Cloning,expression and characterization of glycerol dehydrogenase involved in 2,3-butanediol formation in Serratia marcescens H30

The meso-2,3-butanediol dehydrogenase (meso-BDH) from S. marcescens H30 is responsible for converting acetoin into 2,3-butanediol during sugar fermentation. Inactivation of the meso-BDH encoded by budC gene does not completely abolish 2,3-butanediol production, which suggests that another similar enzyme involved in 2,3-butanediol formation exists in S. marcescens H30. In the present study, a glycerol dehydrogenase (GDH) encoded by gldA gene from S. marcescens H30 was expressed in Escherichia coli BL21(DE3), purified and characterized for its properties. In vitro conversion indicated that the purified GDH could catalyze the interconversion of (3S)-acetoin/meso-2,3-butanediol and (3R)-acetoin/(2R,3R)-2,3-butanediol. (2S,3S)-2,3-Butanediol was not a substrate for the GDH at all. Kinetic parameters of the GDH enzyme showed lower K _m value and higher catalytic efficiency for (3S/3R)-acetoin in comparison to those for (2R,3R)-2,3-butanediol and meso-2,3-butanediol, implying its physiological role in favor of 2,3-butanediol formation. Maximum activity for reduction of (3S/3R)-acetoin and oxidations of meso-2,3-butanediol and glycerol was observed at pH 8.0, while it was pH 7.0 for diacetyl reduction. The enzyme exhibited relative high thermotolerance with optimum temperature of 60 °C in the oxidation–reduction reactions. Over 60 % of maximum activity was retained at 70 °C. Additionally, the GDH activity was significantly enhanced for meso-2,3-BD oxidation in the presence of Fe²⁺ and for (3S/3R)-acetoin reduction in the presence of Mn²⁺, while several cations inhibited its activity, particularly Fe²⁺ and Fe³⁺ for (3S/3R)-acetoin reduction. The properties provided potential application for single configuration production of acetoin and 2,3-butanediol .     

Characterization of a stereospecific acetoin(diacetyl) reductase from Rhodococcus erythropolis WZ010 and its application for the synthesis of (2S,3S)-2,3-butanediol

Rhodococcus erythropolis WZ010 was capable of producing optically pure (2S,3S)-2,3-butanediol in alcoholic fermentation. The gene encoding an acetoin(diacetyl) reductase from R. erythropolis WZ010 (ReADR) was cloned, overexpressed in Escherichia coli, and subsequently purified by Ni-affinity chromatography. ReADR in the native form appeared to be a homodimer with a calculated subunit size of 26,864, belonging to the family of the short-chain dehydrogenase/reductases. The enzyme accepted a broad range of substrates including aliphatic and aryl alcohols, aldehydes, and ketones. It exhibited remarkable tolerance to dimethyl sulfoxide (DMSO) and retained 53.6 % of the initial activity after 4 h incubation with 30 % (v/v) DMSO. The enzyme displayed absolute stereospecificity in the reduction of diacetyl to (2S,3S)-2,3-butanediol via (S)-acetoin. The optimal pH and temperature for diacetyl reduction were pH 7.0 and 30 °C, whereas those for (2S,3S)-2,3-butanediol oxidation were pH 9.5 and 25 °C. Under the optimized conditions, the activity of diacetyl reduction was 11.9-fold higher than that of (2S,3S)-2,3-butanediol oxidation. Kinetic parameters of the enzyme showed lower K _m values and higher catalytic efficiency for diacetyl and NADH in comparison to those for (2S,3S)-2,3-butanediol and NAD⁺, suggesting its physiological role in favor of (2S,3S)-2,3-butanediol formation. Interestingly, the enzyme showed higher catalytic efficiency for (S)-1-phenylethanol oxidation than that for acetophenone reduction. ReADR-catalyzed asymmetric reduction of diacetyl was coupled with stereoselective oxidation of 1-phenylethanol, which simultaneously formed both (2S,3S)-2,3-butanediol and (R)-1-phenylethanol in great conversions and enantiomeric excess values.     

Role of Saccharomyces cerevisiae Oxidoreductases Bdh1p and Ara1p in the Metabolism of Acetoin and 2,3-Butanediol

NAD-dependent butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae reversibly transforms acetoin to 2,3-butanediol in a stereospecific manner. Deletion of BDH1 resulted in an accumulation of acetoin and a diminution of 2,3-butanediol in two S. cerevisiae strains under two different growth conditions. The concentrations of (2R,3R)-2,3-butanediol are mostly dependent on Bdh1p activity, while those of (meso)-2,3-butanediol are also influenced by the activity of NADP(H)-dependent oxidoreductases. One of them has been purified and shown to be d-arabinose dehydrogenase (Ara1p), which converts (R/S)-acetoin to meso-2,3-butanediol and (2S,3S)-2,3-butanediol. Deletion of BDH2, a gene adjacent to BDH1, whose encoded protein is 51% identical to Bdh1p, does not significantly alter the levels of acetoin or 2,3-butanediol in comparison to the wild-type strain. Furthermore, we have expressed Bdh2p with a histidine tag and have shown it to be inactive toward 2,3-butanediol. A whole-genome expression analysis with microarrays demonstrates that BDH1 and BDH2 are reciprocally regulated.Acetoin and 2,3-butanediol are minor products generated by Saccharomyces cerevisiae during alcohol fermentation. Their sensory impacts on wine are poorly documented. Acetoin may affect the wine bouquet, although its perception threshold in wine is relatively high, around 150 mg/liter (21, 31). On the other hand, 2,3-butanediol is odorless (33) and cannot be expected to appreciably affect the sensory quality of wine. However, the compound may contribute to the wine body (28).Acetaldehyde, pyruvate, and α-acetolactate are the main precursors of acetoin in S. cerevisiae. Acetoin can be formed from acetaldehyde and/or pyruvate through an anomalous reaction of pyruvate decarboxylase. Thus, although its main activity is to irreversibly decarboxylate pyruvate to acetaldehyde, it can also catalyze carbon-carbon bond formation, yielding acetoin from pyruvate and/or acetaldehyde (2, 4). In addition, α-acetolactate would produce acetoin through its nonenzymatic decarboxylation to diacetyl and subsequent reduction to acetoin through the action of several NADH- and NADPH-dependent oxidoreductases (12). However, the situation is more complex in wine fermentation, where other yeasts and bacteria display supplementary enzymatic activities capable of producing both acetoin and 2,3-butanediol (1, 27).We have previously characterized a butanediol dehydrogenase (Bdh1p) as a medium-chain dehydrogenase/reductase (MDR) that can reversibly transform R-acetoin and S-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol, respectively, in a NAD(H)-dependent reaction (10). BDH2 is a gene adjacent to BDH1 whose uncharacterized protein product (Bdh2p) shares 51% sequence identity with Bdh1p. To evaluate the in vivo roles of Bdh1p and Bdh2p, we compared the levels of several extracellular metabolites in cultures of wild-type and deficient strains. The results show that, although Bdh1p is the main enzyme in 2,3-butanediol production [essentially the (2R,3R)-2,3-butanediol stereoisomer], some meso-2,3-butanediol is still produced by the bdh1Δ strains. We have characterized Ara1p as an oxidoreductase that can reduce racemic acetoin to meso-2,3-butanediol and (2S,3S)-2,3-butanediol in the presence of NADPH.Furthermore, we have overexpressed Bdh2p with a histidine tag at its carboxyl terminus and have shown it to be inactive toward acetoin and 2,3-butanediol. A microarray study indicated that BDH1 and BDH2 are reciprocally regulated under the conditions studied.     

