Efficient Whole-Cell Biocatalyst for Acetoin Production with NAD+ Regeneration System through Homologous Co-Expression of 2,3-Butanediol Dehydrogenase and NADH Oxidase in Engineered Bacillus subtilis

Acetoin (3-hydroxy-2-butanone), an extensively-used food spice and bio-based platform chemical, is usually produced by chemical synthesis methods. With increasingly requirement of food security and environmental protection, bio-fermentation of acetoin by microorganisms has a great promising market. However, through metabolic engineering strategies, the mixed acid-butanediol fermentation metabolizes a certain portion of substrate to the by-products of organic acids such as lactic acid and acetic acid, which causes energy cost and increases the difficulty of product purification in downstream processes. In this work, due to the high efficiency of enzymatic reaction and excellent selectivity, a strategy for efficiently converting 2,3-butandiol to acetoin using whole-cell biocatalyst by engineered Bacillus subtilis is proposed. In this process, NAD⁺ plays a significant role on 2,3-butanediol and acetoin distribution, so the NADH oxidase and 2,3-butanediol dehydrogenase both from B. subtilis are co-expressed in B. subtilis 168 to construct an NAD⁺ regeneration system, which forces dramatic decrease of the intracellular NADH concentration (1.6 fold) and NADH/NAD⁺ ratio (2.2 fold). By optimization of the enzymatic reaction and applying repeated batch conversion, the whole-cell biocatalyst efficiently produced 91.8 g/L acetoin with a productivity of 2.30 g/(L·h), which was the highest record ever reported by biocatalysis. This work indicated that manipulation of the intracellular cofactor levels was more effective than the strategy of enhancing enzyme activity, and the bioprocess for NAD⁺ regeneration may also be a useful way for improving the productivity of NAD⁺-dependent chemistry-based products.     

A Novel Whole-Cell Mechanism for Long-Term Memory Enhancement

Olfactory-discrimination learning was shown to induce a profound long-lasting enhancement in the strength of excitatory and inhibitory synapses of pyramidal neurons in the piriform cortex. Notably, such enhancement was mostly pronounced in a sub-group of neurons, entailing about a quarter of the cell population. Here we first show that the prominent enhancement in the subset of cells is due to a process in which all excitatory synapses doubled their strength and that this increase was mediated by a single process in which the AMPA channel conductance was doubled. Moreover, using a neuronal-network model, we show how such a multiplicative whole-cell synaptic strengthening in a sub-group of cells that form a memory pattern, sub-serves a profound selective enhancement of this memory. Network modeling further predicts that synaptic inhibition should be modified by complex learning in a manner that much resembles synaptic excitation. Indeed, in a subset of neurons all GABA_A-receptors mediated inhibitory synapses also doubled their strength after learning. Like synaptic excitation, Synaptic inhibition is also enhanced by two-fold increase of the single channel conductance. These findings suggest that crucial learning induces a multiplicative increase in strength of all excitatory and inhibitory synapses in a subset of cells, and that such an increase can serve as a long-term whole-cell mechanism to profoundly enhance an existing Hebbian-type memory. This mechanism does not act as synaptic plasticity mechanism that underlies memory formation but rather enhances the response of already existing memory. This mechanism is cell-specific rather than synapse-specific; it modifies the channel conductance rather than the number of channels and thus has the potential to be readily induced and un-induced by whole-cell transduction mechanisms.     

Development of a Continuous Bioconversion System Using a Thermophilic Whole-Cell Biocatalyst

The heat treatment of recombinant mesophilic cells having heterologous thermophilic enzymes results in the denaturation of indigenous mesophilic enzymes and the elimination of undesired side reactions; therefore, highly selective whole-cell catalysts comparable to purified enzymes can be readily prepared. However, the thermolysis of host cells leads to the heat-induced leakage of thermophilic enzymes, which are produced as soluble proteins, limiting the exploitation of their excellent stability in repeated and continuous reactions. In this study, Escherichia coli cells having the thermophilic fumarase from Thermus thermophilus (TtFTA) were treated with glutaraldehyde to prevent the heat-induced leakage of the enzyme, and the resulting cells were used as a whole-cell catalyst in repeated and continuous reactions. Interestingly, although electron microscopic observations revealed that the cellular structure of glutaraldehyde-treated E. coli was not apparently changed by the heat treatment, the membrane permeability of the heated cells to relatively small molecules (up to at least 3 kDa) was significantly improved. By applying the glutaraldehyde-treated E. coli having TtFTA to a continuous reactor equipped with a cell-separation membrane filter, the enzymatic hydration of fumarate to malate could be operated for more than 600 min with a molar conversion yield of 60% or higher.     

