Reduced Nicotinamide Cofactors Research Articles

Evolution of the catalytic mechanism at the dawn of the Baeyer-Villiger monooxygenases

Enzymes are crucial for the emergence and sustenance of life on earth. How they became catalytically activeduring their evolution is still an open question. Two opposite explanations are plausible: acquiring a mechanism in a series of discrete steps or all at once in a single evolutionary event. Here, we use molecular phylogeny, ancestral sequence reconstruction, and biochemical characterization to follow the evolution of a specialized group of flavoprotein monooxygenases, the bacterial Baeyer-Villiger monooxygenases (BVMOs). These enzymes catalyze an intricate chemical reaction relying on three different elements: a reduced nicotinamide cofactor, dioxygen, and a substrate. Characterization of ancestral BVMOs shows that the catalytic mechanism evolved in a series of steps starting from a FAD-binding protein and further acquiring reactivity and specificity toward each of the elements participating in the reaction. Together, the results of our work portray how an intrinsically complex catalytic mechanism emerged during evolution.

Catalytic Promiscuity of Horseradish Peroxidase: Aerobic Oxidative Regeneration of Oxidized Nicotinamide Cofactors

AbstractNAD(P)+ regeneration is of great importance for dehydrogenase (DH)‐catalyzed oxidations. In this work, we report horseradish peroxidase (HRP)‐catalyzed aerobic oxidation of the reduced nicotinamide cofactors, including natural and non‐natural ones, into the oxidized form at neutral and alkaline pH enabled by external amino acids (AAs). HRP showed promiscuous yet extremely low oxidase activity toward NADH at pH 7 in the absence of both AAs and mediators, as evidenced by the observation of compound III. The mechanistic study revealed that AAs as decoy molecules (likely act as acid‐base catalysts) and mediators significantly promoted the formation of compound III as well as its decay back to the ground state. The applicability of the aerobic cofactor regeneration method was demonstrated by the HRP total turnover number up to 88 000 as well as high yields (91–99 %) in bi‐enzymatic synthesis of carboxylic acids. The present work offers an alternative method to recycle NAD(P)+. Also, it may open up possibilities for HRP‐catalyzed aerobic oxidations.

A Hitchhiker's Guide to Supplying Enzymatic Reducing Power into Synthetic Cells.

The construction from scratch of synthetic cells by assembling molecular building blocks is unquestionably an ambitious goal from a scientific and technological point of view. To realize functional life-like systems, minimal enzymatic modules are required to sustain the processes underlying the out-of-equilibrium thermodynamic status hallmarking life, including the essential supply of energy in the form of electrons. The nicotinamide cofactors NAD(H) and NADP(H) are the main electron carriers fueling reductive redox reactions of the metabolic network of living cells. One way to ensure the continuous availability of reduced nicotinamide cofactors in a synthetic cell is to build a minimal enzymatic module that can oxidize an external electron donor and reduce NAD(P)+. In the diverse world of metabolism there is a plethora of potential electron donors and enzymes known from living organisms to provide reducing power to NAD(P)+ coenzymes. This perspective proposes guidelines to enable the reduction of nicotinamide cofactors enclosed in phospholipid vesicles, while avoiding high burdens of or cross-talk with other encapsulated metabolic modules. By determining key requirements, such as the feasibility of the reaction and transport of the electron donor into the cell-like compartment, we select a shortlist of potentially suitable electron donors. We review the most convenient proteins for the use of these reducing agents, highlighting their main biochemical and structural features. Noting that specificity toward either NAD(H) or NADP(H) imposes a limitation common to most of the analyzed enzymes, we discuss the need for specific enzymes─transhydrogenases─to overcome this potential bottleneck.

