Properties and Role of Glyceraldehyde-3-Phosphate Dehydrogenase in the Control of Fermentation Pattern and Growth in a Ruminal Bacterium, <Emphasis Type="Italic">Streptococcus bovis</Emphasis>

To clarify the control of glycolysis and the fermentation pattern in Streptococcus bovis, the molecular and enzymatic properties of NAD⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined. The GAPDH gene (gapA) was found to cluster with several others, including those that encode phosphoglycerate kinase and translation elongation factor G, however, gapA was transcribed in a monocistronic fashion. Since biochemical properties, such as optimal pH and affinity for glyceraldehyde-3-phosphate (GAP), were not very different between GAPDH- and NADP⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPN), the flux from GAP may be greatly influenced by the relative amounts of these two enzymes. Using S. bovis JB1 as a parent, JB1gapA and JB1ldh, which overproduce GAPDH and lactate dehydrogenase (LDH), respectively, were constructed to examine the control of the glycolytic flux and lactate production. There were no significant differences in growth rates and formate-to-lactate ratios among JB1, JB1gapA, and JB1ldh grown on glucose. When grown on lactose, JB1ldh showed a much lower formate-to-lactate ratio than JB1gapA, which showed the highest NADH-to-NAD⁺ ratio. However, growth rates did not differ among JB1, JB1gapA, and JB1ldh. These results suggest that GAPDH is not involved in the control of the glycolytic flux and that lactate production is mainly controlled by LDH activity.     

Facilitated Transport of Lactate by Rat Jejunal Enterocyte

L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral membrane vesicles l-lactate uptake is stimulated by an inwardly directed H⁺ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective. The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is saturable with respect to external lactate with a K _m of 39.2 ± 4.8 mm and a J _max of 8.9 ± 0.7 nmoles mg protein⁻¹ sec⁻¹. A very small conductive pathway for l-lactate is present in basolateral membranes. In brush border membrane vesicles both Na⁺ and H⁺ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains a H⁺-lactate cotransporter, whereas in the apical membrane both H⁺-lactate and Na⁺-lactate cotransporters are present, even if they exhibit a low transport rate. Received: 22 October 1996/Revised: 11 March 1997     

Role of the pyruvate metabolic network on carbohydrate metabolism and virulence in Streptococcus pneumoniae

Streptococcus pneumoniae is a major human pathogen that must adapt to unique nutritional environments in several host niches. The pneumococcus can metabolize a range of carbohydrates that feed into glycolysis ending in pyruvate, which is catabolized by several enzymes. We investigated how the pneumococcus utilizes these enzymes to metabolize different carbohydrates and how this impacts survival in the host. Loss of ldh decreased bacterial burden in the nasopharynx and enhanced bacteremia in mice. Loss of spxB, pdhC or pfl2 decreased bacteremia and increased host survival. In glucose or galactose, loss of ldh increased capsule production, whereas loss of spxB and pdhC reduced capsule production. The pfl2 mutant exhibited reduced capsule production only in galactose. In glucose, pyruvate was metabolized primarily by LDH to generate lactate and NAD⁺ and by SpxB and PDHc to generate acetyl-CoA. In galactose, pyruvate metabolism was shunted toward acetyl-CoA production. The majority of acetyl-CoA generated by PFL was used to regenerate NAD⁺ with a subset used in capsule production, while the acetyl-CoA generated by SpxB and PDHc was utilized primarily for capsule biosynthesis. These data suggest that the pneumococcus can alter flux of pyruvate metabolism dependent on the carbohydrate present to succeed in distinct host niches.     

