Advances in physiological activities and synthesis of β-nicotinamide mononucleotide / 生物工程学报

Nicotinamide mononucleotide (NMN) is one of the key precursors of coenzyme Ⅰ (NAD+). NMN exists widely in a variety of organisms, and β isomer is its active form. Studies have shown that β-NMN plays a key role in a variety of physiological and metabolic processes. As a potential active substance in anti-aging and improving degenerative and metabolic diseases, the application value of β-NMN has been deeply explored, and it is imminent to achieve large-scale production. Biosynthesis has become the preferred method to synthesize β-NMN because of its high stereoselectivity, mild reaction conditions, and fewer by-products. This paper reviews the physiological activity, chemical synthesis as well as biosynthesis of β-NMN, highlighting the metabolic pathways involved in biosynthesis. This review aims to explore the potential of improving the production strategy of β-NMN by using synthetic biology and provide a theoretical basis for the research of metabolic pathways as well as efficient production of β-NMN.

AIFM1 variants associated with auditory neuropathy spectrum disorder cause apoptosis due to impaired apoptosis-inducing factor dimerization / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Auditory neuropathy spectrum disorder (ANSD) represents a variety of sensorineural deafness conditions characterized by abnormal inner hair cells and/or auditory nerve function, but with the preservation of outer hair cell function. ANSD represents up to 15% of individuals with hearing impairments. Through mutation screening, bioinformatic analysis and expression studies, we have previously identified several apoptosis-inducing factor (AIF) mitochondria-associated 1 (AIFM1) variants in ANSD families and in some other sporadic cases. Here, to elucidate the pathogenic mechanisms underlying each AIFM1 variant, we generated AIF-null cells using the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and constructed AIF-wild type (WT) and AIF-mutant (mut) (p.‍T260A, p.‍R422W, and p.‍R451Q) stable transfection cell lines. We then analyzed AIF structure, coenzyme-binding affinity, apoptosis, and other aspects. Results revealed that these variants resulted in impaired dimerization, compromising AIF function. The reduction reaction of AIF variants had proceeded slower than that of AIF-WT. The average levels of AIF dimerization in AIF variant cells were only 34.5%‍‒‍49.7% of that of AIF-WT cells, resulting in caspase-independent apoptosis. The average percentage of apoptotic cells in the variants was 12.3%‍‒‍17.9%, which was significantly higher than that (6.9%‍‒‍7.4%) in controls. However, nicotinamide adenine dinucleotide (NADH) treatment promoted the reduction of apoptosis by rescuing AIF dimerization in AIF variant cells. Our findings show that the impairment of AIF dimerization by AIFM1 variants causes apoptosis contributing to ANSD, and introduce NADH as a potential drug for ANSD treatment. Our results help elucidate the mechanisms of ANSD and may lead to the provision of novel therapies.

Formate dehydrogenase and its application in biomanufacturing of chiral chemicals / 生物工程学报

The redox biosynthesis system has important applications in green biomanufacturing of chiral compounds. Formate dehydrogenase (FDH) catalyzes the oxidation of formate into carbon dioxide, which is associated with the reduction of NAD(P)+ into NAD(P)H. Due to this property, FDH is used as a crucial enzyme in the redox biosynthesis system for cofactor regeneration. Nevertheless, the application of natural FDH in industrial production is hampered by low catalytic efficiency, poor stability, and inefficient coenzyme utilization. This review summarized the structural characteristics and catalytic mechanism of FDH, as well as the advances in protein engineering of FDHs toward improved enzyme activity, catalytic efficiency, stability and coenzyme preference. The applications of using FDH as a coenzyme regeneration system for green biomanufacturing of chiral compounds were summarized.

Characterization of succinic semialdehyde dehydrogenase from Aspergillus niger.