Mechanism of 2,3-butanediol stereoisomer formation in Klebsiella pneumoniae

Klebsiella pneumoniae is known to produce meso-2,3-butanediol and 2S,3S-butanediol, whereas 2R,3R-butanediol was detected in the culture broth of K. pneumoniae CGMCC 1.6366. The ratio of 2R,3R-butanediol to all isomers obtained using glycerol as the carbon source was higher than that obtained using glucose as the carbon source. Therefore, enzymes involved in glycerol metabolism are likely related to 2R,3R-butanediol formation. In vitro reactions show that glycerol dehydrogenase catalyzes the stereospecific conversion of R-acetoin to 2R,3R-butanediol and S-acetoin to meso-2,3-butanediol. Butanediol dehydrogenase exhibits high (S)-enantioselectivity in ketone reduction. Genes encoding glycerol dehydrogenase, α-acetolactate decarboxylase, and butanediol dehydrogenase were individually disrupted in K. pneumoniae CGMCC 1.6366, and the 2,3-butanediol synthesis characteristics of these mutants were investigated. K. pneumoniae ΔdhaD lost the ability to synthesize 2R,3R-butanediol. K. pneumoniae ΔbudA showed reduced 2R,3R-butanediol synthesis. However, K. pneumoniae ΔbudC produced a high level of 2R,3R-butanediol, and R-acetoin was accumulated in the broth. The metabolic characteristics of these mutants and in vitro experiment results demonstrated the mechanism of the 2,3-butanediol stereoisomer synthesis pathway. Glycerol dehydrogenase, encoded by dhaD, exhibited 2R,3R-butanediol dehydrogenase activity and was responsible for 2R,3R-butanediol synthesis from R-acetoin. This enzyme also contributed to meso-2,3-butanediol synthesis from S-acetoin. Butanediol dehydrogenase, encoded by budC, was the only enzyme that catalyzed the conversion of diacetyl to S-acetoin and further to 2S,3S-butanediol.     

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.     

Characterization and functional role of Saccharomyces cerevisiae 2,3-butanediol dehydrogenase

Using a conserved sequence motif, a new gene (YAL060W) of the MDR family has been identified in Saccharomyces cerevisiae. The expressed protein was a stereoespecific (2R,3R)-2,3-butanediol dehydrogenase (BDH). The best substrates were (2R,3R)-2,3-butanediol for the oxidation and (3R/3S)-acetoin and 1-hydroxy-2-propanone for the reduction reactions. The enzyme is extremely specific for NAD(H) as cofactor, probably because the presence of Glu223 in the cofactor binding site, instead of the highly conserved Asp223. BDH is inhibited competitively by 4-methylpyrazole with a K_i of 34 μM. Yeast could grow on 2,3-butanediol or acetoin as a sole energy and carbon sources, and a 3.6-fold increase in BDH activity was observed when cells were grown in 2,3-butanediol, suggesting a role of the enzyme in 2,3-butanediol metabolism. However, the disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions, and the disrupted strain could also grow in 2,3-butanediol and acetoin. This suggests that other enzymes, in addition to BDH, can also metabolize 2,3-butanediol in yeast.     

Glycerol Dehydrogenase Plays a Dual Role in Glycerol Metabolism and 2,3-Butanediol Formation in Klebsiella pneumoniae

Glycerol dehydrogenase (GDH) is an important polyol dehydrogenase for glycerol metabolism in diverse microorganisms and for value-added utilization of glycerol in the industry. Two GDHs from Klebsiella pneumoniae, DhaD and GldA, were expressed in Escherichia coli, purified and characterized for substrate specificity and kinetic parameters. Both DhaD and GldA could catalyze the interconversion of (3R)-acetoin/(2R,3R)-2,3-butanediol or (3S)-acetoin/meso-2,3-butanediol, in addition to glycerol oxidation. Although purified GldA appeared more active than DhaD, in vivo inactivation and quantitation of their respective mRNAs indicate that dhaD is highly induced by glycerol and plays a dual role in glycerol metabolism and 2,3-butanediol formation. Complementation in K. pneumoniae further confirmed the dual role of DhaD. Promiscuity of DhaD may have vital physiological consequences for K. pneumoniae growing on glycerol, which include balancing the intracellular NADH/NAD⁺ ratio, preventing acidification, and storing carbon and energy. According to the kinetic response of DhaD to modified NADH concentrations, DhaD appears to show positive homotropic interaction with NADH, suggesting that the physiological role could be regulated by intracellular NADH levels. The co-existence of two functional GDH enzymes might be due to a gene duplication event. We propose that whereas DhaD is specialized for glycerol utilization, GldA plays a role in backup compensation and can turn into a more proficient catalyst to promote a survival advantage to the organism. Revelation of the dual role of DhaD could further the understanding of mechanisms responsible for enzyme evolution through promiscuity, and guide metabolic engineering methods of glycerol metabolism.     