Highly Enantioselective Production of Chiral Secondary Alcohols with Candida zeylanoides as a New Whole Cell Biocatalyst

The increasing demand for biocatalysts in synthesizing enantiomerically pure chiral alcohols results from the outstanding characteristics of biocatalysts in reaction, economic, and ecological issues. Herein, fifteen yeast strains belonging to three food originated yeast species Candida zeylanoides, Pichia fermentans, and Saccharomyces uvarum were tested for their capability for asymmetric reduction of acetophenone to 1‐phenylethanol as biocatalysts. Of these strains, C. zeylanoides P1 showed an effective asymmetric reduction ability. Under optimized conditions, substituted acetophenones were converted to corresponding optically active secondary alcohols in up to 99% enantiomeric excess and at high yields. The preparative scale asymmetric bioreduction of 4‐nitroacetophenone ( 1m ) by C. zeylanoides P1 gave (S)‐1‐(4‐nitrophenyl)ethanol ( 2m ) with 89% yield and > 99% enantiomeric excess. Compound 2m has been obtained in an enantiomerically pure and inexpensive form. Additionally, these results indicate that C. zeylanoides P1 is a promising biocatalyst for the synthesis of chiral alcohols in industry.     

新生物催化剂的筛选与开发

生物催化剂是具有催化作用的游离或固定化细胞和游离或固定化酶的统称.目前,筛选新生物催化剂的方法有两种:一是从环境样品中筛选全新的生物催化剂;二是探索现有生物催化剂的非天然新活力.本文详细综述了筛选新生物催化剂的方法及策略,并着重介绍了从微生物源开发新生物催化剂的方法.     

L-茶氨酸新型酶法制备及其催化工艺优化

L-茶氨酸是茶叶中游离氨基酸的主要组成部分,关于其良好的生理活性已有广泛报道。首次报道了来源于Cunnighamella echinulata 9980的L-氨基酰化酶用于高光学纯度的L-茶氨酸的酶法制备。该酶在pH 6.5,底物N-乙酰-DL-茶氨酸浓度为50 mM,且有40 mM CoCl2时催化效果较好。结果表明,在上述条件下,50℃作用12 h得L-茶氨酸22.5 mM,转化率90%。     

Efficient Repeated Use of Alcohol Dehydrogenase with NAD + Regeneration in an Aqueous-organic Two-phase System

Bioconversion of cinnamyl alcohol to cinnamaldehyde was carried out in an aqueous-organic two-phase reaction system by the repeated use of horse liver alcohol dehydrogenase (HLADH) and NAD + with coenzyme regeneration. Both HLADH and the coenzyme were efficiently entrapped in the aqueous phase, while the substrate was supplied successively from the organic phase and the product was accumulated in the organic phase. Optimum conditions for cinnamaldehyde production in the aqueous-organic two-phase system were also examined, including substrate concentration, pH, and organic solvent type. Under suitable conditions, both HLADH and NAD + in the aqueous-organic two-phase system could be reused, and NAD + cycling numbers of 3040 were obtained after repeated operation for 40 &#117 h.     

Efficient Repeated Use of Alcohol Dehydrogenase with NAD + Regeneration in an Aqueous-organic Two-phase System

Bioconversion of cinnamyl alcohol to cinnamaldehyde was carried out in an aqueous-organic two-phase reaction system by the repeated use of horse liver alcohol dehydrogenase (HLADH) and NAD + with coenzyme regeneration. Both HLADH and the coenzyme were efficiently entrapped in the aqueous phase, while the substrate was supplied successively from the organic phase and the product was accumulated in the organic phase. Optimum conditions for cinnamaldehyde production in the aqueous-organic two-phase system were also examined, including substrate concentration, pH, and organic solvent type. Under suitable conditions, both HLADH and NAD + in the aqueous-organic two-phase system could be reused, and NAD + cycling numbers of 3040 were obtained after repeated operation for 40 λh.     

