was also characterized through Fourier-transform infrared spectroscopy. This analysis revealed interesting differences among the NPs produced by Ochrobactrum sp. MPV1 and suggested a possible different role of phospholipids and proteins in both biosynthesis and stabilization of such chalcogen-NPs.ConclusionsIn conclusion, Ochrobactrum sp. MPV1 has demonstrated to be an ideal candidate for the bioconversion of toxic oxyanions such as selenite and tellurite to their respective elemental forms, producing intracellular Se- and TeNPs possibly exploitable in biomedical and industrial applications.

Isolation, Characterization and Bioactivities of an Extracellular Polysaccharide Produced from Streptomyces sp. MOE6.

A Streptomyces strain was isolated from soil and the sequence of 1471 nucleotides of its 16S rDNA showed 99% identity to Streptomyces sp. HV10. This newly isolated Streptomyces strain produced an extracellular polysaccharide (EPS) composed mainly of glucose and mannose in a ratio of 1:4.1, as was characterized by Fourier transform infrared spectroscopy (FTIR), HPLC and 1H-NMR. The antioxidant activities of the partially purified MOE6-EPS were determined by measuring the hydroxyl free radical scavenging activity and the scavenging of 2,2-diphenyl-2-picryl-hydrazyl (DPPH) radicals. In addition, the partially purified MOE6-EPS showed high ferrous ion (Fe2+) chelation activity which is another antioxidant activity. Interestingly, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays that were colorimetric assays for NAD(P)H-dependent cellular oxidoreductases and a proxy of the number of viable cells, showed that the partially purified MOE6-EPS inhibited the proliferation of the human breast cancer cells (MDA-MB-231). The scratch wound assay showed that MOE6-EPS reduced the migration of mouse breast cancer cells (4T1). This study reports the production of EPS from Streptomyces species with promising antioxidant, metal chelating and mammalian cell inhibitory activities.

Aldo-Keto Reductase Regulation by the Nrf2 System: Implications for Stress Response, Chemotherapy Drug Resistance, and Carcinogenesis.

Human aldo-keto reductases (AKRs) are NAD(P)H-dependent oxidoreductases that convert aldehydes and ketones to primary and secondary alcohols for subsequent conjugation reactions and can be referred to as "phase 1" enzymes. Among all the human genes regulated by the Keap1/Nrf2 pathway, they are consistently the most overexpressed in response to Nrf2 activators. Although these enzymes play clear cytoprotective roles and deal effectively with carbonyl stress, their upregulation by the Keap1/Nrf2 pathway also has a potential dark-side, which can lead to chemotherapeutic drug resistance and the metabolic activation of lung carcinogens (e.g., polycyclic aromatic hydrocarbons). They also play determinant roles in 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone metabolism to R- and S-4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol. The overexpression of AKR genes as components of the "smoking gene" battery raises the issue as to whether this is part of a smoking stress response or acquired susceptibility to lung cancer. Human AKR genes also regulate retinoid, prostaglandin, and steroid hormone metabolism and can regulate the local concentrations of ligands available for nuclear receptors (NRs). The prospect exists that signaling through the Keap1/Nrf2 system can also effect NR signaling, but this has remained largely unexplored. We present the case that chemoprevention through the Keap1/Nrf2 system may be context dependent and that the Nrf2 "dose-response curve" for electrophilic and redox balance may not be monotonic.

Identification of catalysis, substrate, and coenzyme binding sites and improvement catalytic efficiency of formate dehydrogenase from Candida boidinii.

Formate dehydrogenases (FDHs) are continually used for the cofactor regeneration in biocatalysis and biotransformation with hiring NAD(P)H-dependent oxidoreductases. Major weaknesses of most native FDHs are their low activity and operational stability in the catalytic reaction. In this work, the FDH from Candida boidinii (CboFDH) was engineered in order to gain an enzyme with high activity and better operational stability. Through comparing and analyzing its spatial structure with other FDHs, the catalysis, substrate, and coenzyme binding sites of the CboFDH were identified. To improve its performance, amino acids, which concentrated on the enzyme active site or in the conserved NAD(+) and substrate binding motif, were mutated. The mutant V120S had the highest catalytic efficiency (k cat/K m ) with COONH4 as it enhanced the catalytic velocity (k cat) and k cat/K m 3.48-fold and 1.60-fold, respectively, than that of the wild type. And, the double-mutant V120S-N187D had the highest k cat/K m with NAD(+) as it displayed an approximately 1.50-fold increase in k cat/K m . The mutants showed higher catalytic efficiency than other reported FDHs, suggesting that the mutation has achieved good results. The single and double mutants exhibited higher thermostability than the wild type. The structure-function relationship of single and double mutants was analyzed by homology models and site parsing. Asymmetric synthesis of L-tert-leucine was executed to evaluate the ability of cofactor regeneration of the mutants with about 100% conversion rates. This work provides a helpful theoretical reference for the evolution of an enzyme in vitro and promotion of the industrial production of chiral compounds, e.g., amino acid and chiral amine.