Enantioselective Synthesis of Vicinal (R,R)-Diols by Saccharomyces cerevisiae Butanediol Dehydrogenase

Butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae belongs to the superfamily of the medium-chain dehydrogenases and reductases and converts reversibly R-acetoin and S-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol, respectively. It is specific for NAD(H) as a coenzyme, and it is the main enzyme involved in the last metabolic step leading to (2R,3R)-2,3-butanediol in yeast. In this study, we have used the activity of Bdh1p in different forms—purified enzyme, yeast extracts, permeabilized yeast cells, and as a fusion protein (with yeast formate dehydrogenase, Fdh1p)—to transform several vicinal diketones to the corresponding diols. We have also developed a new variant of the delitto perfetto methodology to place BDH1 under the control of the GAL1 promoter, resulting in a yeast strain that overexpresses butanediol dehydrogenase and formate dehydrogenase activities in the presence of galactose and regenerates NADH in the presence of formate. While the use of purified Bdh1p allows the synthesis of enantiopure (2R,3R)-2,3-butanediol, (2R,3R)-2,3-pentanediol, (2R,3R)-2,3-hexanediol, and (3R,4R)-3,4-hexanediol, the use of the engineered strain (as an extract or as permeabilized cells) yields mixtures of the diols. The production of pure diol stereoisomers has also been achieved by means of a chimeric fusion protein combining Fdh1p and Bdh1p. Finally, we have determined the selectivity of Bdh1p toward the oxidation/reduction of the hydroxyl/ketone groups from (2R,3R)-2,3-pentanediol/2,3-pentanedione and (2R,3R)-2,3-hexanediol/2,3-hexanedione. In conclusion, Bdh1p is an enzyme with biotechnological interest that can be used to synthesize chiral building blocks. A scheme of the favored pathway with the corresponding intermediates is proposed for the Bdh1p reaction.     

A Membrane-Bound NAD(P)+-Reducing Hydrogenase Provides Reduced Pyridine Nucleotides during Citrate Fermentation by Klebsiella pneumoniae

During anaerobic growth of Klebsiella pneumoniae on citrate, 9.4 mmol of H₂/mol of citrate (4-kPa partial pressure) was formed at the end of growth besides acetate, formate, and CO₂. Upon addition of NiCl₂ (36 μM) to the growth medium, hydrogen formation increased about 36% to 14.8 mmol/mol of citrate (6 kPa), and the cell yield increased about 15%. Cells that had been harvested and washed under anoxic conditions exhibited an H₂-dependent formation of NAD(P)H in vivo. The reduction of internal NAD(P)⁺ was also achieved by the addition of formate. In crude extracts, the H₂:NAD⁺ oxidoreductase activity was 0.13 μmol min⁻¹ mg⁻¹, and 76% of this activity was found in the washed membrane fraction. The highest specific activities of the membrane fraction were observed in 50 mM potassium phosphate, with 1.6 μmol of NADPH formed min⁻¹ mg⁻¹ at pH 7.0 and 1.7 μmol of NADH formed min⁻¹ mg⁻¹ at pH 9.5. In the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the Na⁺/H⁺ antiporter monensin, the H₂-dependent reduction of NAD⁺ by membrane vesicles decreased only slightly (about 16%). The NADP⁺- or NAD⁺-reducing hydrogenases were solubilized from the membranes with the detergent lauryldimethylamine-N-oxide or Triton X-100. NAD(P)H formation with H₂ as electron donor, therefore, does not depend on an energized state of the membrane. It is proposed that hydrogen which is formed by K. pneumoniae during citrate fermentation is recaptured by a novel membrane-bound, oxygen-sensitive H₂:NAD(P)⁺ oxidoreductase that provides reducing equivalents for the synthesis of cell material.     

NADP+ Reduction with Reduced Ferredoxin and NADP+ Reduction with NADH Are Coupled via an Electron-Bifurcating Enzyme Complex in Clostridium kluyveri