Novel photocatalytic NAD+ recycling system with a semiconductor in organic solvent

A novel, simple NAD⁺ recycling system in an organic solvent is described. Alcohol dehydrogenase and NAD⁺ adsorbed on a semiconductor, TIO₂, catalyzed dehydrogenation of cinnamyl alcohol through photocatalytic regeneration of NAD⁺. The merit of this system in an organic solvent is discussed.     

Engineering of a Stable Whole-Cell Biocatalyst Capable of (S)-Styrene Oxide Formation for Continuous Two-Liquid-Phase Applications

Recombinant strains of Pseudomonas putida KT2440 carrying genetic expression cassettes with xylene oxygenase- and styrene monooxygenase-encoding genes on their chromosomes could be induced in shaking-flask experiments to specific activities that rivaled those of multicopy-plasmid-based Escherichia coli recombinants. Such strains maintained the introduced styrene oxidation activity in continuous two-liquid-phase cultures for at least 100 generations, although at a lower level than in the shaking-flask experiments. The data suggest that placement of target genes on the chromosome might be a suitable route for the construction of segregationally stable and highly active whole-cell biocatalysts.     

Whole-Cell Biocatalysis for 1-Naphthol Production in Liquid-Liquid Biphasic Systems

Whole-cell biocatalysis to oxidize naphthalene to 1-naphthol in liquid-liquid biphasic systems was performed. Escherichia coli expressing TOM-Green, a variant of toluene ortho-monooxygenase (TOM), was used for this oxidation. Three different solvents, dodecane, dioctyl phthalate, and lauryl acetate, were screened for biotransformations in biphasic media. Of the solvents tested, lauryl acetate gave the best results, producing 0.72 ± 0.03 g/liter 1-naphthol with a productivity of 0.46 ± 0.02 g/g (dry weight) cells after 48 h. The effects of the organic phase ratio and the naphthalene concentration in the organic phase were investigated. The highest 1-naphthol concentration (1.43 g/liter) and the highest 1-naphthol productivity (0.55 g/g [dry weight] cells) were achieved by optimization of the organic phase. The ability to recycle both free cells and cells immobilized in calcium alginate was tested. Both free and immobilized cells lost more than ∼60% of their activity after the first run, which could be attributed to product toxicity. On a constant-volume basis, an eightfold improvement in 1-naphthol production was achieved using biphasic media compared to biotransformation in aqueous media.Biocatalysis has emerged as an important technology in industrial organic synthesis for the production of chemical synthons and high-value products (29, 34, 37). Biocatalysis offers the advantage of performing reactions under mild conditions and provides an environmentally benign approach for chemical reactions (1, 38). Oxygenases are a class of enzymes that have great potential and versatility for catalyzing reactions that are generally not accessible by chemical routes with high regio-, stereo-, and enantioselectivities (6, 27, 42, 43). Oxygenases introduce either one or two atoms of molecular oxygen into organic molecules using NADH or NADPH as a cofactor. To eliminate the addition of a costly cofactor, whole cells expressing oxygenases are generally used (34, 43).One of the potential applications of biocatalysis utilizing oxygenases is the oxidation of naphthalene to 1-naphthol. 1-Naphthol has wide applications in the manufacture of dyes, drugs, insecticides, perfumes, and surfactants (2, 7, 17). Tao et al. (39) have compared the reaction rates and regioselectivities of various wild-type and modified monooxygenases for the oxidation of naphthalene to 1-naphthol. Of the monooxygenases tested, the best enzyme for the oxidation of naphthalene to 1-naphthol was a toluene ortho-monooxygenase (TOM) variant, TomA3(V106A), also known as TOM-Green. TOM was isolated from Burkholderia cepacia G4 and consists of an α₂β₂γ₂ hydroxylase (encoded by tomA1, tomA3, and tomA4) with two