The new insight into extracellular NAD+ degradation-the contribution of CD38 and CD73 in calcific aortic valve disease

Nicotinamide adenine dinucleotide (NAD⁺) is crucial for cell energy metabolism and many signalling processes. Recently, we proved the role of ecto-enzymes in controlling adenine nucleotide–dependent pathways during calcific aortic valve disease (CAVD). This study aimed to investigate extracellular hydrolysis of NAD⁺ and mononucleotide nicotinamide (NMN) in aortic valves and aorta fragments of CAVD patients and on the inner aortic surface of ecto-5′-nucleotidase knockout mice (CD73−/−). Human non-stenotic valves (n = 10) actively converted NAD⁺ and NMN via both CD73 and NAD⁺-glycohydrolase (CD38) according to our analysis with RP-HPLC and immunofluorescence. In stenotic valves (n = 50), due to reduced CD73 activity, NAD⁺ was degraded predominantly by CD38 and additionally by ALP and eNPP1. CAVD patients had significantly higher hydrolytic rates of NAD⁺ (0.81 ± 0.07 vs 0.56 ± 0.10) and NMN (1.12 ± 0.10 vs 0.71 ± 0.08 nmol/min/cm²) compared with controls. CD38 was also primarily engaged in human vascular NAD⁺ metabolism. Studies using specific ecto-enzyme inhibitors and CD73−/− mice confirmed that CD73 is not the only enzyme involved in NAD⁺ and NMN hydrolysis and that CD38 had a significant contribution to these pathways. Modifications of extracellular NAD⁺ and NMN metabolism in aortic valve cells may be particularly important in valve pathology and could be a potential therapeutic target.     

Molecular Cloning and Characterization of a Malic Enzyme Gene from the Oleaginous Yeast <Emphasis Type="Italic">Lipomyces starkeyi</Emphasis>

The malic enzyme-encoding cDNA (GQ372891) from the oleaginous yeast Lipomyces starkeyi AS 2.1560 was isolated, which has an 1719-bp open reading frame flanked by a 290-bp 5′ untranslated sequence and a 92-bp 3′ untranslated sequence. The proposed gene, LsME1, encoded a protein with 572 amino acid residues. The protein presented 58% sequence identity with the malic enzymes from Yarrowia lipolytica CLIB122 and Aspergillus fumigatus Af293. The LsME1 gene was cloned into the vector pMAL-p4x to express a fusion protein (MBP-LsME1) in Escherichia coli TB1. The fusion protein was purified and then cleaved by Factor Xa to give the recombinant LsME1. This purified enzyme took either NAD⁺ or NADP⁺ as the coenzyme but preferred NAD⁺. The K _m values for malic acid, NAD⁺ and NADP⁺ were 0.85 ± 0.05 mM, 0.34 ± 0.08 mM, and 7.4 ± 0.32 mM, respectively, at pH 7.3.     

<Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> as a novel biocatalyst for pyruvate production from<Emphasis Type="SmallCaps"> DL</Emphasis>-lactate

Pseudomonas stutzeri SDM oxidized dl-lactic acid (25.5 g l^-1) into pyruvic acid (22.6 g l^-1) over 24 h. Both NAD⁺-independent d-lactate dehydrogenase and NAD⁺-independent l-lactate dehydrogenase were found for the first time in the bioconversion of lactate to pyruvate based on the enzyme activity assay and proteomic analysis. Jianrong Hao and Cuiqing Ma contributed equally to this work     

Kinetic Parameters and Lactate Dehydrogenase Isozyme Activities Support Possible Lactate Utilization by Neurons

