scholarly journals Effect of Different NADH Oxidase Levels on Glucose Metabolism by Lactococcus lactis: Kinetics of Intracellular Metabolite Pools Determined by In Vivo Nuclear Magnetic Resonance

2002 ◽  
Vol 68 (12) ◽  
pp. 6332-6342 ◽  
Author(s):  
Ana Rute Neves ◽  
Ana Ramos ◽  
Helena Costa ◽  
Iris I. van Swam ◽  
Jeroen Hugenholtz ◽  
...  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Glucose Metabolism ◽  
Lactococcus Lactis ◽  
Oxidase Activity ◽  
Nadh Oxidase ◽  
Glycolytic Flux ◽  
Aerobic Conditions ◽  
Nadh Oxidase Activity

ABSTRACT Three isogenic strains of Lactococcus lactis with different levels of H2O-forming NADH oxidase activity were used to study the effect of oxygen on glucose metabolism: the parent strain L. lactis MG1363, a NOX− strain harboring a deletion of the gene coding for H2O-forming NADH oxidase, and a NOX+ strain with the NADH oxidase activity enhanced by about 100-fold. A comprehensive description of the metabolic events was obtained by using 13C nuclear magnetic resonance in vivo. The most noticeable results of this study are as follows: (i) under aerobic conditions the level of fructose 1,6-bisphosphate [Fru(1,6)P2] was lower than the level under anaerobic conditions, and the rate of Fru(1,6)P2 depletion was very high; (ii) the levels of 3-phosphoglycerate and phosphoenolpyruvate were considerably enhanced under aerobic conditions and significantly lower in the NOX− strain; and (iii) the glycolytic flux decreased in the presence of saturating levels of oxygen, but it was not altered in response to changes in the NADH oxidase activity. In particular, the observation that the glycolytic flux was not enhanced in the NOX+ strain indicated that glycolytic flux was not primarily determined by the level of NADH in the cell. The patterns of end products were identical for the NOX− and parent strains; in the NOX+ strain the carbon flux was diverted to the production of α-acetolactate-derived compounds, and at a low pH this strain produced diacetyl at concentrations up to 1.6 mM. The data were integrated with the goal of identifying the main regulatory aspects of glucose metabolism in the presence of oxygen.

scholarly journals Cofactor Engineering: a Novel Approach to Metabolic Engineering in Lactococcus lactis by Controlled Expression of NADH Oxidase

1998 ◽  
Vol 180 (15) ◽  
pp. 3804-3808 ◽  
Author(s):  
Felix Lopez de Felipe ◽  
Michiel Kleerebezem ◽  
Willem M. de Vos ◽  
Jeroen Hugenholtz
Keyword(s):  
Lactococcus Lactis ◽  
Oxidase Activity ◽  
Nadh Oxidase ◽  
Metabolic Effects ◽  
Initial Growth ◽  
Acid Fermentation ◽  
Aerobic Conditions ◽  
Mixed Acid Fermentation ◽  
Mixed Acid ◽  
Nadh Oxidase Activity

ABSTRACT NADH oxidase-overproducing Lactococcus lactis strains were constructed by cloning the Streptococcus mutans nox-2gene, which encodes the H2O-forming NADH oxidase, on the plasmid vector pNZ8020 under the control of the L. lactis nisA promoter. This engineered system allowed a nisin-controlled 150-fold overproduction of NADH oxidase at pH 7.0, resulting in decreased NADH/NAD ratios under aerobic conditions. Deliberate variations on NADH oxidase activity provoked a shift from homolactic to mixed-acid fermentation during aerobic glucose catabolism. The magnitude of this shift was directly dependent on the level of NADH oxidase overproduced. At an initial growth pH of 6.0, smaller amounts of nisin were required to optimize NADH oxidase overproduction, but maximum NADH oxidase activity was twofold lower than that found at pH 7.0. Nonetheless at the highest induction levels, levels of pyruvate flux redistribution were almost identical at both initial pH values. Pyruvate was mostly converted to acetoin or diacetyl via α-acetolactate synthase instead of lactate and was not converted to acetate due to flux limitation through pyruvate dehydrogenase. The activity of the overproduced NADH oxidase could be increased with exogenously added flavin adenine dinucleotide. Under these conditions, lactate production was completely absent. Lactate dehydrogenase remained active under all conditions, indicating that the observed metabolic effects were only due to removal of the reduced cofactor. These results indicate that the observed shift from homolactic to mixed-acid fermentation under aerobic conditions is mainly modulated by the level of NADH oxidation resulting in low NADH/NAD+ratios in the cells.

scholarly journals Identification of bioactive compounds from Fraxinus angustifolia extracts with anti-NADH oxidase activity of bovine milk xanthine oxidoreductase

2019 ◽  
pp. 133-147 ◽  
Author(s):  
Nadjia AHMANE ◽  
Dina ATMANI-KILANI ◽  
Nassima CHAHER ◽  
Karima AYOUNI ◽  
Meriem RAHMANI-BERBOUCHA ◽  
...  
Keyword(s):  
Oxidase Activity ◽  
Nadh Oxidase ◽  
Bovine Milk ◽  
Ros Generation ◽  
Xanthine Oxidoreductase ◽  
Fraxinus Angustifolia ◽  
Activity Of Enzymes ◽  
Nadh Oxidase Activity