catalytic oxygen-bridged binuclear iron centers, an NADH-oxidoreductase (encoded by tomA5), a protein (encoded by tomA2) involved in electron transfer between oxidoreductase and hydroxylase, and a relatively unknown subunit (encoded by tomA0) (26, 36). TOM-Green was produced by directed evolution of TOM with one amino acid change in the alpha-subunit of the hydroxylase (7, 33). TOM-Green retained high regioselectivity (98%) and was sevenfold faster than wild- type TOM.There has been considerable effort to identify and characterize oxidative biocatalysts for 1-naphthol production (7, 11, 26, 33, 36, 40, 41). However, this process is not economically feasible owing to the very low optimum concentration of naphthalene (0.1 mM [7], which is less than the solubility of naphthalene, 0.23 mM [28]) and the toxicity of both naphthalene and 1-naphthol (38, 44). Substrate loading has to be increased, and the toxicities of both naphthalene and 1-naphthol have to be minimized to make the process feasible. As a consequence, biotransformations in water-organic solvent biphasic media have been developed (8, 9, 12, 21, 45, 46). The use of a second phase consisting of an organic solvent not only increases substrate loading but also maintains low concentrations of toxic compounds in the aqueous phase (4). The organic solvent chosen is critical for achieving the benefits of biphasic media. Two main criteria for solvent selection are a high distribution coefficient for the product and biocompatibility with microorganisms (3, 4). Biocompatibility is generally correlated with the logP of the solvent, which is the logarithm of the partition coefficient in an octanol-water system, and organic solvents with logP values greater than 4 are generally biocompatible with microorganisms (19). However, the correlation of activity with logP is specific to the microorganism, and the critical logP above which solvents are biocompatible has to be identified for each microorganism (8, 16).Biphasic systems have been widely used for reactions involving a toxic substrate and/or product to enhance productivity or to improve recovery of the product (22-25, 31, 38). Oxidation of naphthalene has also been improved using biphasic reactions (13, 23, 35, 38). Tao et al. (38) used a biphasic system for 2-naphthol and phenol production using toluene 4-moooxygenase and its variant TmoA(I100A). They obtained 10- to 21-fold increases in 2-naphthol and phenol concentrations using dioctyl phthalate as the organic solvent. McIver et al. (23) used naphthalene dioxygenase to oxidize naphthalene to cis-(1R,2S)-1,2-naphthalene dihydrodiol using dodecane as the organic solvent and obtained a productivity of 1.7 g/g (dry weight) cells/h in the first 6 h. In spite of the significant improvements achieved by using a biphasic system for various reactions, application of this strategy to 1-naphthol production has not been explored yet. Considering the high toxicities of naphthalene and 1-naphthol (38), biphasic reactions can enhance the productivities. In this work, a biphasic system was used to increase 1-naphthol productivities with whole cells of Escherichia coli expressing the TOM-Green enzyme. Organic solvents were screened, and solvents suitable for high 1-naphthol productivity were identified. The organic phase was optimized by studying the effects of the naphthalene concentration and the organic phase ratio. The stability of the biocatalyst for recycling was also tested.     

Micro method for determination of borohydride with NAD+

A spectrophotometric method for the determination of borohydride is described. It involves the reduction of NAD⁺ to a number of isomeric forms of NADH by borohydride. In the standard assay procedure herewith presented, there is a direct proportionality between the absorption at 340 nm and the amount of borohydride in solution over the range 10–100 nmoles, with an effective “molar extinction coefficient” of 12.2 × 10³. The method is simple, rapid, and sensitive.     