Lactate is potentially a major energy source in brain, particularly following hypoxia/ischemia; however, the regulation of brain lactate metabolism is not well understood. Lactate dehydrogenase (LDH) isozymes in cytosol from primary cultures of neurons and astrocytes, and freshly isolated synaptic terminals (synaptosomes) from adult rat brain were separated by electrophoresis, visualized with an activity-based stain, and quantified. The activity and kinetics of LDH were determined in the same preparations. In synaptosomes, the forward reaction (pyruvate + NADH + H⁺ → lactate + NAD⁺), which had a V _max of 1,163 μmol/min/mg protein was 62% of the rate in astrocyte cytoplasm. In contrast, the reverse reaction (lactate + NAD⁺ → pyruvate + NADH + H⁺), which had a V _max of 268 μmol/min/mg protein was 237% of the rate in astrocytes. Although the relative distribution was different, all five isozymes of LDH were present in synaptosomes and primary cultures of cortical neurons and astrocytes from rat brain. LDH1 was 14.1% of the isozyme in synaptic terminals, but only 2.6% and 2.4% in neurons and astrocytes, respectively. LDH5 was considerably lower in synaptic terminals than in neurons and astrocytes, representing 20.4%, 37.3% and 34.8% of the isozyme in these preparations, respectively. The distribution of LDH isozymes in primary cultures of cortical neurons does not directly reflect the kinetics of LDH and the capacity for lactate oxidation. However, the kinetics of LDH in brain are consistent with the possible release of lactate by astrocytes and oxidative use of lactate for energy in synaptic terminals. Special issue dedicated to John P. Blass.     

A novel enzyme-immobilization method for a biofuel cell

Biofuel cells utilizing biocatalysts are attractive alternatives to metal catalyst-based cells because of environmentally friendly cells and their renewability and good operations at room temperatures, even though they provide a low level of electrical power. In this study, the effect of a novel enzyme immobilization method on anodic electrical properties was evaluated under ambient conditions for increasing the power of an enzyme-based biofuel cell. The anodic system employed in the cell contained a gold electrode, pyrroloquinoline quinone (PQQ) as the electron transfer mediator, lactate dehydrogenase (LDH), β-nicotinamide adenine dinucleotide (NAD⁺) as the cofactor, and lactate as the substrate. The anodic electrical properties increased as a result of the novel enzyme-immobilization method. Furthermore, lactate, NAD⁺, or CaCl₂, which can all influence enzyme activation, were used to prevent covalent bond formation near the active site of the LDH during enzyme-immobilization. Protection of the active site of the LDH using this novel enzyme-immobilization method increased its stability, which enabled to increase power production (142 μW/cm²) in a basic enzymatic fuel cell (EFC).     

A novel mode of lactate metabolism in strictly anaerobic bacteria

Lactate is a common substrate for major groups of strictly anaerobic bacteria, but the biochemistry and bioenergetics of lactate oxidation is obscure. The high redox potential of the pyruvate/lactate pair of E₀′ = ?190 mV excludes direct NAD⁺ reduction (E₀′ = ?320 mV). To identify the hitherto unknown electron acceptor, we have purified the lactate dehydrogenase (LDH) from the strictly anaerobic, acetogenic bacterium Acetobacterium woodii. The LDH forms a stable complex with an electron‐transferring flavoprotein (Etf) that exhibited NAD⁺ reduction only when reduced ferredoxin (Fd^2?) was present. Biochemical analyses revealed that the LDH/Etf complex of A. woodii uses flavin‐based electron confurcation to drive endergonic lactate oxidation with NAD⁺ as oxidant at the expense of simultaneous exergonic electron flow from reduced ferredoxin (E₀′ ≈ –500 mV) to NAD⁺ according to: lactate + Fd^2? + 2 NAD⁺ → pyruvate + Fd + 2 NADH. The reduced Fd^2? is regenerated from NADH by a sequence of events that involves conversion of chemical (ATP) to electrochemical and finally redox energy (Fd^2? from NADH) via reversed electron transport catalysed by the Rnf complex. Inspection of genomes revealed that this metabolic scenario for lactate oxidation may also apply to many other anaerobes.     

Identification of small-molecule urea derivatives as novel NAMPT inhibitors via pharmacophore-based virtual screening