It was recently found that the cytoplasmic butyryl-coenzyme A (butyryl-CoA) dehydrogenase-EtfAB complex from Clostridium kluyveri couples the exergonic reduction of crotonyl-CoA to butyryl-CoA with NADH and the endergonic reduction of ferredoxin with NADH via flavin-based electron bifurcation. We report here on a second cytoplasmic enzyme complex in C. kluyveri capable of energetic coupling via this novel mechanism. It was found that the purified iron-sulfur flavoprotein complex NfnAB couples the exergonic reduction of NADP⁺ with reduced ferredoxin (Fd_red) and the endergonic reduction of NADP⁺ with NADH in a reversible reaction: Fd_red²⁻ + NADH + 2 NADP⁺ + H⁺ = Fd_ox + NAD⁺ + 2 NADPH. The role of this energy-converting enzyme complex in the ethanol-acetate fermentation of C. kluyveri is discussed.Clostridium kluyveri is unique in fermenting ethanol and acetate to butyrate, caproate, and H₂ (reaction 1) and in deriving a large (30%) portion of its cell carbon from CO₂. Both the energy metabolism and the pathways of biosynthesis have therefore been the subject of many investigations (for relevant literature, see references 12 and 27). (1)During growth of C. kluyveri on ethanol and acetate, approximately five ethanol and four acetate molecules are converted to three butyrate molecules and one caproate molecule (reaction 1a), and one ethanol molecule is oxidized to one acetate⁻, one H⁺, and two H₂ (reaction 1b) molecules (23, 31). How exergonic reaction 1a is coupled with endergonic reaction 1b and with ATP synthesis from ADP and P_i (ΔG^o′ = +32 kJ/mol) has remained unclear for many years. (1a) (1b)We recently showed (12) that, in Clostridium kluyveri, the exergonic reduction of crotonyl-coenzyme A (crotonyl-CoA) (E_o′ = −10 mV) with NADH (E_o′ = −320 mV) involved in reaction 1a is coupled with the endergonic reduction of ferredoxin (Fd_ox) (E_o′ = −420 mV) with NADH (E_o′ = −320 mV) involved in reaction 1b via the recently proposed mechanism of flavin-based electron bifurcation (7). The coupling reaction is catalyzed by the cytoplasmic butyryl-CoA dehydrogenase-EtfAB complex (reaction 2) (12): (2)The reduced ferredoxin (Fd_red²⁻) is assumed to be used for rereduction of NAD⁺ via a membrane-associated, proton-translocating ferredoxin:NAD oxidoreductase (RnfABCDEG) (reaction 3), and the proton motive force thus generated is assumed to drive the phosphorylation of ADP via a membrane-associated F₁F₀ ATP synthetase (reaction 4): (3) (4)The novel coupling mechanism represented by reactions 2 and 3 allowed for the first time the possibility of formulating a metabolic scheme for the ethanol-acetate fermentation that could account for the observed fermentation products and growth yields and thus for the observed ATP gains (27). One issue, however, remained open, namely, why the formation of butyrate from ethanol and acetate in the fermentation involves both an NADP⁺- and an NAD⁺-specific β-hydroxybutyryl-CoA dehydrogenase (16), considering that, in the oxidative part of the fermentation (ethanol oxidation to acetyl-CoA), only NADH is generated (8, 9, 13).The presence of a reduced ferredoxin:NADP⁺ oxidoreductase was proposed based on results of enzymatic studies performed 40 years ago. Cell extracts of Clostridium kluyveri were found to catalyze the formation of H₂ from NADPH in a ferredoxin- and NAD⁺-dependent reaction (34). The results were interpreted to indicate that C. kluyveri contains a ferredoxin-dependent hydrogenase and an NADPH:ferredoxin oxidoreductase with transhydrogenase activity. H₂ formation from NADPH was strictly dependent on the presence of NAD⁺ and was inhibited by NADH, inhibition being competitive with the presence of NAD⁺, indicating that ferredoxin reduction with NADPH is under the allosteric control of the NAD⁺/NADH couple. The cell extracts also catalyzed the NADH-dependent reduction of NADP⁺ with reduced ferredoxin (21, 34). Purification of the enzyme catalyzing these reactions was not achieved, and no function in the energy metabolism of C. kluyveri was assigned.In this communication, we report on the properties of the recombinant enzyme that catalyzes the NAD⁺-dependent reduction of ferredoxin with NADPH and the NADH-dependent reduction of NADP⁺ with reduced ferredoxin and show that the cytoplasmic heterodimeric enzyme couples the exergonic reduction of NADP⁺ with reduced ferredoxin with the endergonic reduction of NADP⁺ with NADH in a fully reversible reaction. The transhydrogenation reaction is endergonic, because in vivo the NADH/NAD⁺ ratio is generally near 0.3 and the NADPH/NADP⁺ ratio is generally above 1 (2, 30). (5)NADP⁺ reduction is most probably the physiological function of the enzyme, which is why we chose the abbreviation NfnAB (for NADH-dependent reduced ferredoxin:NADP⁺ oxidoreductase).     

NADH Enzyme-Dependent Fluorescence Recovery after Photobleaching (ED-FRAP): Applications to Enzyme and Mitochondrial Reaction Kinetics, In Vitro

NADH enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) was evaluated for studying enzyme kinetics in vitro and in isolated mitochondria. Mass, optical, and nuclear magnetic resonance spectroscopy data were consistent with the UV NADH photolysis reaction being NADH → NAD^· + H⁺ + e⁻. The overall net reaction was O₂ + 2NADH + 2H⁺ → 2NAD⁺ + 2H₂O, or in the presence of other competing electron acceptors such as cytochrome c, NADH + 2Cyt_ox → NAD⁺ + H⁺ + 2Cyt_red. Solution pH could differentiate between these free-radical scavenging pathways. These net reactions represent the photooxidation of NADH to NAD⁺. Kinetic models and acquisition schemes were developed, varying [NADH] and [NAD] by altering NADH photolysis levels, for extracting kinetic parameters. UV irradiation levels used did not damage mitochondrial function or enzymatic activity. In mitochondria, [NADH] is a high affinity product inhibitor that significantly reduced the NADH regeneration rate. Matrix NADH regeneration only slightly exceeded the net rate of NADH consumption, suggesting that the NADH regeneration process is far from equilibrium. Evaluation of NADH regeneration in active mitochondria, in comparison to rotenone-treated preparations, revealed other regulatory elements in addition to matrix [NADH] and [NAD] that have yet to be fully characterized. These studies demonstrate that the rapid UV photolysis of NADH to NAD is an effective tool in evaluating the steady-state kinetic properties of enzyme systems. Initial data support the notion that the NADH regeneration process is far from equilibrium in mitochondria and is potentially controlled by NADH levels as well as several other matrix factors.     