The catabolism of fungal 4-aminobutyrate (GABA) occurs via succinic semialdehyde (SSA). Succinic semialdehyde dehydrogenase (SSADH) from the acidogenic fungus Aspergillus niger was purified from GABA grown mycelia to the highest specific activity of 277 nmol min-1 mg-1, using phenyl Sepharose and DEAE Sephacel chromatography. The purified enzyme was specific for its substrates SSA and NAD+. The substrate inhibition observed with SSA was uncompetitive with respect to NAD+. While product inhibition by succinate was not observed, NADH inhibited the enzyme competitively with respect to NAD+ and noncompetitively with respect to SSA. Dead-end inhibition by AMP and p-hydroxybenzaldehyde (pHB) was analyzed. The pHB inhibition was competitive with SSA and uncompetitive with NAD+; AMP competed with NAD+. Consistent with the kinetic data, a sequential, ordered Bi Bi mechanism is proposed for this enzyme.

Anthocyanin Induces Apoptosis of DU-145 Cells In Vitro and Inhibits Xenograft Growth of Prostate Cancer

PURPOSE: To investigate the effects of anthocyanins extracted from black soybean, which have antioxidant activity, on apoptosis in vitro (in hormone refractory prostate cancer cells) and on tumor growth in vivo (in athymic nude mouse xenograft model). MATERIALS AND METHODS: The growth and viability of DU-145 cells treated with anthocyanins were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and apoptosis was assessed by DNA laddering. Immunoblotting was conducted to evaluate differences in the expressions of p53, Bax, Bcl, androgen receptor (AR), and prostate specific antigen (PSA). To study the inhibitory effects of anthocyanins on tumor growth in vivo, DU-145 tumor xenografts were established in athymic nude mice. The anthocyanin group was treated with daily oral anthocyanin (8 mg/kg) for 14 weeks. After 2 weeks of treatment, DU-145 cells (2x106) were inoculated subcutaneously into the right flank to establish tumor xenografts. Tumor dimensions were measured twice a week using calipers and volumes were calculated. RESULTS: Anthocyanin treatment of DU-145 cells resulted in 1) significant increase in apoptosis in a dose-dependent manner, 2) significant decrease in p53 and Bcl-2 expressions (with increased Bax expression), and 3) significant decrease in PSA and AR expressions. In the xenograft model, anthocyanin treatment significantly inhibit tumor growth. CONCLUSION: This study suggests that anthocyanins from black soybean inhibit the progression of prostate cancer in vitro and in a xenograft model.

Purification and Partial Characterization of a Non-specific Acid Phosphatase Degrading NAD from Aspergillus oryzae NRRL447.

A non specific acid phosphatase from Aspergillus oryzae NRRL447 catalyzes the phosphate hydrolysis from nicotinamide adenine dinucleotide forming nicotinamide riboside, adenosine and Pi as the final products of the reaction. The enzyme was purified to homogeneity by a sequential treatment of acetone fractionation, DEAE-cellulose chromatography and gel filtration chromatography. The enzyme was purified 400-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of MW 52 kDa. The enzyme displayed maximum activity at pH 5.0 and 40 °C with NAD as substrate. The enzyme activity appeared to be stable over pH 2.0–5.0 and up to 40 °C. The enzyme activity was enhanced slightly by Mg2+, Ca2+ whereas inhibited strongly by F-, Mo04 -, Cu2+ and Fe2+. The enzyme hydrolyzes several phosphate esters, suggesting a probable non-specific nature. The substrate concentration-activity relationship is the hyperbolic type and the apparent Km for NAD+ was 6.25 x 10-4 M.

Presence of bound substrate in lactate dehydrogenase from carp liver.

While attempting to purify UDP-galactose 4-epimerase from carp liver extract at pH 8.0, it was observed that the preparation even after dialysis could reduce NAD to NADH, interfering epimerase assay. The NAD reduction activity and the epimerase were co-eluted in a series of chromatographic steps. Mass spectrometric analysis of semi-purified fraction revealed that carp liver lactate dehydrogenase (LDH) contained bound lactate which was converted to pyruvate in the presence of NAD. The enzyme-bound lactate and the association with epimerase stabilized LDH from trypsin digestion and thermal inactivation at 45°C by factors of 2.7 and 4.2 respectively, as compared to substrate-free LDH. LDH and epimerase do not belong to any one pathway, but are the rate-limiting enzymes of two different pathways of carbohydrate metabolism. Typically, strongly associated enzymes work in combination, such as two enzymes of the same metabolic pathway. In that background, co-purification of LDH and epimerase as reloaded in this study was an unusual phenomenon.