Native amine dehydrogenases can catalyze the direct reduction of carbonyl compounds to alcohols in the absence of ammonia

Native amine dehydrogenases (nat-AmDHs) catalyze the (S)-stereoselective reductive amination of various ketones and aldehydes in the presence of high concentrations of ammonia. Based on the structure of CfusAmDH from Cystobacter fuscus complexed with Nicotinamide adenine dinucleotide phosphate (NADP+) and cyclohexylamine, we previously hypothesized a mechanism involving the attack at the electrophilic carbon of the carbonyl by ammonia followed by delivery of the hydride from the reduced nicotinamide cofactor on the re-face of the prochiral ketone. The direct reduction of carbonyl substrates into the corresponding alcohols requires a similar active site architecture and was previously reported as a minor side reaction of some native amine dehydrogenases and variants. Here we describe the ketoreductase (KRED) activity of a set of native amine dehydrogenases and variants, which proved to be significant in the absence of ammonia in the reaction medium but negligible in its presence. Conducting this study on a large set of substrates revealed the heterogeneity of this secondary ketoreductase activity, which was dependent upon the enzyme/substrate pairs considered. In silico docking experiments permitted the identification of some relationships between ketoreductase activity and the structural features of the enzymes. Kinetic studies of MsmeAmDH highlighted the superior performance of this native amine dehydrogenases as a ketoreductase but also its very low activity towards the reverse reaction of alcohol oxidation.

H2-Driven Reduction of Flavin by Hydrogenase Enables Cleaner Operation of Nitroreductases for Nitro-Group to Amine Reductions

Hydrogenase-mediated reduction of flavin mononucleotide by H2 is exploited to enable cleaner application of nitroreductase enzymes for reduction of aromatic nitro functional groups. This turns the overall reaction into a biocatalytic hydrogenation. Use of flavin-containing nitroreductases in industrial biotechnology typically relies upon NADH or NADPH as reductant, together with glucose dehydrogenase and glucose as a regeneration system for the reduced nicotinamide cofactor, with 3 equivalents of the carbon-intensive glucose required for a single 6-electron nitro to amine conversion. We show here that reduced flavin mononucleotide is an alternative reductant for nitroreductases, and by combining this with H2-driven recycling of reduced flavin, we avoid glucose, thereby enabling atom-efficient biocatalytic nitro reductions. We compare this biocatalytic system, via green chemistry metrics, to existing strategies for biocatalytic nitro-group reductions, particularly with respect to replacing glucose with H2 gas. We take steps towards demonstrating industrial viability: we report an overexpression system for E. coli hydrogenase 1, giving a 12-fold improvement in enzyme yield; we show a reaction in which the hydrogenase exhibits > 26,000 enzyme turnovers; and we demonstrate reasonable solvent tolerance of the hydrogenase and flavin reduction system which would enable reaction intensification.

Insight into redox regulation of apoptosis in cancer cells with multiparametric live-cell microscopy

Cellular redox status and the level of reactive oxygen species (ROS) are important regulators of apoptotic potential, playing a crucial role in the growth of cancer cell and their resistance to apoptosis. However, the relationships between the redox status and ROS production during apoptosis remain poorly explored. In this study, we present an investigation on the correlations between the production of ROS, the redox ratio FAD/NAD(P)H, the proportions of the reduced nicotinamide cofactors NADH and NADPH, and caspase-3 activity in cancer cells at the level of individual cells. Two-photon excitation fluorescence lifetime imaging microscopy (FLIM) was applied to monitor simultaneously apoptosis using the genetically encoded sensor of caspase-3, mKate2-DEVD-iRFP, and the autofluorescence of redox cofactors in colorectal cancer cells upon stimulation of apoptosis with staurosporine, cisplatin or hydrogen peroxide. We found that, irrespective of the apoptotic stimulus used, ROS accumulation correlated well with both the elevated pool of mitochondrial, enzyme-bound NADH and caspase-3 activation. Meanwhile, a shift in the contribution of bound NADH could develop independently of the apoptosis, and this was observed in the case of cisplatin. An increase in the proportion of bound NADPH was detected only in staurosporine-treated cells, this likely being associated with a high level of ROS production and their resulting detoxification. The results of the study favor the discovery of new therapeutic strategies based on manipulation of the cellular redox balance, which could help improve the anti-tumor activity of drugs and overcome apoptotic resistance.

Lignin-Induced CaCO3 Vaterite Structure for Biocatalytic Artificial Photosynthesis.