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the condensation of nicotinamide (NAM) with 5-phosphoribosyl-1-prophosphate (PRPP) to yield nicotinamide mononucleotide (NMN), a rate limiting enzyme in a mammalian salvage pathway of nicotinamide adenine dinucleotide (NAD⁺) synthesis. Recently, intracellular NAD⁺ has received substantial attention due to the recent discovery that several enzymes including poly(ADP-ribose) polymerases (PARPs), mono(ADP-ribose) transferases (ARTs), and sirtuins (SIRTs), use NAD⁺ as a substrate, suggesting that intracellular NAD⁺ level may regulate cytokine production, metabolism, and aging through these enzymes. NAMPT is found to be upregulated in various types of cancer, and given its importance in the NAD⁺ salvage pathway, NAMPT is considered as an attractive target for the development of new cancer therapies. In this study, the reported NAMPT inhibitors bearing amide, cyanoguanidine, and urea scaffolds were used to generate pharmacophore models and pharmacophore-based virtual screening studies were performed against ZINC database. Following the filtering steps, ten hits were identified and evaluated for their in vitro NAMPT inhibitory effects. Compounds GF4 (NAMPT IC₅₀ = 2.15 ± 0.22 μM) and GF8 (NAMPT IC₅₀ = 7.31 ± 1.59 μM) were identified as new urea-typed inhibitors of NAMPT which also displayed cytotoxic activities against human HepG2 hepatocellular carcinoma cell line with IC₅₀ values of 15.20 ± 1.28 and 24.28 ± 6.74 μM, respectively.     

Double mutation of the <Emphasis Type="Italic">PDC1</Emphasis> and <Emphasis Type="Italic">ADH1</Emphasis> genes improves lactate production in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing the bovine lactate dehydrogenase gene

Expression of a heterologous l-lactate dehydrogenase (l-ldh) gene enables production of optically pure l-lactate by yeast Saccharomyces cerevisiae. However, the lactate yields with engineered yeasts are lower than those in the case of lactic acid bacteria because there is a strong tendency for ethanol to be competitively produced from pyruvate. To decrease the ethanol production and increase the lactate yield, inactivation of the genes that are involved in ethanol production from pyruvate is necessary. We conducted double disruption of the pyruvate decarboxylase 1 (PDC1) and alcohol dehydrogenase 1 (ADH1) genes in a S. cerevisiae strain by replacing them with the bovine l-ldh gene. The lactate yield was increased in the pdc1/adh1 double mutant compared with that in the single pdc1 mutant. The specific growth rate of the double mutant was decreased on glucose but not affected on ethanol or acetate compared with in the control strain. The aeration rate had a strong influence on the production rate and yield of lactate in this strain. The highest lactate yield of 0.75 g lactate produced per gram of glucose consumed was achieved at a lower aeration rate.     

Metabolism in 1,3‐propanediol fed‐batch fermentation by a D‐lactate deficient mutant of Klebsiella pneumoniae

Klebsiella pneumoniae HR526, a new isolated 1,3‐propanediol (1,3‐PD) producer, exhibited great productivity. However, the accumulation of lactate in the late‐exponential phase remained an obstacle of 1,3‐PD industrial scale production. Hereby, mutants lacking D ‐lactate pathway were constructed by knocking out the ldhA gene encoding fermentative D ‐lactate dehydrogenase (LDH) of HR526. The mutant K. pneumoniae LDH526 with the lowest LDH activity was studied in aerobic fed‐batch fermentation. In experiments using pure glycerol as feedstock, the 1,3‐PD concentrations, conversion, and productivity increased from 95.39 g L^?1, 0.48 and 1.98 g L^?1 h^?1 to 102. 06 g L^?1, 0.52 mol mol^?1 and 2.13 g L^?1 h^?1, respectively. The diol (1,3‐PD and 2,3‐butanediol) conversion increased from 0.55 mol mol^?1 to a maximum of 0.65 mol mol^?1. Lactate would not accumulate until 1,3‐PD exceeded 84 g L^?1, and the final lactate concentration decreased dramatically from more than 40 g L^?1 to <3 g L^?1. Enzymic measurements showed LDH activity decreased by 89–98% during fed‐batch fermentation, and other related enzyme activities were not affected. NADH/NAD⁺ enhanced more than 50% in the late‐exponential phase as the D ‐lactate pathway was cut off, which might be the main reason for the change of final metabolites concentrations. The ability to utilize crude glycerol from biodiesel process and great genetic stability demonstrated that K. pnemoniae LDH526 was valuable for 1,3‐PD industrial production. Biotechnol. Bioeng. 2009; 104: 965–972. © 2009 Wiley Periodicals, Inc.     