Inhibition of chlorophyll biosynthesis by mercury in excised etiolated maize leaf segments during greening: effect of 2-oxoglutarate.

Mercury (0.01-1.0 mM) inhibited chlorophyll formation in greening maize leaf segments. However, supplementing incubation medium with 2-oxoglutarate, maintained substantially higher level of chlorophyll in absence of metal after an initial period of 8 hr. On preincubation of leaf segments with HgCl2, per cent inhibition of chlorophyll synthesis by metal was same in the presence and absence of 2-oxoglutarate. Supply of 2-oxoglutarate (0.1-10.0 mM) exerted concentration dependent effect on chlorophyll formation in absence or presence of metal. Increase in delta-amino levulinic acid dehydratase as well as NADH-glutamate synthase activity and decrease in NADH-glutamate dehydrogenase activity by 2-oxoglutarate in the presence of Hg suggested that glutamate for delta-amino levulinic acid synthesis could be made available from NH4+ assimilation via., glutamine synthetase/glutamate synthase pathway during mercury toxicity.

Enzymatic method for assaying calcium in serum with Ca++-ATPase

A kinetic assay for total calcium in serum was developed which is based on the activation of Ca++-ATPase by free Ca++ [Ca++]f maintained by EGTA in the reaction mixture. The concentration of Caf++ was dependent on total reference calcium added or serum calcium. Ca++-ATPase activity was coupled to the reduction of NADH by pyruvate kinase (PK) and lactate dehydrogenase (LDH) and monitored by change in absorbance at 340 nm. The calcium in normal serum was 10.08 +/- 0.24 mg/ dl (n = 35) by our method while with o-cresolphthalein complexone (CPC) method, the total calcium in the same 35 serum samples was 10.14 +/- 0.54 mg/dl. The range of within-run coefficient of variations (CVs) by this method was 0.9-2.87% at 8-12 mg/dl and day-to-day CVs were 0.72-3.17%. The presence of other ions and standard clinical interfering agents did not affect this assay system. The correlation between values obtained with our method (y) and CPC method (x) for normal serum was: y = 1.064x-0.580 mg/dl (r = 0.912, n = 59).

UDP-galactose 4-epimerase from Escherichia coli: equilibrium unfolding studies.

UDP-galactose 4-epimerase from Escherichia coli is a homodimer of 39 kDa subunit with non-covalently bound NAD acting as cofactor. The enzyme can be reversibly reactivated after denaturation and dissociation using 8 M urea at pH 7.0. There is a strong affinity between the cofactor and the refolded molecule as no extraneous NAD is required for its reactivation. Results from equilibrium denaturation using parameters like catalytic activity, circular-dichroism, fluorescence emission (both intrinsic and with extraneous fluorophore 1-aniline 8-naphthalene sulphonic acid), 'reductive inhibition' (associated with orientation of NAD on the native enzyme surface), elution profile from size-exclusion HPLC and light scattering have been compiled here. These show that inactivation, integrity of secondary, tertiary and quaternary structures have different transition mid-points suggestive of non-cooperative transition. The unfolding process may be broadly resolved into three parts: an active dimeric holoenzyme with 50% of its original secondary structure at 2.5 M urea; an active monomeric holoenzyme at 3 M urea with only 40% of secondary structure and finally further denaturation by 6 M urea leads to an inactive equilibrium unfolded state with only 20% of residual secondary structure. Thermodynamical parameters associated with some transitions have been quantitated. The results have been discussed with the X-ray crystallographic structure of the enzyme.

Blue light-sensitive plasma membrane bound exogenous NADH oxidase in Cuscuta reflexa.