Threonine 188 is critical for interaction with NAD+ in human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme in the inactivation pathway of prostaglandins. It is a member of the short-chain dehydrogenase family of enzymes. A relatively conserved threonine residue corresponding to threonine 188 of 15-PGDH is proposed to be involved in the interaction with the carboxamide group of NAD+. Site-directed mutagenesis was used to examine the important role of this residue. Threonine 188 was changed to alanine (T188A), serine (T188S) or tyrosine (T188Y) and the mutant proteins were expressed in E. coli. Western blot analysis showed that the expression levels of mutant proteins were similar to that of the wild type protein. Mutants T188A and T188Y were found to be inactive. Mutant T188S still retained substantial activity and the Km value for PGE2 was similar to the wild enzyme; however, the Km value for NAD+ was increased over 100 fold. These results suggest that threonine 188 is critical for interaction with NAD+ and contributes to the full catalytic activity of 15-PGDH.     

Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates

We have developed a sensitive and highly efficient whole-cell methodology for quantitative analysis and screening of protease activity in vivo. The method is based on the ability of a genetically encoded protease to rescue a coexpressed short-lived fluorescent substrate reporter from cytoplasmic degradation and thereby confer increased whole-cell fluorescence in proportion to the protease''s apparent activity in the Escherichia coli cytoplasm. We demonstrated that this system can reveal differences in the efficiency with which tobacco etch virus (TEV) protease processes different substrate peptides. In addition, when analyzing E. coli cells expressing TEV protease variants that differed in terms of their in vivo solubility, cells containing the most-soluble protease variant exhibited the highest fluorescence intensity. Furthermore, flow cytometry screening allowed for enrichment and subsequent identification of an optimal substrate peptide and protease variant from a large excess of cells expressing suboptimal variants (1:100,000). Two rounds of cell sorting resulted in a 69,000-fold enrichment and a 22,000-fold enrichment of the superior substrate peptide and protease variant, respectively. Our approach presents a new promising path forward for high-throughput substrate profiling of proteases, engineering of novel protease variants with desired properties (e.g., altered substrate specificity and improved solubility and activity), and identification of protease inhibitors.Proteases constitute a group of enzymes that irreversibly catalyze the cleavage of peptide bonds and represent approximately 2% of all protein-encoding genes in living organisms (39). Besides acting as virulence factors for many pathogens (16), proteases are crucial for the regulation of numerous biological processes that influence the life and death of a cell (4). These enzymes also underlie several pathological conditions, such as cancer (13) and neurodegenerative (20) and cardiovascular (8) diseases. A key issue for increasing our knowledge about such complex biological processes, and thereby hopefully also providing possibilities for new therapeutic strategies, is to deduce the proteases’ substrate repertoires. Consequently, a lot of efforts around the world are dedicated to the characterization of proteases and their substrates (2, 31). In addition to their biological importance, proteases have attracted much interest in several biotechnological and industrial applications, such as removal of “fusion tags” from recombinant target proteins (38), as supplements in dishwashing and laundry detergents, or for bating of hides and skin in the leather industry (41, 44). Sometimes, however, their use is hindered due to limitations inherent to a specific protease: for example, low solubility, poor enzyme stability and specificity, or limited activity. It would therefore be of great aid to have powerful and straightforward methods available that facilitate the engineering of novel protease variants not suffering from such limitations.Traditionally, protease substrate specificity has been studied by comparison and alignment of naturally occurring substrate peptide sequences (7) or through biochemical analysis of cleavage products with synthetic peptides (47). More recent and powerful methods instead rely on the use of combinatorial substrate libraries, which can either be chemically or biologically generated (6, 15). Although all of these methods have proven useful in determining protease function, many suffer from being laborious and of limited throughput capacity, having an insufficient dynamic range, and resulting in limited information on the substrate profile. Moreover, only a small fraction of all proteases have been studied to date, and there is a need for novel approaches that allow for determination of protease specificity in a rapid, accurate, and quantitative manner.Concerning the engineering of enzymes toward novel desired properties, like altered substrate specificity and improved activity, solubility, and stability; researchers have relied on the use of rational design and/or directed-evolution methods in combination with appropriate screening and selection procedures (1, 10, 11, 22). For instance, various mutagenesis procedures and subsequent screening via assays that report on the successful folding of a protein of interest (9, 32, 45) have been used to engineer protein variants exhibiting improved solubility (35, 37, 46). Despite the obvious success of using such folding reporters in solubility/folding engineering projects, there is a risk that the engineered protein may lose its inherent activity since these screening procedures in general do not select for retained activity but only improved solubility/folding. Therefore, as in the case of a protease, it would be advantageous to establish a screening or selection system that has the ability to simultaneously address traits such as improved folding/solubility without loss of proteolytic activity. However, directed evolution of desired catalytic properties has proven quite a challenge. A popular strategy has been to use phage display technologies, often in combination with transition state analogues (18) or mechanism-based suicide inhibitors, for selection (30). Although successful, the enrichment conferred by these methods is generally based on binding rather than catalysis. Georgiou and coworkers circumvented this potential problem by developing an interesting system that actually enables function-based isolation of novel protease variants from large libraries (34, 42, 43). However, their methodology is dependent on the use of cell surface-displayed proteases, which is not applicable to all proteases and therefore may limit its usefulness.Herein, we present a novel, function-based, and highly efficient fluorescence-assisted whole-cell assay for characterization and engineering of proteases and their cognate substrate peptides. The method takes advantage of genetically encoded short-lived fluorescent substrates that upon coexpression of a substrate-specific protease result in a fluorescence signal, which can easily be monitored on a flow cytometer. Cells having a desired fluorescence profile can then be collected through sorting and sequenced to identify the protease-sensitive substrate peptide or protease capable of processing a particular peptide. Using this approach, we show that it is possible to analyze the efficiency with which the highly sequence-specific tobacco etch virus protease (TEVp) processes different substrate peptides and in model experiments also identify and enrich cells expressing the most favorable substrate peptide or protease from a large background of cells harboring less-efficient variants.     