The vaterite phase of CaCO3 exhibits unique characteristics, such as high porosity, surface area, dispersivity, and low specific gravity, but it is the most unstable polymorph. Here, we report lignin-induced stable vaterite as a support matrix for integrated artificial photosynthesis through the encapsulation of key active components such as the photosensitizer (eosin y, EY) and redox enzyme (l-glutamate dehydrogenase, GDH). The lignin-vaterite/EY/GDH photobiocatalytic platform enabled the regeneration of the reduced nicotinamide cofactor under visible light and facilitated the rapid conversion of α-ketoglutarate into l-glutamate (initial conversion rate, 0.41 mM h-1; turnover frequency, 1060 h-1; and turnover number, 39,750). The lignin-induced vaterite structure allowed for long-term protection and recycling of the active components while facilitating the photosynthesis reaction due to the redox-active lignin. Succession of stability tests demonstrated a significant improvement of GDH's robustness in the lignin-vaterite structure against harsh environments. This work provides a simple approach for solar-to-chemical conversion using a sustainable, integrated light-harvesting system.

Hemoprotein Catalyzed Oxygenations: P450s, UPOs, and Progress toward Scalable Reactions.

The selective oxygenation of nonactivated carbon atoms is an ongoing synthetic challenge, and biocatalysts, particularly hemoprotein oxygenases, continue to be investigated for their potential, given both their sustainable chemistry credentials and also their superior selectivity. However, issues of stability, activity, and complex reaction requirements often render these biocatalytic oxygenations problematic with respect to scalable industrial processes. A continuing focus on Cytochromes P450 (P450s), which require a reduced nicotinamide cofactor and redox protein partners for electron transport, has now led to better catalysts and processes with a greater understanding of process requirements and limitations for both in vitro and whole-cell systems. However, the discovery and development of unspecific peroxygenases (UPOs) has also recently provided valuable complementary technology to P450-catalyzed reactions. UPOs need only hydrogen peroxide to effect oxygenations but are hampered by their sensitivity to peroxide and also by limited selectivity. In this Perspective, we survey recent developments in the engineering of proteins, cells, and processes for oxygenations by these two groups of hemoproteins and evaluate their potential and relative merits for scalable reactions.

Characterization and Application of a Robust Glucose Dehydrogenase from Paenibacillus pini for Cofactor Regeneration in Biocatalysis.

Glucose dehydrogenases are important auxiliary enzymes in biocatalysis, employed in the regeneration of reduced nicotinamide cofactors for oxidoreductase catalysed reactions. Here we report the identification and characterization of a novel glucose-1-dehydrogenase (GDH) from Paenibacillus pini that prefers NAD+ as cofactor over NADP+. The purified recombinant P. pini GDH displayed a specific activity of 247.5U/mg. The enzyme was stable in the pH range 4-8.5 and exhibited excellent thermostability till 50°C for 24h, even in the absence of NaCl or glycerol. Paenibacillus pini GDH was also tolerant to organic solvents, demonstrating its potential for recycling cofactors for biotransformation. The potential application of the enzyme was evaluated by coupling with a NAD+-dependent alcohol dehydrogenase for the reduction of acetophenone and ethyl-4-chloro-3-oxo-butanoate. Conversions higher than 95% were achieved within 2h with low enzyme loading using lyophilized cell lysate, suggesting that P. pini GDH could be highly effective for recycling NADH in redox biocatalysis.

Myoglobin-Catalyzed Efficient In Situ Regeneration of NAD(P)+and Their Synthetic Biomimetic for Dehydrogenase-Mediated Oxidations

On the basis of its catalytic promiscuity, wild-type myoglobin (Mb) from horse heart was used for in situ regeneration of oxidized nicotinamide cofactors. Mb proved to be a versatile catalyst, since it was capable of efficient oxidation of both natural cofactors NAD(P)H and their synthetic biomimetic BNAH with H2O2. Mb-catalyzed oxidation of reduced nicotinamide cofactors was promoted significantly in the presence of mediators such as scopoletin and acetaminophen. A cofactor total turnover number (TTN) up to 50 000 was obtained in glucose dehydrogenase-catalyzed oxidation of glucose coupled with the Mb/scopoletin regeneration system. Approximately 4 mol of glucose was enzymatically oxidized with 1 mol of H2O2. Besides, this system is well compatible with various dehydrogenases. The desired products were afforded with yields of 93–97% in dehydrogenase-catalyzed oxidations. A radical mechanism was proposed for Mb/scopoletin-catalyzed recycling of NAD(P)+. The Mb-based recycling system may create opportuniti...