Protoplasts isolated from Cuscuta reflexa exhibited a higher rate of exogenous NADH oxidation as compared to NADPH in the dark. NAD(P)H oxidation was monitored by measuring the rate of oxygen consumption and this oxidase system was sensitive to blue light. Both NADH oxidase and its blue light sensitivity were inhibited by -SH group reacting agents. The corresponding changes occurring in H+-extrusion activity and intracellular ATP levels were also monitored. Stimulation of NADH oxidation under blue light corresponded to increased rate of H+-extrusion and intracellular ATP level, the converse was also true under NADH oxidase inhibitory conditions. These observations suggested a close functional association between blue light-sensitive plasma membrane bound redox activity and H+-ATPase in this tissue. Further, concanavalin A binding of protoplasts resulted in a loss in NADH oxidase activity and its blue light sensitivity suggesting apoplastic location and glycoprotein nature of the blue light sensitive NADH oxidase system in Cuscuta.

Hepatic pyridine nucleotides content in rat--a better indicator for determining available niacin values of food.

After 10 days depletion period with niacin free diet, weanling rats were repleted with either reference diets containing niacin at four levels (2, 4, 8 and 12 mg/kg diet) or test diets containing test material at two levels as source of niacin. Gains in body weight and hepatic pyridine nucleotides content increased with increase in niacin intake. The correlation coefficient for hepatic pyridine nucleotides content and niacin intake was 0.98, whereas for weight gain and niacin intake was 0.89. Dried skim milk and pearl millet were taken as test materials. Dried skim milk with most of its niacin in free form showed 98% niacin equivalent to be bioavailable whereas in pearl millet niacin was in bound form and bioavailability equivalent was only 48 per cent.

Characterization of yeast deoxyhypusine synthase: PKC-dependent phosphorylation in vitro and functional domain identification

The biosynthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl)-lysine] occurs in the eIF-5A precursor protein through two step posttranslational modification involving deoxyhypusine synthase which catalyzes transfer of the butylamine moiety of spermidine to the epsilon-amino group of a designated lysine residue and subsequent hydroxylation of this intermediate. This enzyme is exclusively required for cell viability and growth of yeast (Park, M.H. et al., J. Biol. Chem. 273: 1677-1683, 1998). In an effort to understand structure-function relationship of deoxyhypusine synthase, posttranslational modification(s) of the enzyme by protein kinases were carried out for a possible cellular modulation of this enzyme. And also twelve deletion mutants were constructed, expressed in E. coli system, and enzyme activities were examined. The results showed that deoxyhypusine synthase was phosphorylated by PKC in vitro but not by p56lck and p60c-src. Treatment with PMA specifically increased the relative phosphorylation of the enzyme supporting PKC was involved. Phosphoamino acid analysis of this enzyme revealed that deoxyhypusine synthase is mostly phosphorylated on serine residue and weakly on threonine. Removal of Met1-Glu10 (deltaMet1-Glu10) residues from amino terminal showed no effect on the catalytic activity but further deletion (deltaMet1-Ser20) caused loss of enzyme activity. The enzyme with internal deletion, deltaGln197-Asn212 (residues not present in the human enzyme) was found to be inactive. Removal of 5 residues from carboxyl terminal, deltaLys383-Asn387, retained only slight activity. These results suggested that deoxyhypusine synthase is substrate for PKC dependent phosphorylation and requires most of the polypeptide chains for enzyme activity except the first 15 residues of N-terminal despite of N- and C-terminal residues of the enzyme consist of variable regions. Copyright 2000 Academic Press.

LDH-C4-substrate binary complexes studied by intrinsic fluorescence method.