Expression of the Escherichia coli pntA and pntB Genes, Encoding Nicotinamide Nucleotide Transhydrogenase, in Saccharomyces cerevisiae and Its Effect on Product Formation during Anaerobic Glucose Fermentation

We studied the physiological effect of the interconversion between the NAD(H) and NADP(H) coenzyme systems in recombinant Saccharomyces cerevisiae expressing the membrane-bound transhydrogenase from Escherichia coli. Our objective was to determine if the membrane-bound transhydrogenase could work in reoxidation of NADH to NAD⁺ in S. cerevisiae and thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recombinant strains exhibited reduction of 3-acetylpyridine-NAD⁺ by NADPH and by NADH in the presence of NADP⁺, which demonstrated that an active enzyme was present. Unlike the situation in E. coli, however, most of the transhydrogenase activity was not present in the yeast plasma membrane; rather, the enzyme appeared to remain localized in the membrane of the endoplasmic reticulum. During anaerobic glucose fermentation we observed an increase in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expressing a high level of transhydrogenase, which indicated that increased NADPH consumption and NADH production occurred. The intracellular concentrations of NADH, NAD⁺, NADPH, and NADP⁺ were measured in cells expressing transhydrogenase. The reduction of the NADPH pool indicated that the transhydrogenase transferred reducing equivalents from NADPH to NAD⁺.     

Guanine Nucleotides in the Meiotic Maturation of Starfish Oocytes: Regulation of the Actin Cytoskeleton and of Ca2+ Signaling

Background

Starfish oocytes are arrested at the first prophase of meiosis until they are stimulated by 1-methyladenine (1-MA). The two most immediate responses to the maturation-inducing hormone are the quick release of intracellular Ca²⁺ and the accelerated changes of the actin cytoskeleton in the cortex. Compared with the later events of oocyte maturation such as germinal vesicle breakdown, the molecular mechanisms underlying the early events involving Ca²⁺ signaling and actin changes are poorly understood. Herein, we have studied the roles of G-proteins in the early stage of meiotic maturation.

Methodology/Principal Findings

By microinjecting starfish oocytes with nonhydrolyzable nucleotides that stabilize either active (GTPγS) or inactive (GDPβS) forms of G-proteins, we have demonstrated that: i) GTPγS induces Ca²⁺ release that mimics the effect of 1-MA; ii) GDPβS completely blocks 1-MA-induced Ca²⁺; iii) GDPβS has little effect on the amplitude of the Ca²⁺ peak, but significantly expedites the initial Ca²⁺ waves induced by InsP₃ photoactivation, iv) GDPβS induces unexpectedly striking modification of the cortical actin networks, suggesting a link between the cytoskeletal change and the modulation of the Ca²⁺ release kinetics; v) alteration of cortical actin networks with jasplakinolide, GDPβS, or actinase E, all led to significant changes of 1-MA-induced Ca²⁺ signaling.

Conclusions/Significance

Taken together, these results indicate that G-proteins are implicated in the early events of meiotic maturation and support our previous proposal that the dynamic change of the actin cytoskeleton may play a regulatory role in modulating intracellular Ca²⁺ release.     

NAMPT inhibition sensitizes pancreatic adenocarcinoma cells to tumor-selective,PAR-independent metabolic catastrophe and cell death induced by β-lapachone

Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (e.g., FK866) target the most active pathway of NAD⁺ synthesis in tumor cells, but lack tumor-selectivity for use as a single agent. Reducing NAD⁺ pools by inhibiting NAMPT primed pancreatic ductal adenocarcinoma (PDA) cells for poly(ADP ribose) polymerase (PARP1)-dependent cell death induced by the targeted cancer therapeutic, β-lapachone (β-lap, ARQ761), independent of poly(ADP ribose) (PAR) accumulation. β-Lap is bioactivated by NADPH:quinone oxidoreductase 1 (NQO1) in a futile redox cycle that consumes oxygen and generates high levels of reactive oxygen species (ROS) that cause extensive DNA damage and rapid PARP1-mediated NAD⁺ consumption. Synergy with FK866+β-lap was tumor-selective, only occurring in NQO1-overexpressing cancer cells, which is noted in a majority (∼85%) of PDA cases. This treatment strategy simultaneously decreases NAD⁺ synthesis while increasing NAD⁺ consumption, reducing required doses and treatment times for both drugs and increasing potency. These complementary mechanisms caused profound NAD(P)⁺ depletion and inhibited glycolysis, driving down adenosine triphosphate levels and preventing recovery normally observed with either agent alone. Cancer cells died through an ROS-induced, μ-calpain-mediated programmed cell death process that kills independent of caspase activation and is not driven by PAR accumulation, which we call NAD⁺-Keresis. Non-overlapping specificities of FK866 for PDA tumors that rely heavily on NAMPT-catalyzed NAD⁺ synthesis and β-lap for cancer cells with elevated NQO1 levels affords high tumor-selectivity. The concept of reducing NAD⁺ pools in cancer cells to sensitize them to ROS-mediated cell death by β-lap is a novel strategy with potential application for pancreatic and other types of NQO1+ solid tumors.An emerging metabolic target for the treatment of recalcitrant cancers, such as pancreatic adenocarcinoma (PDA), is their reliance on NAD⁺ synthesis, particularly through the nicotinamide-recycling pathway.^{1, 2, 3} Rapid and efficient NAD⁺ synthesis is critical to sustain signaling processes, such as deacetylation by sirtuins and adenosine diphosphate (ADP) ribosylation by poly(ADP ribose) polymerase 1 (PARP1). NAD(P)⁺ pools are also necessary to support anabolic metabolism and proliferation of cancer cells. In an attempt to leverage increased tumor-cell reliance on NAD⁺ synthesis, small molecule inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) were developed (e.g., FK866).⁴ NAMPT catalyzes the rate-limiting step of the most active pathway of NAD⁺ synthesis. Inhibitors of NAMPT, such as FK866, reduce NAD⁺ levels, induce canonical apoptosis preferentially in cancer cells in vitro, inhibit tumor growth, and increase overall survival in preclinical cancer models.^{1, 5, 6, 7} FK866 (APO866) was relatively well tolerated in humans and advanced to phase II clinical trials. However, owing to its short half-life in circulation, prolonged treatment regimens were required and toxicity to normal, rapidly proliferating hematopoietic cells was noted. Accordingly, FK866 and other NAMPT inhibitors did not demonstrate sufficient tumor-selectivity to achieve clinical success as single agents.⁸To increase the specificity and efficacy of NAMPT inhibition, we combined FK866 with β-lapachone (β-lap), a targeted cancer therapeutic that causes tumor-selective PARP1 hyperactivation and NAD⁺ depletion in an NADPH:quinone oxidoreductase 1 (NQO1)-specific manner.⁹ β-Lap is a substrate for two-electron oxidoreduction by NQO1, a Phase II quinone-detoxifying enzyme.⁹ The resulting hydroquinone form of β-lap is highly unstable and spontaneously reacts with oxygen to revert back to the parent compound, generating two moles of superoxide per mole of NAD(P)H used in the process. This results in a futile cycle that occurs rapidly in NQO1-overexpressing cells, causing marked NADH/NADPH oxidation. DNA damage in the form of base oxidation and DNA single-strand breaks results from H₂O₂ generated from the futile redox cycle. In an attempt to repair this damage, PARP1 becomes hyperactivated, generating extensive branched poly(ADP ribose) (PAR) polymer. Hyperactivated PARP1 substantially depletes NAD⁺ and ultimately adenosine triphosphate (ATP) levels, thereby inhibiting subsequent repair of β-lap-induced DNA lesions. The observed cell death is caspase-independent and driven by nuclear translocation of apoptosis-inducing factor (AIF), activation of μ-calpain, and post-translational modification of GAPDH.^{10, 11, 12, 13} NQO1 is highly expressed in many types of cancer, and the therapeutic window provided by NQO1 bioactivation of β-lap has advanced its use to phase I clinical trials (ARQ761).¹⁴ Elevated NQO1 expression (≥10-fold) has been identified in ~85% of patient tissue from pancreatic ductal adenocarcinoma (PDA), making pancreatic cancer an especially appealing target for therapy using NQO1 bioactivatable drugs, such as β-lap.^{15, 16, 17, 18} However, dose-limiting methemoglobinemia caused by nonspecific reactive oxygen species (ROS) generation at high β-lap doses may limit the efficacy of β-lap as monotherapy.¹⁹ Strategies for increasing cancer cell cytotoxicity while maintaining NQO1 specificity could enhance use of β-lap for therapy against PDAs, as well as other solid cancers that overexpress NQO1.We found that examining cell death pathways induced by β-lap, with or without FK866 treatment, is a novel means to elucidate general mechanisms of lethality mediated by NAD⁺ loss, as cell death by PARP1 hyperactivation occurs in other contexts. Notably, cell death induced by ischemia/reperfusion shares many of the same characteristics: ROS induction, PARP1 hyperactivation, calcium release, AIF translocation, and caspase-independence.^{20, 21} Similarly, treatment with methylnitronitrosoguanidine (MNNG; a DNA alkylating agent) or induction of neuronal excitotoxicty induces PARP1 hyperactivation and cell death, but without futile cycle-induced ROS production.^{22, 23, 24} Recent studies suggest an important role for accumulated free PAR polymer that can directly activate μ-calpain, activate and release AIF, and inhibit glycolysis.^{22, 25, 26, 27, 28} By combining β-lap and FK866, we uncouple NAD⁺ and ATP depletion from the robust formation of PAR noted with β-lap alone, allowing us to define the function of PAR formation in β-lap-induced cell death.β-Lap and FK866 have distinct, but highly complementary mechanisms of action. β-Lap induces tumor-selective NAD⁺ depletion specifically in cancer cells that express high levels of NQO1. FK866 primes cancer cells for cell death by lowering NAD⁺/NADH pools and prevents recovery by inhibiting NAD⁺ synthesis from nicotinamide liberated by activated PARP1. We show that the increased dependence of PDA cells on glycolysis is specifically targeted by ROS-induced, NAD⁺ depletion caused by exposure to both drugs. Glycolytic inhibition, ATP depletion, and cell death is independent of PAR formation, strongly suggesting that PAR accumulation is not directly involved. The use of β-lap with NAMPT inhibitors results in synergistic NQO1- and PARP1-dependent cancer cell death, allowing the use of lower doses and shorter treatment times for both therapeutics.     

Improved Production of 2,3-Butanediol in Bacillus amyloliquefaciens by Over-Expression of Glyceraldehyde-3-Phosphate Dehydrogenase and 2,3-butanediol Dehydrogenase

Background

Previously, a safe strain, Bacillus amyloliquefaciens B10-127 was identified as an excellent candidate for industrial-scale microbial fermentation of 2,3-butanediol (2,3-BD). However, B. amyloliquefaciens fermentation yields large quantities of acetoin, lactate and succinate as by-products, and the 2,3-BD yield remains prohibitively low for commercial production.

Methodology/Principal Findings

In the 2,3-butanediol metabolic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of 3-phosphate glyceraldehyde to 1,3-bisphosphoglycerate, with concomitant reduction of NAD⁺ to NADH. In the same pathway, 2,3-BD dehydrogenase (BDH) catalyzes the conversion of acetoin to 2,3-BD with concomitant oxidation of NADH to NAD⁺. In this study, to improve 2,3-BD production, we first over-produced NAD⁺-dependent GAPDH and NADH-dependent BDH in B. amyloliquefaciens. Excess GAPDH reduced the fermentation time, increased the 2,3-BD yield by 12.7%, and decreased the acetoin titer by 44.3%. However, the process also enhanced lactate and succinate production. Excess BDH increased the 2,3-BD yield by 16.6% while decreasing acetoin, lactate and succinate production, but prolonged the fermentation time. When BDH and GAPDH were co-overproduced in B. amyloliquefaciens, the fermentation time was reduced. Furthermore, in the NADH-dependent pathways, the molar yield of 2,3-BD was increased by 22.7%, while those of acetoin, lactate and succinate were reduced by 80.8%, 33.3% and 39.5%, relative to the parent strain. In fed-batch fermentations, the 2,3-BD concentration was maximized at 132.9 g/l after 45 h, with a productivity of 2.95 g/l·h.