Dual-Functional Small Molecules for Generating an Efficient Cytochrome P450BM3 Peroxygenase.

We report a unique strategy for the development of a H2 O2 -dependent cytochrome P450BM3 system, which catalyzes the monooxygenation of non-native substrates with the assistance of dual-functional small molecules (DFSMs), such as N-(ω-imidazolyl fatty acyl)-l-amino acids. The acyl amino acid group of DFSM is responsible for bounding to enzyme as an anchoring group, while the imidazolyl group plays the role of general acid-base catalyst in the activation of H2 O2 . This system affords the best peroxygenase activity for the epoxidation of styrene, sulfoxidation of thioanisole, and hydroxylation of ethylbenzene among those P450-H2 O2 system previously reported. This work provides the first example of the activation of the normally H2 O2 -inert P450s through the introduction of an exogenous small molecule. This approach improves the potential use of P450s in organic synthesis as it avoids the expensive consumption of the reduced nicotinamide cofactor NAD(P)H and its dependent electron transport system. This introduces a promising approach for exploiting enzyme activity and function based on direct chemical intervention in the catalytic process.

Dual‐Functional Small Molecules for Generating an Efficient Cytochrome P450BM3 Peroxygenase

AbstractWe report a unique strategy for the development of a H2O2‐dependent cytochrome P450BM3 system, which catalyzes the monooxygenation of non‐native substrates with the assistance of dual‐functional small molecules (DFSMs), such as N‐(ω‐imidazolyl fatty acyl)‐l‐amino acids. The acyl amino acid group of DFSM is responsible for bounding to enzyme as an anchoring group, while the imidazolyl group plays the role of general acid–base catalyst in the activation of H2O2. This system affords the best peroxygenase activity for the epoxidation of styrene, sulfoxidation of thioanisole, and hydroxylation of ethylbenzene among those P450–H2O2 system previously reported. This work provides the first example of the activation of the normally H2O2‐inert P450s through the introduction of an exogenous small molecule. This approach improves the potential use of P450s in organic synthesis as it avoids the expensive consumption of the reduced nicotinamide cofactor NAD(P)H and its dependent electron transport system. This introduces a promising approach for exploiting enzyme activity and function based on direct chemical intervention in the catalytic process.

Complete Enzymatic Oxidation of Methanol to Carbon Dioxide: Towards More Eco‐Efficient Regeneration Systems for Reduced Nicotinamide Cofactors

AbstractA novel system for in situ regeneration of reduced nicotinamide cofactors (NADH) is proposed: through a cascade of alcohol dehydrogenase (ADH), formaldehyde dismutase (FDM) and formate dehydrogenase (FDH) complete oxidation of methanol to carbon dioxide (CO2) is coupled to the regeneration of NADH. As a consequence, from one equivalent of methanol three equivalents of NADH can be obtained. The feasibility of this cascade is demonstrated at the examples of an NADH‐dependent reduction of conjugated CC‐double bonds (catalysed by an enoate reductase) and the NADH‐dependent hydroxylation of phenols (catalysed by a monooxygenase). The major limitation of the current regeneration system is the comparably poor catalytic efficiency of the methanol oxidation step (low kcat and high KM value of the ADH used) necessitating higher than theoretical methanol concentrations.magnified image

Trapping of a Cross-Link Formed by a Major Purine Adduct of a Metabolite of the CarcinogenN-Nitrosomorpholine by Inorganic and Biological Reductants