A Whole-Cell Biosensor for the Detection of Gold

Geochemical exploration for gold (Au) is becoming increasingly important to the mining industry. Current processes for Au analyses require sampling materials to be taken from often remote localities. Samples are then transported to a laboratory equipped with suitable analytical facilities, such as Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) or Instrumental Neutron Activation Analysis (INAA). Determining the concentration of Au in samples may take several weeks, leading to long delays in exploration campaigns. Hence, a method for the on-site analysis of Au, such as a biosensor, will greatly benefit the exploration industry. The golTSB genes from Salmonella enterica serovar typhimurium are selectively induced by Au(I/III)-complexes. In the present study, the golTSB operon with a reporter gene, lacZ, was introduced into Escherichia coli. The induction of golTSB::lacZ with Au(I/III)-complexes was tested using a colorimetric β-galactosidase and an electrochemical assay. Measurements of the β-galactosidase activity for concentrations of both Au(I)- and Au(III)-complexes ranging from 0.1 to 5 µM (equivalent to 20 to 1000 ng g⁻¹ or parts-per-billion (ppb)) were accurately quantified. When testing the ability of the biosensor to detect Au(I/III)-complexes_(aq) in the presence of other metal ions (Ag(I), Cu(II), Fe(III), Ni(II), Co(II), Zn, As(III), Pb(II), Sb(III) or Bi(III)), cross-reactivity was observed, i.e. the amount of Au measured was either under- or over-estimated. To assess if the biosensor would work with natural samples, soils with different physiochemical properties were spiked with Au-complexes. Subsequently, a selective extraction using 1 M thiosulfate was applied to extract the Au. The results showed that Au could be measured in these extracts with the same accuracy as ICP-MS (P<0.05). This demonstrates that by combining selective extraction with the biosensor system the concentration of Au can be accurately measured, down to a quantification limit of 20 ppb (0.1 µM) and a detection limit of 2 ppb (0.01 µM).     

A New Biocatalyst for Production of Optically Pure Aryl Epoxides by Styrene Monooxygenase from Pseudomonas fluorescens ST

We developed a biocatalyst by cloning the styrene monooxygenase genes (styA and styB) from Pseudomonas fluorescens ST responsible for the oxidation of styrene to its corresponding epoxide. Recombinant Escherichia coli was able to oxidize different aryl vinyl and aryl ethenyl compounds to their corresponding optically pure epoxides. The results of bioconversions indicate the broad substrate preference of styrene monooxygenase and its potential for the production of several fine chemicals.     