Fraxinus angustifolia leaves and bark are used in traditional medicine against various inflammatory-related pathologies incumbent to reactive oxygen species (ROS) generation by the NADH oxidase activity of enzymes such as xanthine oxidoreductase (XOR). This study was designed to investigate the in vitro and in vivo inhibitory activities of this enzyme by Fraxinus angustifolia extracts. The leaf organic phase of ethyl acetate (LFA) and its bark aqueous counterpart (BFA) showed the strongest anti-NADH oxidase activity in vitro (IC50 = 38.51 and 42.04 μg mL-1, respectively). They consequently suppressed superoxide generation both enzymatically (53% and 19%, respectively) and nonenzymatically (34% and 19%, respectively). These results were corroborated in vivo, with high anti- NADH oxidase potential of the leaves and bark extracts (75.32% and 51.32%, respectively) concomitant with moderate hypouricemic activities (36.84% and 38.59%, respectively). Bio-guided fractionation led to the identification, by LC-DAD-MS/MS, of esculin and calcelarioside in bark and kaempferol glucoside in leaves as the main compounds responsible for the anti-NADH oxidase activity of XOR. These results plead in favor of the use of F. angustifolia as a source of potentially interesting therapeutic substances.

scholarly journals Competence Regulation by Oxygen Availability and by Nox Is Not Related to Specific Adjustment of Central Metabolism inStreptococcus pneumoniae

2001 ◽  
Vol 183 (9) ◽  
pp. 2957-2962 ◽  
Author(s):  
Sabine Chapuy-Regaud ◽  
Frédérique Duthoit ◽  
Laurence Malfroy-Mastrorillo ◽  
Pierre Gourdon ◽  
Nic D. Lindley ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Oxidase Activity ◽  
Nadh Oxidase ◽  
Lactate Production ◽  
Oxygen Availability ◽  
Glycolytic Flux ◽  
Mrna Levels ◽  
Central Metabolism ◽  
Loss Of Function ◽  
Nadh Oxidase Activity

ABSTRACT In Streptococcus pneumoniae oxygen availability is a major determinant for competence development in exponentially growing cultures. NADH oxidase activity is required for optimal competence in cultures grown aerobically. The implication of oxidative metabolism and more specifically of Nox on central metabolism has been examined. Glycolytic flux throughout exponential growth revealed homolactic fermentation with a lactate production/glucose utilization ratio close to 2, whatever the aerobiosis level of the culture. Loss-of-function mutations in nox, which encodes NADH oxidase, did not change this trait. Consistently, mRNA levels of glyceraldehyde-3-phosphate dehydrogenase, l-lactate dehydrogenase, pyruvate oxidase, and NADH oxidase remained comparable to wild-type levels, as did the specific activities of key enzymes which control central metabolism. Competence regulation by oxygen involving the NADH oxidase activity is not due to significant modification of carbon flux through glycolysis. Failure to obtain loss-of-function mutation in L-ldh, which encodes thel-lactate dehydrogenase, indicates its essential role in pneumococci whatever their growth status.

scholarly journals The NADH oxidase system (external) of muscle mitochondria and its role in the oxidation of cytoplasmic NADH

1985 ◽  
Vol 229 (3) ◽  
pp. 631-641 ◽  
Author(s):  
U F Rasmussen ◽  
H N Rasmussen
Keyword(s):  
Oxidase Activity ◽  
Nadh Oxidase ◽  
Specific Activity ◽  
Reductase Activity ◽  
Heart Mitochondria ◽  
Freezing And Thawing ◽  
Oxidase System ◽  
Muscle Mitochondria ◽  
Nadh Oxidase Activity

An exo-NADH oxidase system [NADH oxidase system (external)], effecting intact-mitochondrial oxidation of added NADH, was studied in pigeon heart mitochondria. Breast muscle mitochondria showed an equal specific activity of the system. The exo-NADH oxidase activity (200 micron mol of NADH/min per g of protein) equalled two-thirds of the State-3 respiratory activity with malate + pyruvate or one-seventh of the total NADH oxidase activity of heart mitochondria. The activity was not caused by use of proteinase in the preparation procedure and all measured parameters were very reproducible from preparation to preparation. The activity is therefore most likely not due to preparation artefacts. The exo-NADH oxidase system is present in all mitochondria in the preparation and is not confined to a subpopulation. The system reduced all cytochrome anaerobically and direct interaction with all cytochrome oxidase was demonstrated by interdependent cyanide inhibition. The exo-NADH oxidase system seems to be located at the outer surface of the mitochondrial inner membrane because, for instance, only this system was rapidly inhibited by rotenone, and ferricyanide could act as acceptor in the rotenone-inhibited system (reductase activity = 20 times oxidase activity). In the presence of antimycin, added NADH reduced only a part of the b-cytochromes. Freezing and thawing the mitochondria, one of the methods used for making them permeable to NADH, destroyed this functional compartmentation. The characteristics of the exo-NADH oxidase system and the malate-aspartate shuttle are compared and the evidence for the shuttle's function in heart in vivo is re-evaluated. It is proposed that oxidation of cytoplasmic NADH in red muscles primarily is effected by the exo-NADH oxidase system.

Biological NMR Spectroscopy

1997 ◽  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Nmr Spectroscopy ◽  
Magnetic Resonance ◽  
Magnetic Resonance Spectroscopy ◽  
State Of The Art ◽  
Critical Assessment ◽  
Resonance Spectroscopy ◽  
History Of ◽  
Structure Of Proteins

This book presents a critical assessment of progress on the use of nuclear magnetic resonance spectroscopy to determine the structure of proteins, including brief reviews of the history of the field along with coverage of current clinical and in vivo applications. The book, in honor of Oleg Jardetsky, one of the pioneers of the field, is edited by two of the most highly respected investigators using NMR, and features contributions by most of the leading workers in the field. It will be valued as a landmark publication that presents the state-of-the-art perspectives regarding one of today's most important technologies.

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