3-Hydroperoxy-N-nitrosomorpholine in buffered aqueous media in the presence of calf thymus DNA was treated with a phosphine reductant to generate the transient α-hydroxynitrosamine and subsequent diazonium ion that alkylated the DNA, as previously reported. Subsequent addition of hydride donors, for 30 min, followed by acid hydrolysis of the mixture allowed detection and quantification of 6-(2-{2-[(9H-purin-6-yl)amino]ethoxy}ethoxy)-9H-purin-2-amine, the reduced cross-link formed from deposition, via the diazonium ion, of a 3-oxapentanal fragment on O(6)-Gua, and condensation with N(6)-Ade, presumably in the vicinity. Decreasing the temperature of the reaction mixtures and decreasing the pH modestly increased the yields of the trapped cross-link. Among three borohydride reductants, NaNCBH3 is superior, being ∼4 times more effective on a molar basis, as opposed to a hydride equivalent basis, than NaBH4 or Na(AcO)3BH. For trapping with NaNCBH3, it is deduced that the reaction likely occurs with the iminium ion that is in protonic equilibrium with its conjugate base imine. In an experiment in which the hydroperoxide was decomposed and NaNCBH3 was introduced after various periods of time, the amount of cross-link was observed to increase, nearly linearly, by ∼4-fold over 1 week. These data indicate that there are a minimum of two populations of cross-links, one that forms rapidly, in minutes, and another that grows in with time, over days. Reduced nicotinamide cofactors and ascorbate are observed to effect reduction (over 3 days) of the cross-links, confirming the possibility that otherwise reversible cross-links might be immortalized under biological conditions.

A modular system for regeneration of NADcofactors using graphite particles modified with hydrogenase and diaphorase moieties

Pyrolytic graphite particles modified with hydrogenase and an NAD(+)/NADH cycling enzyme provide a modular heterogeneous catalyst system for regeneration of oxidised or reduced nicotinamide cofactors using H(2) and H(+) as electron source or sink. Particles can be tuned for cofactor supply under different conditions by appropriate choice of hydrogenase.

Photoenzymatic Reduction of CC Double Bonds

AbstractA simplified procedure for cell‐free biocatalytic reductions of conjugated CC double bonds using old yellow enzymes (OYEs) is reported. Instead of indirectly regenerating YqjM (an OYE homologue from B. subtilis) or NemA (N‐ethylmaleimide reductase from E. coli) via regeneration of reduced nicotinamide cofactors, we demonstrate that direct regeneration of catalytically active reduced flavins is an efficient and convenient approach. Reducing equivalents are provided from simple sacrificial electron donors such as ethylenediaminetetraacetate (EDTA), formate, or phosphite via photocatalytic oxidation. This novel photoenzymatic reaction scheme was characterized. Up to 65% rates of the NADH‐driven reaction were obtained while preserving enantioselectivity. The chemoselectivity of the novel approach was exclusive. Even when using crude cell extracts as biocatalyst preparations, only CC bond reduction was observed while ketone and aldehyde groups remained unaltered. Overall, a simple and practical approach for photobiocatalytic reductions is presented.

Iron oxide/carbon black (Fe 2O 3/CB) composite electrode for the detection of reduced nicotinamide cofactors using an amperometric method under a low overpotential

An amperometric biosensor for the detection of the reduced nicotinamide cofactors NADH and NADPH was designed, based on the electrochemical oxidation of NAD(P)H with an iron oxide/carbon black composite (Fe 2O 3/CB) electrode. The electrode exhibited excellent performances in that it led to a substantial decrease in the overpotential of electrochemical NADH oxidation. Iron oxide plays a significant role as a catalyst for NADH oxidation and the reaction occurs at +0.00 V (vs. Ag/AgCl). The method of the sensor construction is very simple and the sensor performed well, giving high sensitivity, high stability, and a broad detection range. The sensitivity of this system is 2.54 μA mM −1 and the limit of detection (S/N = 3) is 10 μM. A linear range was observed between 10 μM and 1000 μM of NADH ( R 2 = 0.993), which is preferable to that of the previous studies. The Fe 2O 3/CB electrode also oxidizes NADPH under the same condition and can be applied as an NADPH sensor. Moreover, when the sensor system was integrated into a dehydrogenase-based sensor system, it also showed a good sensing performance.