Conclusions/Significance

Co-overexpression of bdh and gapA genes proved an effective method for enhancing 2,3-BD production and inhibiting the accumulation of unwanted by-products (acetoin, lactate and succinate). To our knowledge, we have attained the highest 2,3-BD fermentation yield thus far reported for safe microorganisms.     

High-yield production of (R)-acetoin in Saccharomyces cerevisiae by deleting genes for NAD(P)H-dependent ketone reductases producing meso-2,3-butanediol and 2,3-dimethylglycerate

Acetoin is widely used in food and cosmetics industries as a taste and fragrance enhancer. To produce (R)-acetoin in Saccharomyces cerevisiae, acetoin biosynthetic genes encoding α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD) from Bacillus subtilis and water-forming NADH oxidase (NoxE) from Lactococcus lactis were integrated into delta-sequences in JHY605 strain, where the production of ethanol, glycerol, and (R,R)-2,3-butanediol (BDO) was largely eliminated. We further improved acetoin production by increasing acetoin tolerance by adaptive laboratory evolution, and eliminating other byproducts including meso-2,3-BDO and 2,3-dimethylglycerate, a newly identified byproduct. Ara1, Ypr1, and Ymr226c (named Ora1) were identified as (S)-alcohol-forming reductases, which can reduce (R)-acetoin to meso-2,3-BDO in vitro. However, only Ara1 and Ypr1 contributed to meso-2,3-BDO production in vivo. We elucidate that Ora1, having a substrate preference for (S)-acetoin, reduces (S)-α-acetolactate to 2,3-dimethylglycerate, thus competing with AlsD-mediated (R)-acetoin production. By deleting ARA1, YPR1, and ORA1, 101.3 g/L of (R)-acetoin was produced with a high yield (96% of the maximum theoretical yield) and high stereospecificity (98.2%).     

Intracellular nicotinamide adenine dinucleotide promotes TNF-induced necroptosis in a sirtuin-dependent manner

Cellular necrosis has long been regarded as an incidental and uncontrolled form of cell death. However, a regulated form of cell death termed necroptosis has been identified recently. Necroptosis can be induced by extracellular cytokines, pathogens and several pharmacological compounds, which share the property of triggering the formation of a RIPK3-containing molecular complex supporting cell death. Of interest, most ligands known to induce necroptosis (including notably TNF and FASL) can also promote apoptosis, and the mechanisms regulating the decision of cells to commit to one form of cell death or the other are still poorly defined. We demonstrate herein that intracellular nicotinamide adenine dinucleotide (NAD⁺) has an important role in supporting cell progression to necroptosis. Using a panel of pharmacological and genetic approaches, we show that intracellular NAD⁺ promotes necroptosis of the L929 cell line in response to TNF. Use of a pan-sirtuin inhibitor and shRNA-mediated protein knockdown led us to uncover a role for the NAD⁺-dependent family of sirtuins, and in particular for SIRT2 and SIRT5, in the regulation of the necroptotic cell death program. Thus, and in contrast to a generally held view, intracellular NAD⁺ does not represent a universal pro-survival factor, but rather acts as a key metabolite regulating the choice of cell demise in response to both intrinsic and extrinsic factors.Nicotinamide adenine dinucleotide (NAD⁺) has been long recognized as a key intermediate in cellular metabolism. By accepting and donating electrons in reactions catalyzed by dehydrogenases, NAD⁺ has, for example, a central role in the generation of ATP, a molecule required for most energy-consuming cellular reactions. The recognition of NAD⁺ as a substrate in a number of regulatory processes has shed a new light on its role in cell physiology. Indeed, NAD⁺ represents a substrate for a wide range of enzymes including cADP-ribose synthases, poly (ADP-ribose) polymerases (PARPs) and the sirtuin family of NAD⁺-dependent deacylases (SIRTs). In marked contrast to its role in energy metabolism, the involvement of NAD⁺ in these enzymatic reactions is based on its ability to function as a donor of ADP-ribose, a reaction that, if sustained, can lead to the depletion of the intracellular NAD⁺ pool.^{1, 2, 3, 4, 5}The pro-survival role of NAD⁺ has been particularly well described in cells exposed to genotoxic/oxidative stress. In response to DNA damage, PARP1, the founding and most abundant member of the PARP family, binds to DNA strand breaks and initiates a repair response by catalyzing the post-translational modification of several nuclear proteins, including itself. This protective response is characterized by the transfer of successive units of the ADP-ribose moiety (up to 200 units) from NAD⁺ to other proteins, compromising therefore both energy production (slowing the rate of glycolysis, electron transport and ATP formation) and activity of other NAD⁺-dependent enzymes through NAD⁺ depletion.^{6, 7} Moreover, PARP1-synthesized PAR polymers can be degraded into free oligomers, known to translocate to the mitochondria where they can trigger the release of AIF from mitochondria to the nucleus.^{8, 9, 10, 11} The precise molecular steps linking PARP1 activation to this form of stress-induced cell death, termed parthanatos, have not been fully elucidated, and probably depend on the particular metabolic status of the cell examined (i.e., anerobic glycolysis in most in vitro cell lines versus oxidative metabolism of neuronal cells, see Welsby et al.¹² for review). In any instances, and independently of the fine molecular events at work, virtually all studies have identified the brisk loss of intracellular NAD⁺ as the critical step initiating this specific form of cell death. The protective role of NAD⁺ in PARP1-dependent cell death has been indeed amply documented.^{13, 14, 15, 16, 17, 18} In mammals, nicotinamide (Nam) acts as the main precursor for NAD⁺ biosynthesis and nicotinamide phosphoribosyl tranferase (NAMPT) is the rate-limiting enzyme for NAD⁺ biosynthesis from Nam.¹⁹ Nampt deficiency in mice leads to lethality and the heterozygous animals suffer from significant perturbations related to their NAD⁺ metabolism.²⁰ In keeping with the general role of NAD⁺ as a survival factor in cells exposed to genotoxic stress, genetic ablation of Nampt and/or treatment with a specific NAMPT inhibitor (FK866) sensitized cells to the toxic effects of alkylating agents.^{16, 18} Similarly, overexpression of a catalytically active recombinant NAMPT protected the NIH-3T3 cell line from the toxicity of the same DNA alkylating agents,¹⁸ further establishing a functional link between NAD⁺ biosynthesis and sensitivity to stress-induced, PARP1-dependent cell death.While analyzing the influence of NAD metabolism on survival of NIH3T3 cells exposed to genotoxic agents, we observed that overexpression of NAMPT prolonged cell survival of cells exposed to the alkylating agent N-methyl-N''-nitro-N-nitrosoguanidine (MNNG), and unexpectedly, led to increased sensitivity to cell death induced by the pro-inflammatory cytokine TNF.¹⁸ TNF is a pleiotropic cytokine regulating many cellular functions and known to induce several forms of cell death, including apoptosis and the recently uncovered regulated form of necrosis termed necroptosis.^{21, 22} In contrast to apoptosis, necroptosis is largely independent of the so-called executioner caspase (such as caspase-3, 6 and 7) activity and is initiated by the formation of a signaling complex comprising the receptor-interacting serine-threonine kinase 1 (RIPK1), RIPK3 and the recently identified mixed lineage kinase domain-like protein MLKL. Although necroptosis often appears to occur when apoptosis is abortive (such as in situations of caspase inhibition), the cellular factors regulating the choice between these two forms of regulated cell death have not been fully uncovered. Using a model cell line engineered to respond to both apoptosis and necroptosis, we demonstrate herein that intracellular NAD⁺ represents a critical factor in promoting cell death by necroptosis. In keeping with the well-described role of sirtuins as intracellular NAD⁺ sensors, we also demonstrate that sirtuins, and in particular SIRT2 and SIRT5, are required for adequate completion of the necroptotic program in response to TNF. Accordingly, a pan-sirtuin inhibitor was found to attenuate organ damage induced by transient ischemia. Thus, intracellular NAD⁺, rather than acting as a general cell survival factor, appears to promote cell necroptosis in a sirtuin-dependent manner, a finding that may suggest novel therapeutic approaches to attenuate in vivo necrotic insults in several pathological settings.     