Lactate dehydrogenase-C4 (LDH-C4) has been studied in presence of substrates using intrinsic fluorescence measurements. Excitation maximum of LDH-C4 occurred at 282 nm whereas fluorescence emission maximum was obtained at 340 nm. Fluorescence intensities at 340 nm showed that ligands viz. NAD+, NADH, pyruvate and lactate quench the relative fluorescence intensities of LDH-C4 in a concentration dependent manner. NAD+ and NADH produced a maximum quenching between 92-93% while pyruvate and lactate quenched the fluorescence of LDH up to 29% and 21% respectively. Association constants (Ka) based on fluorescence measurements were 6.05 x 10(4)M-1, 20 x 10(4)M-1, 0.113 x 10(4)M-1 and 0.3 x 10(4)M-1, for NAD+, NADH, lactate and pyruvate respectively. Stern-Volmer constants (Ksv) show that NAD+ and NADH have single Ksv of 4.07 x 10(4)M-1 and 1.47 x 10(5)M-1, whereas lactate and pyruvate indicated quenching reaction to be made up of two components. Ksv at low and high concentration of lactate respectively were 0.645 x 10(2)M-1 and 0.05 x 10(2)M-1, whereas corresponding Ksv with pyruvate were 1.008 x 10(3)M-1 and 0.408 x 10(3)M-1. Low Ksv at higher concentrations suggested that the aromatic chromophores are located within a hydrophobic environment. Red shift in fluorescence maximum (lambda max) by 2nm with lactate and 6nm with pyruvate showed that interaction of these ligands with LDH-C4 exposes some buried chromophores of the enzyme to the surface.

Fatty acid synthesis by isolated leucoplasts from developing Brassica seeds: role of glycolytic intermediates as the source of carbon and energy.

Fatty acid synthesis from Na [1-14C] acetate in leucoplasts isolated from developing seeds of Brassica campestris was completely dependent on exogenous supply of ATP. None of the intermediates of glycolysis or pentose phosphate pathway tested could replace ATP in the reaction mixture. In absence of exogenously supplied ATP, maximum activity was obtained with glu-6-P (68%) followed by fru-6-P (50%) and PEP (44%), respectively. With other intermediates as energy sources, the activity ranged from 1 to 38%. In complementary experiments (presence of ATP), none of the metabolites gave activity higher than the ATP control activity. Under optimum conditions for fatty acid synthesis from acetate, Brassica leucoplasts readily utilized labelled glucose as the substrate for fatty acid synthesis. Omission of NADH and NADPH individually from the reaction mixtures containing labelled glucose resulted only in 46 and 20% loss in activity, respectively, compared to the corresponding losses of 56 and 50%, when labelled acetate was used as the substrate. Similarly, deletion of ATP from the reaction mixture containing glucose as the substrate decreased the rate of fatty acid synthesis by about 65%, while the corresponding decrease with acetate as the substrate was 96%. Inclusion of 5 mM cold acetate, pyruvate, malate and glu-6-P in the reaction mixture containing glucose as the labelled substrate reduced label incorporation into fatty acids by 38 to 69%, maximum reduction being observed with pyruvate followed by glu-6-P, acetate and malate, respectively. With labelled acetate as the substrate, maximum reduction in label incorporation was obtained with cold glucose (5 mM) followed by glu-6-P, pyruvate and malate, respectively. The study demonstrated the operation of complete glycolytic pathway in Brassica leucoplasts, allowing the plastids to use glucose as a source of carbon, reducing power and energy for fatty acid synthesis.

Redox state of pyridine nucleotides, but not glutathione, regulate Ca2+ release by H2O2 from mitochondria of pulmonary smooth muscle.

Treatment of bovine pulmonary artery smooth muscle mitochondria with H2O2 stimulated oxidation of GSH and NAD(P)H along with an increase in Ca2+ release. Addition of oxaloacetate to mitochondrial suspension stimulated Ca2+ release and oxidation of NAD(P)H while GSH level remained unchanged. Subsequently, addition of beta-hydroxybutyrate which reduced mitochondrial pyridine nucleotides caused reuptake of the released Ca2+ without causing appreciable alteration of GSH level. Treatment of the mitochondria with 1,3-bis(2-dichloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase, significantly decreased GSH level without producing discernible change in Ca2+ release and NAD(P)H oxidation.

Effect of tamoxifen, estradiol 17 beta on coenzymes NAD, NADPH and the metabolism of estradiol and estrone in rabbit uterus in vivo.