Physical and Functional Association of Lactate Dehydrogenase (LDH) with Skeletal Muscle Mitochondria

The intracellular lactate shuttle hypothesis posits that lactate generated in the cytosol is oxidized by mitochondrial lactate dehydrogenase (LDH) of the same cell. To examine whether skeletal muscle mitochondria oxidize lactate, mitochondrial respiratory oxygen flux (JO₂) was measured during the sequential addition of various substrates and cofactors onto permeabilized rat gastrocnemius muscle fibers, as well as isolated mitochondrial subpopulations. Addition of lactate did not alter JO₂. However, subsequent addition of NAD⁺ significantly increased JO₂, and was abolished by the inhibitor of mitochondrial pyruvate transport, α-cyano-4-hydroxycinnamate. In experiments with isolated subsarcolemmal and intermyofibrillar mitochondrial subpopulations, only subsarcolemmal exhibited NAD⁺-dependent lactate oxidation. To further investigate the details of the physical association of LDH with mitochondria in muscle, immunofluorescence/confocal microscopy and immunoblotting approaches were used. LDH clearly colocalized with mitochondria in intact, as well as permeabilized fibers. LDH is likely localized inside the outer mitochondrial membrane, but not in the mitochondrial matrix. Collectively, these results suggest that extra-matrix LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria.     

Heterotrophic nitrification by <Emphasis Type="Italic">Achromobacter xylosoxidans</Emphasis> S18 isolated from a small-scale slaughterhouse wastewater

Heterotrophic carbon utilizing microbes were acclimatized in the laboratory by inoculating sludge collected from the waste discharge pond of a small-scale rural abattoir in India in a nutrient solution intermittently fed with glucose and ammonium chloride. Cultures of 10 well-developed isolates were selected and grown in a basal medium containing glucose and ammonium chloride. Culture supernatants were periodically analyzed for ammonium nitrogen (NH₄ ⁺-N) and chemical oxygen demand (COD). Polyphasic taxonomic study of the most active nitrifier (S18) was done. Half saturation concentration (K _s), maximum rate of substrate utilization (k), yield coefficient (Y) and decay coefficient (K _d) were determined from the Lineweaver–Burk plot using the modified Monod equation. S18 was able to remove 97 ± 2% of (NH₄ ⁺-N) and 88 ± 3% of COD. Molecular phylogenetic study supported by physiological and biochemical characteristics assigned S18 as Achromobacter xylosoxidans. Nitrification activity of A. xylosoxidans was demonstrated for the first time, while interestingly, the distinctive anaerobic denitrification property was preserved in S18. K _s values were determined as 232.13 ± 1.5 mg/l for COD reduction and 2.131 ± 1.9 mg/l for NH₄ ⁺-N utilization. Yield coefficients obtained were 0.4423 ± 0.1134 mg of MLVSS/mg of COD and 0.2461 ± 0.0793 mg of MLVSS/mg of NH₄ ⁺-N while the decay coefficients were 0.0627 ± 0.0013 per day and 0.0514 ± 0.0008 per day, respectively. After a contact period of 24 h, 650 ± 5 mg/l solids were produced when the initial concentration of COD and NH₄ ⁺-N were 1820 ± 10 mg/l and 120 ± 5.5 mg/l, respectively. This is the first report on the kinetic coefficients for carbon oxidation and nitrification by a single bacterium isolated from slaughterhouse wastewater.     