NAD-Linked Isocitrate Dehydrogenase: Isolation, Purification, and Characterization of the Protein from Pea Mitochondria

The NAD⁺-dependent isocitrate dehydrogenase from etiolated pea (Pisum sativum L.) mitochondria was purified more than 200-fold by dye-ligand binding on Matrix Gel Blue A and gel filtration on Superose 6. The enzyme was stabilized during purification by the inclusion of 20% glycerol. In crude matrix extracts, the enzyme activity eluted from Superose 6 with apparent molecular masses of 1400 ± 200, 690 ± 90, and 300 ± 50 kD. During subsequent purification steps the larger molecular mass species disappeared and an additional peak at 94 ± 16 kD was evident. The monomer for the enzyme was tentatively identified at 47 kD by sodium dodecyl-polyacrylamide gel electrophoresis. The NADP⁺-specific isocitrate dehydrogenase activity from mitochondria eluted from Superose 6 at 80 ± 10 kD. About half of the NAD⁺ and NADP⁺-specific enzymes remained bound to the mitochondrial membranes and was not removed by washing. The NAD⁺-dependent isocitrate dehydrogenase showed sigmodial kinetics in response to isocitrate (S_0.5 = 0.3 mm). When the enzyme was aged at 4°C or frozen, the isocitrate response showed less allosterism, but this was partially reversed by the addition of citrate to the reaction medium. The NAD⁺ isocitrate dehydrogenase showed standard Michaelis-Menten kinetics toward NAD⁺ (K_m = 0.2 mm). NADH was a competitive inhibitor (K_i = 0.2 mm) and, unexpectedly, NADPH was a noncompetitive inhibitor (K_i = 0.3 mm). The regulation by NADPH may provide a mechanism for coordination of pyridine nucleotide pools in the mitochondria.     

<Emphasis Type="Italic">R</Emphasis>-acetoin accumulation and dissimilation in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Klebsiella pneumoniae is a 2,3-butanediol producer, and R-acetoin is an intermediate of 2,3-butanediol production. R-acetoin accumulation and dissimilation in K. pneumoniae was studied here. A budC mutant, which has lost 2,3-butanediol dehydrogenase activity, accumulated high levels of R-acetoin in culture broth. However, after glucose was exhausted, the accumulated R-acetoin could be reused by the cells as a carbon source. Acetoin dehydrogenase enzyme system, encoded by acoABCD, was responsible for R-acetoin dissimilation. acoABCD mutants lost the ability to grow on acetoin as the sole carbon source, and the acetoin accumulated could not be dissimilated. However, in the presence of another carbon source, the acetoin accumulated in broth of acoABCD mutants was converted to 2,3-butanediol. Parameters of R-acetoin production by budC mutants were optimized in batch culture. Aerobic culture and mildly acidic conditions (pH 6–6.5) favored R-acetoin accumulation. At the optimized conditions, in fed-batch fermentation, 62.3 g/L R-acetoin was produced by budC and acoABCD double mutant in 57 h culture, with an optical purity of 98.0 %, and a substrate conversion ratio of 28.7 %.     

The MTCY428.08 Gene of Mycobacterium tuberculosis Codes for NAD+ Synthetase

The product of the MTCY428.08 gene of Mycobacterium tuberculosis shows sequence homology with several NAD⁺ synthetases. The MTCY428.08 gene was cloned into the expression vectors pGEX-4T-1 and pET-15b. Expression in Escherichia coli led to overproduction of glutathione S-transferase fused and His₆-tagged gene products, which were enzymatically assayed for NAD synthetase activity. Our results demonstrate that the MTCY428.08 gene of M. tuberculosis is the structural gene for NAD⁺ synthetase.