Detection of NAD(P)H-dependent enzyme activity by time-domain ratiometry of terbium luminescence

NAD(P)H-dependent oxidoreductases play important roles in biology. Recently, we reported that the luminescence lifetime of some Tb3+ complexes is sensitive to NAD(P)H, and we used this phenomenon to detect activities of these enzymes. However, conventional time-resolved luminescence assays are susceptible to static quenchers such as ATP. Herein we describe a detection methodology that overcomes this issue: the intensity of the sample is measured twice with different delay times and the intensity ratio value is used as an index of NAD(P)H concentration. The method is more robust than single-point measurement, and is compatible with high-throughput assays using conventional microplate readers.

Enzyme-Modified Particles for Selective Biocatalytic Hydrogenation by Hydrogen-Driven NADH Recycling.

We describe a new approach to selective H2-driven hydrogenation that exploits a sequence of enzymes immobilised on carbon particles. We used a catalyst system that comprised alcohol dehydrogenase, hydrogenase and an NAD+ reductase on carbon black to demonstrate a greater than 98 % conversion of acetophenone to phenylethanol. Oxidation of H2 by the hydrogenase provides electrons through the carbon for NAD+ reduction to recycle the NADH cofactor required by the alcohol dehydrogenase. This biocatalytic system operates over the pH range 6–8 or in un-buffered water, and can function at low concentrations of the cofactor (10 μm NAD+) and at H2 partial pressures below 1 bar. Total turnover numbers >130 000 during acetophenone reduction indicate high enzyme stability, and the immobilised enzymes can be recovered by a simple centrifugation step and re-used several times. This offers a route to convenient, atom-efficient operation of NADH-dependent oxidoreductases for selective hydrogenation catalysis.

Stereoselective synthesis of phenylpropane 1,2-diols from (S)-2-hydroxypropiophenone by NADH-dependent oxidoreductases

In this study, alcohol dehydrogenase (EC 1.1.1.2) from Lactobacillus brevis and glycerol dehydrogenase (EC 1.1.1.6) from Cellulomonas sp. were used for the reduction of (S)-2-hydroxypropiophenone to (1S, 2S)- and (1R, 2S)-1-phenylpropane-1,2-diol, respectively. As both dehydrogenases belong to NADH-dependent oxidoreductases, due to high coenzyme cost, the possibility of NADH regeneration was considered by using the enzyme-coupled regeneration system with formate dehydrogenase (EC 1.2.1.2) from Candida boidinii. All three enzymes were kinetically characterized and the kinetics of all reactions were determined using the method of initial rates. Based on kinetic results mathematical models were developed for the synthesis of both phenylpropane 1,2-diols for the systems with and without coenzyme regeneration and were validated in a batch reactor. The equilibrium conversions of 60% and 33% were achieved without coenzyme regeneration using alcohol and glycerol dehydrogenase, respectively, while with coenzyme regeneration a complete (S)-2-hydroxypropiophenone conversion was achieved with both enzymes.

Crystallographic analysis of a novel aldo-keto reductase from Thermotoga maritima in complex with NADP⁺.

Aldo-keto reductases (AKRs) are a superfamily of NAD(P)H-dependent oxidoreductases that catalyse the asymmetric reduction of aldehydes and ketones to chiral alcohols in various organisms. The novel aldo-keto reductase Tm1743 from Thermotoga maritima was identified to have a broad substrate specificity and high thermostability, serving as an important enzyme in biocatalysis and fine-chemical synthesis. In this study, Tm1743 was overexpressed in Escherichia coli BL21(DE3) cells with an N-terminal His6 tag and was purified by Ni(2+)-chelating affinity and size-exclusion chromatography. Purified recombinant enzyme was incubated with its cofactor NADP(+) and its substrate ethyl 2-oxo-4-phenylbutyrate (EOPB) for crystallization. Two X-ray diffraction data sets were collected at 2.0 and 1.7 Å resolution from dodecahedral crystals grown from samples containing Tm1743-NADP(+)-EOPB and Tm1743-NADP(+), respectively. Both crystals belonged to space group P3121, with similar unit-cell parameters. However, in the refined structure model only NADP(+) was observed in the active site of the full-length Tm1743 enzyme. Degradation of the N-terminal vector-derived amino acids during crystallization was confirmed by Western blot and mass-spectrometric analyses.