Evaluation of endogenous acidic metabolic products associated with carbohydrate metabolism in tumor cells

Tumor cells have a high tolerance for acidic and hypoxic microenvironments, also producing abundant lactic acid through accelerated glycolysis in the presence or absence of O₂. While the accumulation of lactate is thought to be a major contributor to the reduction of pH-circumscribing aggressive tumors, it is not known if other endogenous metabolic products contribute this acidity. Furthermore, anaerobic metabolism in cancer cells bears similarity to homo-fermentative lactic acid bacteria, however very little is known about an alternative pathway that may drive adenosine triphosphate (ATP) production independent of glycolysis. In this study, we quantify over 40 end-products (amines, acids, alcohols, aldehydes, or ketones) produced by malignant neuroblastoma under accelerated glycolysis (+glucose (GLU) supply 1–10 mM) ± mitochondrial toxin; 1-methyl-4-phenylpyridinium (MPP⁺) to abate aerobic respiration to delineate differences between anaerobic vs. aerobic cell required metabolic pathways. The data show that an acceleration of anaerobic glycolysis prompts an expected reduction in extracellular pH (pH_ex) from neutral to 6.7 ± 0.006. Diverse metabolic acids associated with this drop in acidity were quantified by ionic exchange liquid chromatography (LC), showing concomitant rise in lactate (Ctrls 7.5 ± 0.5 mM; +GLU 12.35 ± 1.3 mM; +GLU + MPP 18.1 ± 1.8 mM), acetate (Ctrl 0.84 ± 0.13 mM: +GLU 1.3 ± 0.15 mM; +GLU + MPP 2.7 ± 0.4 mM), fumarate, and a-ketoglutarate (<10 μM) while a range of other metabolic organic acids remained undetected. Amino acids quantified by o-phthalaldehyde precolumn derivatization/electrochemical detection–LC show accumulation of l-alanine (1.6 ± .052 mM), l-glutamate (285 ± 9.7 μM), l-asparagine (202 ± 2.1 μM), and l-aspartate (84.2 ± 4.9 μM) produced during routine metabolism, while other amino acids remain undetected. In contrast, the data show no evidence for accumulation of acetaldehyde, aldehydes, or ketones (Purpald/2,4-dinitrophenylhydrazine—Brady's reagent), acetoin (Voges–Proskauer test), or alcohols (NAD⁺-linked alcohol dehydrogenase). In conclusion, these results provide preliminary evidence to suggest the existence of an active pyruvate–alanine transaminase or phosphotransacetylase/acetyl-CoA synthetase pathway to be involved with anaerobic energy metabolism of cancer cells.     

Effect of oxygenation and sulfate concentrations on pyruvate and lactate formation inKlebsiella oxytoca ZS growing in chemostat cultures

Klebsiella oxytoca ZS fermented glucose to ethanol and lactic, formic, and acetic acids, but, in contrast to many strains, accumulates pyruvic and acetic acids as the principal end products in aerobic growth conditions. This strain was grown in sulfate-limited chemostat at a fixed low dilution rate (D=0.033 h^–1) with glucose present in excess. When oxygen was supplied at a high level, pyruvate and acetate were produced, and the ratio NADH/NAD⁺ was low (0.04) while the internal pyruvate concentration increased to 100 mol (g dry wt)^–1. A shortage of oxygen supply was accompanied by lactate production, an increase of the ratio NADH/NAD⁺ (0.53), and an undetectable level in internal pyruvate concentration. The observed changes in LDH activity found in vitro in extracts of the cells are not strictly related to those found in vivo. In fact, the specific activity of LDH was essentially stable at 30% of dissolved oxygen tension (d.o.t.) and decreased slightly at 60% of d.o.t., whereas specific lactic acid production decreased rapidly. The in vitro LDH activity was strongly affected by the NADH/NAD⁺ ratio.     