A New Regeneration System for Oxidized Nicotinamide Cofactors

Abstractmagnified imageA novel regeneration system for oxidized nicotinamide cofactors (NAD+ and NADP+) is presented. By combining 2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulphonic acid (ABTS)‐catalyzed oxidation of NAD(P)H with laccase‐catalyzed utilization of molecular oxygen as terminal oxidant, a simple chemo‐enzymatic NAD(P)+ regeneration method is achieved. Thus, the advantages of both worlds, chemical oxidation of reduced nicotinamide cofactors and laccase‐catalyzed utilization of oxygen from air are combined in a simple and generally applicable new approach for biooxidation catalysis. This new application of the well‐known laccase‐mediator system (LMS) is successfully used to promote alcohol dehydrogenase‐catalyzed oxidation reactions of primary and secondary alcohols. Already under non‐optimized conditions, high turnover numbers of >300 and >16000 were obtained for the nicotinamide cofactor and ABTS, respectively. In this communication, we present the proof‐of‐principle and initial characterization of the proposed new regeneration system.

Interactions of Substrates at the Surface of P450s Can Greatly Enhance Substrate Potency,

Cytochrome P450s are a superfamily of heme containing enzymes that use molecular oxygen and electrons from reduced nicotinamide cofactors to monooxygenate organic substrates. The fatty acid hydroxylase P450BM-3 has been particularly widely studied due to its stability, high activity, similarity to mammalian P450s, and presence of a cytochrome P450 reductase domain that allows the enzyme to directly receive electrons from NADPH without a requirement for additional redox proteins. We previously characterized the substrate N-palmitoylglycine, which found extensive use in studies of P450BM-3 due to its high affinity, high turnover number, and increased solubility as compared to fatty acid substrates. Here, we report that even higher affinity substrates can be designed by acylation of other amino acids, resulting in P450BM-3 substrates with dissociation constants below 100 nM. N-Palmitoyl-l-leucine and N-palmitoyl-l-methionine were found to have the highest affinity, with dissociation constants of less than 8 nM and turnover numbers similar to palmitic acid and N-palmitoylglycine. The interactions of the amino acid side chains with a hydrophobic pocket near R47, as revealed by our crystal structure determination of N-palmitoyl-l-methionine bound to the heme domain of P450BM-3, appears to be responsible for increasing the affinity of substrates. The side chain of R47, previously shown to be important in interactions with negatively charged substrates, does not interact strongly with N-palmitoyl-l-methionine and is found positioned at the enzyme-solvent interface. These are the tightest binding substrates for P450BM-3 reported to date, and the affinity likely approaches the maximum attainable affinity for the binding of substrates of this size to P450BM-3.

Further improvement of phosphite dehydrogenase thermostability by saturation mutagenesis

Phosphite dehydrogenase represents a new enzymatic system for regenerating reduced nicotinamide cofactors for industrial biocatalysis. We previously engineered a variant of phosphite dehydrogenase with relaxed cofactor specificity and significantly increased activity and stability. Here we performed one round of random mutagenesis followed by comprehensive saturation mutagenesis to further improve the enzyme thermostability while maintaining its activity. Two new thermostabilizing mutations were identified. These, along with the 12 mutations previously identified, were subjected to saturation mutagenesis using the parent enzyme or the engineered thermostable variant 12x as a template, followed by screening of variants with increased thermostability. Of the 12 previously identified sites, 6 yielded new variants with improved stability over the parent enzyme. Several mutations were found to be context-dependent. On the basis of molecular modeling and biochemical analysis, various mechanisms of thermostabilization were identified. Combining the most thermostabilizing mutation at each site resulted in a variant that showed a 100-fold increase in half-life at 62 degrees C over the 12x mutant. The final mutant has improved the half-life of thermal inactivation at 45 degrees C by 23,000-fold over the parent enzyme. The engineered phosphite dehydrogenase will be useful in NAD(P)H regeneration.