NAD+ metabolism: A therapeutic target for age-related metabolic disease

Abstract

Nicotinamide adenine dinucleotide (NAD) is a central metabolic cofactor by virtue of its redox capacity, and as such regulates a wealth of metabolic transformations. However, the identification of the longevity protein silent regulator 2 (Sir2), the founding member of the sirtuin protein family, as being NAD⁺-dependent reignited interest in this metabolite. The sirtuins (SIRT1-7 in mammals) utilize NAD⁺ to deacetylate proteins in different subcellular compartments with a variety of functions, but with a strong convergence on optimizing mitochondrial function. Since cellular NAD⁺ levels are limiting for sirtuin activity, boosting its levels is a powerful means to activate sirtuins as a potential therapy for mitochondrial, often age-related, diseases. Indeed, supplying excess precursors, or blocking its utilization by poly(ADP-ribose) polymerase (PARP) enzymes or CD38/CD157, boosts NAD⁺ levels, activates sirtuins and promotes healthy aging. Here, we discuss the current state of knowledge of NAD⁺ metabolism, primarily in relation to sirtuin function. We highlight how NAD⁺ levels change in diverse physiological conditions, and how this can be employed as a pharmacological strategy.     

Adriamycin-catalyzed aerobic photooxidation of NAD dimers to NAD+

The photooxidation of the dimers of nicotinamide adenine dinucleotide, (NAD)2, is catalyzed by adriamycin under aerobic conditions. (NAD)2 and O2 react in 1:1 molar ratio to yield 2 mol of NAD+. Experiments carried out by irradiating at 340 and 485 nm, corresponding to the absorption maxima of (NAD)2 and adriamycin, respectively, clearly indicate that the process is primed by photoexcitation of adriamycin. The key step of the process is the redox reaction between (NAD)2 and adriamycin with formation of the semiquinone radical anion, identified by the EPR spectrum. The semiquinone is then oxidized back to adriamycin by oxygen with formation of the superoxide radical.     

A Novel Type II NAD+-Specific Isocitrate Dehydrogenase from the Marine Bacterium Congregibacter litoralis KT71

In most living organisms, isocitrate dehydrogenases (IDHs) convert isocitrate into ɑ-ketoglutarate (ɑ-KG). Phylogenetic analyses divide the IDH protein family into two subgroups: types I and II. Based on cofactor usage, IDHs are either NAD⁺-specific (NAD-IDH) or NADP⁺-specific (NADP-IDH); NADP-IDH evolved from NAD-IDH. Type I IDHs include NAD-IDHs and NADP-IDHs; however, no type II NAD-IDHs have been reported to date. This study reports a novel type II NAD-IDH from the marine bacterium Congregibacter litoralis KT71 (ClIDH, GenBank accession no. EAQ96042). His-tagged recombinant ClIDH was produced in Escherichia coli and purified; the recombinant enzyme was NAD⁺-specific and showed no detectable activity with NADP⁺. The K _m values of the enzyme for NAD⁺ were 262.6±7.4 μM or 309.1±11.2 μM with Mg²⁺ or Mn²⁺ as the divalent cation, respectively. The coenzyme specificity of a ClIDH Asp487Arg/Leu488His mutant was altered, and the preference of the mutant for NADP⁺ was approximately 24-fold higher than that for NAD⁺, suggesting that ClIDH is an NAD⁺-specific ancestral enzyme in the type II IDH subgroup. Gel filtration and analytical ultracentrifugation analyses revealed the homohexameric structure of ClIDH, which is the first IDH hexamer discovered thus far. A 163-amino acid segment of CIIDH is essential to maintain its polymerization structure and activity, as a truncated version lacking this region forms a non-functional monomer. ClIDH was dependent on divalent cations, the most effective being Mn²⁺. The maximal activity of purified recombinant ClIDH was achieved at 35°C and pH 7.5, and a heat inactivation experiment showed that a 20-min incubation at 33°C caused a 50% loss of ClIDH activity. The discovery of a NAD⁺-specific, type II IDH fills a gap in the current classification of IDHs, and sheds light on the evolution of type II IDHs.