Biotransformation of estradiol (E2) and estrone (E1) and the concentrations of NAD, NADPH and 17 beta-estradiol dehydrogenase (E2DH) were measured in the uterus of rabbits treated with tamoxifen (Tam) in two doses; 100 micrograms/day, (Tam 100) and 500 micrograms/day, (Tam 500), E2 (10 micrograms/day) and combination of E2 + Tam 500 for 4 days. The concentration of NAD in Tam 500 treated group was significantly higher than E2, low dose Tam and E2 + Tam 500 treated groups (P < 0.01). The concentration of NAD in E2+ Tam 500 uteri was also significantly higher than E2 treated uteri. The concentration of NADPH was not significantly different from each other amongst the various treatment groups. The studies have shown that E2DH in E2 treated uteri was less than control and Tam 500 treated groups. A significant rise in the enzyme estradiol oxidoreductase (E2OR) activity (P < 0.02) was observed in E2 + Tam 500 treated uteri over control and other treated groups whereas high dose Tam decreased the E2OR activity significantly over the E2 treated group. The rate of conversion of E1 to E2 in Tam 500 treated group was significantly less than the other treatment groups except E2 + Tam 500 treated group (P < 0.04). This study showed that E2 decreases the uterine biosynthesis of NAD and E2DH and the biotransformation of E2 to E1, while high dose Tam increases uterine NAD, E2DH activity and E2 to E1 conversion.

Purification of two azoreductases from Escherichia coli K12.

Two azoreductases (I and II) were purified to homogeneity from extracts of E. coli K12. Azoreductase I was a dimer of two identical subunits of molecular weight 28000 whereas azoreductase II was a monomer of 12,000 molecular weight. Both NADH and NADPH functioned as electron donors for the azoreductases. Azoreductase I and II used Ponceau SX, Tartrazine, Amaranth and Orange II as substrates. Ponceau SX was the best substrate for both the enzymes. However, azoreductase II utilized tartrazine, amaranth and orange II less efficiently than azoreductase I.

Ca2+ transport and oxidative damage of mitochondria

1. Mitochondria from a wide range of sources have the ability to accumulate Ca2+ down their electrochemical gradient mediated by a uniport mechanism. 2. Ca2+ efflux occurs via two separate pathways: a Na+/Ca2+ exchanger that predominates in mitochondria from excitable tissues and a Na(+)-independent pathway that predominates in mitochondria from non-excitable tissues. 3. The kinetic characteristics of these calcium influx-efflux pathways appear to be incompatible with any role for mitochondria as cytosolic Ca2+ buffers, under resting normal physiological conditions. Instead, the biological role of this Ca(2+)-transporting system seems to be the regulation of matrix Ca2+ in a range that permits the regulation of three intramitochondrial Ca(2+)-dependent dehydrogenases which catalyze rate-limiting reactions of the Krebs cycle. 4. Under conditions in which a high cytosolic Ca2+ concentration is sustained, the matrix Ca2+ concentration may attain levels that lead to impairment of mitochondrial functions such as inhibition of oxidative phosphorylation and increase in inner membrane permeability. 5. Accumulation of Ca2+ by mitochondria under conditions of oxidative stress induces an increase in inner membrane permeability by a mechanism that appears to be mediated by protein polymerization due to thiol cross-linking

Inhibition of mitochondrial oxidative phosphorylation and its electron transport pathway by a polycation in vitro.

Effect of the polycation on oxidative phosphorylation in the rat liver mitochondria has been studied. Both oxygen uptake and coupled phosphorylation were progressively inhibited by increasing concentration of the polycation, as observed with NAD-linked substrates, succinate and ascorbate+TMPD which activates the terminal part of the respiratory chain. NADH oxidase, NADH dehydrogenase and cytochrome oxidase were strongly inhibited by the polycation, 80-90% of the activity being lost at an inhibitor concentration of 100 microM. Succinate oxidase and succinate dehydrogenase were inhibited 60-66% at 100 microM concentration of the polycation. The polycation inhibited the uncoupler 2,4-dinitrophenol stimulated ATPase activity both in presence and absence of Mg2+ ions. The polycation also inhibited salt-induced volume change.