低温冷藏提高家蚕滞育卵NAD含量和胞质苹果酸脱氢酶活性

为了调查5℃低温处理是否改变家蚕Bombyx mori卵滞育NAD代谢, 本研究利用HPLC和分光光度法测定了经25℃和5℃分别处理的滞育卵中NADH 含量、 NAD⁺含量、 乳酸脱氢酶（LDH）活性和胞质苹果酸脱氢酶（cMDH）活性。结果表明： 5℃处理的NAD（NADH + NAD⁺）含量和cMDH活性分别增加了106%和53%, 并且显著高于25℃处理（P< 0.01）; 但是两种处理的NADH/NAD⁺比值和LDH活性没有显著差异（P> 0.05）。据此推测, 5℃低温处理加强了家蚕滞育卵NAD⁺合成和再生能力。     

Two malate dehydrogenases in Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum (strain Marburg) was found to contain two malate dehydrogenases, which were partially purified and characterized. One was specific for NAD⁺ and catalyzed the dehydrogenation of malate at approximately one-third of the rate of oxalacetate reduction, and the other could equally well use NAD⁺ and NADP⁺ as coenzyme and catalyzed essentially only the reduction of oxalacetate. Via the N-terminal amino acid sequences, the encoding genes were identified in the genome of M. thermoautotrophicum (strain ΔH). Comparison of the deduced amino acid sequences revealed that the two malate dehydrogenases are phylogenetically only distantly related. The NAD⁺-specific malate dehydrogenase showed high sequence similarity to l-malate dehydrogenase from Methanothermus fervidus, and the NAD(P)⁺-using malate dehyrogenase showed high sequence similarity to l-lactate dehydrogenase from Thermotoga maritima and l-malate dehydrogenase from Bacillus subtilis. A function of the two malate dehydrogenases in NADPH:NAD⁺ transhydrogenation is discussed. Received: 29 December 1997 / Accepted: 4 March 1998     

Biochemical characterization of glyceraldehyde-3-phosphate dehydrogenase from <Emphasis Type="Italic">Thermococcus kodakarensis</Emphasis> KOD1

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an essential role in glycolysis by catalyzing the conversion of d-glyceraldehyde 3-phosphate (d-G3P) to 1,3-diphosphoglycerate using NAD⁺ as a cofactor. In this report, the GAPDH gene from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (GAPDH-tk) was cloned and the protein was purified to homogeneity. GAPDH-tk exists as a homotetramer with a native molecular mass of 145 kDa; the subunit molecular mass was 37 kDa. GAPDH-tk is a thermostable protein with a half-life of 5 h at 80–90°C. The apparent K _m values for NAD⁺ and d-G3P were 77.8 ± 7.5 μM and 49.3 ± 3.0 μM, respectively, with V _max values of 45.1 ± 0.8 U/mg and 59.6 ± 1.3 U/mg, respectively. Transmission electron microscopy (TEM) and image processing confirmed that GAPDH-tk has a tetrameric structure. Interestingly, GAPDH-tk migrates as high molecular mass forms (~232 kDa and ~669 kDa) in response to oxidative stress.     

Metabolic engineering to improve ethanol production in Thermoanaerobacter mathranii

Thermoanaerobacter mathranii can produce ethanol from lignocellulosic biomass at high temperatures, but its biotechnological exploitation will require metabolic engineering to increase its ethanol yield. With a cofactor-dependent ethanol production pathway in T. mathranii, it may become crucial to regenerate cofactor to increase the ethanol yield. Feeding the cells with a more reduced carbon source, such as mannitol, was shown to increase ethanol yield beyond that obtained with glucose and xylose. The ldh gene coding for lactate dehydrogenase was previously deleted from T. mathranii to eliminate an NADH oxidation pathway. To further facilitate NADH regeneration used for ethanol formation, a heterologous gene gldA encoding an NAD⁺-dependent glycerol dehydrogenase was expressed in T. mathranii. One of the resulting recombinant strains, T. mathranii BG1G1 (Δldh, P _xyl GldA), showed increased ethanol yield in the presence of glycerol using xylose as a substrate. With an inactivated lactate pathway and expressed glycerol dehydrogenase activity, the metabolism of the cells was shifted toward the production of ethanol over acetate, hence restoring the redox balance. It was also shown that strain BG1G1 acquired the capability to utilize glycerol as an extra carbon source in the presence of xylose, and utilization of the more reduced substrate glycerol resulted in a higher ethanol yield.