Anaerobic Enzyme Research Articles

Nitrate regulation of anaerobic respiratory gene expression in narX deletion mutants of Escherichia coli K-12

Previous studies have shown that narL+ is required for nitrate regulation of anaerobic respiratory enzyme synthesis, including formate dehydrogenase-N, nitrate reductase, and fumarate reductase. Insertions in the closely linked narX gene decrease, but do not abolish, nitrate regulation of anaerobic enzyme synthesis. Analysis of sequence similarities suggests that NarX and NarL comprise a two-component regulatory pair. We constructed lacZ operon and gene fusions to investigate the operon structure of narXL. We found evidence for a complex operon with at least two promoters; PXL-narX-PL-narL. We also investigated the role of NarX in nitrate regulation of anaerobic respiratory enzyme synthesis by constructing nonpolar loss of function narX alleles. These deletions were studied on narL+ lambda specialized transducing bacteriophage. The narX deletions had no effect on nitrate regulation in delta (narXL) strains. This finding suggest that the subtle effects of previously studied narX insertions are due to decreased expression of narL and that narX+ is not essential for normal nitrate regulation. The role of NarX in nitrate regulation remains to be determined.

Multiple regulatory elements for the glpA operon encoding anaerobic glycerol-3-phosphate dehydrogenase and the glpD operon encoding aerobic glycerol-3-phosphate dehydrogenase in Escherichia coli: further characterization of respiratory control

In Escherichia coli, sn-glycerol-3-phosphate can be oxidized by two different flavo-dehydrogenases, an anaerobic enzyme encoded by the glpACB operon and an aerobic enzyme encoded by the glpD operon. These two operons belong to the glp regulon specifying the utilization of glycerol, sn-glycerol-3-phosphate, and glycerophosphodiesters. In glpR mutant cells grown under conditions of low catabolite repression, the glpA operon is best expressed anaerobically with fumarate as the exogenous electron acceptor, whereas the glpD operon is best expressed aerobically. Increased anaerobic expression of glpA is dependent on the fnr product, a pleiotropic activator of genes involved in anaerobic respiration. In this study we found that the expression of a glpA1(Oxr) (oxygen-resistant) mutant operon, selected for increased aerobic expression, became less dependent on the FNR protein but more dependent on the cyclic AMP-catabolite gene activator protein complex mediating catabolite repression. Despite the increased aerobic expression of glpA1(Oxr), a twofold aerobic repressibility persisted. Moreover, anaerobic repression by nitrate respiration remained normal. Thus, there seems to exist a redox control apart from the FNR-mediated one. We also showed that the anaerobic repression of the glpD operon was fully relieved by mutations in either arcA (encoding a presumptive DNA recognition protein) or arcB (encoding a presumptive redox sensor protein). The arc system is known to mediate pleiotropic control of genes of aerobic function.

Profiles of enzyme activity in larvae of two cyprinid species with contrasting life styles (Cyprinidae; Teleostei)

Growth and the development of gills, muscle fibres and 10 enzymes serving different metabolic functions were studied in larvae of Rutilus rutilus (L.) and Chalcalburnus chalcoides (Agassiz, 1832). R. rutilus starts swimming and feeding one to three days after hatching, whereas in C. chalcoides this process is delayed by about 10 days. This difference in behaviour is reflected in the time‐course of growth, the differentiation of the red muscle fibres and the activity of the enzymes of aerobic energy metabolism. On the other hand, the activity of the enzymes of anaerobic energy metabolism increases steadily throughout the period of observation (up to 60 days post‐hatch), this trend being more pronounced in C. chalcoides than in R. rutilus. A weight‐independent and a weight‐dependent phase of development can be distinguished in the enzymes of aerobic energy metabolism. It is suggested that, in accord with previous findings, the early phase of locomotor activity of cyprinid larvae is fuelled mainly by aerobic processes, and that the central muscle mass of the larvae is more aerobic than the white muscle fibres of the adults. The delayed development of aerobic enzyme activity in C. chalcoides is compensated by the accelerated development of anaerobic enzyme activity, particularly of creatine kinase and lactate dehydrogenase. This difference between the two species studied suggests differences in the metabolic basis of burst activity.

Binding of Isotopically Labeled Substrates, Inhibitors, and Cyanide by Protocatechuate 3,4-dioxygenase

Binding of ligands to the active site Fe3+ of protocatechuate 3,4-dioxygenase is investigated using EPR-detected transferred hyperfine coupling from isotopically labeled substrates, inhibitors, and cyanide. Broadening is observed in EPR resonances from the anaerobic enzyme complex with homoprotocatechuate (3,4-dihydroxyphenylacetate), a slow substrate, enriched with 17O (I = 5/2) in either the 3-OH or the 4-OH group. This shows that this substrate binds directly to the Fe3+ and strongly suggests that an iron chelate can be formed. Cyanide is known to bind to the enzyme in at least two steps, forming first a high spin and then a low spin complex (Whittaker, J. W., and Lipscomb, J. D. (1984) J. Biol. Chem. 259, 4487-4495). Hyperfine broadening from [13C]cyanide (I = 1/2) is observed in the EPR spectra of both complexes, showing that cyanide is an Fe3+ ligand in each case. Cyanide binding is also at least biphasic in the presence of protocatechuate (PCA). The initial high spin enzyme-PCA-cyanide complex forms rapidly and exhibits a unique EPR spectrum. Broadening from PCA enriched with 17O in either the 3-OH or the 4-OH group is detected showing that PCA binds to the iron, probably as a chelate complex. In contrast, no broadening from [13C]cyanide is detected for this complex suggesting that cyanide binds at a site away from the Fe3+. Steady state kinetic measurements of cyanide inhibition of PCA turnover are consistent with two rapidly exchanging cyanide binding sites that inhibit PCA binding and which can be simultaneously occupied. Formation of the nearly irreversible, low spin enzyme-PCA-cyanide complex is competitively inhibited by PCA. Transient kinetics of the formation of this complex are second order in cyanide implying that two cyanides bind. Broadening in the EPR spectrum of this complex is detected from [13C]cyanide, but not from [17O]PCA, suggesting that PCA is displaced. This study provides the first direct evidence for chelation of the active site Fe3+ by substrates and for a small molecule binding site away from the iron in intradiol dioxygenases.

Oxygen-sensitive ribonucleoside triphosphate reductase is present in anaerobic Escherichia coli.

Ribonucleoside diphosphate reductase from Escherichia coli and mammalian cells provides the deoxyribonucleoside triphosphates for DNA synthesis. The active enzyme contains a tyrosyl free radical whose formation requires oxygen. Earlier genetic evidence suggested that the enzyme is not required for anaerobic growth of E. coli, implicating the activity of a different enzyme or enzyme system for deoxyribonucleotide synthesis in the absence of oxygen. We now conclude from isotope incorporation experiments that E. coli during anaerobiosis obtains its deoxyribonucleotides by reduction of ribonucleotides. Extracts from anaerobically grown bacteria contain a different enzyme activity capable of reducing CTP to dCTP. To obtain an active enzyme, strict anaerobiosis must be maintained during extract preparation and during assay of the enzyme. The reaction is stimulated by NADPH, Mg2+, and ATP. Inhibition by deoxyribonucleoside triphosphates suggests that the anaerobic enzyme has allosteric properties. Antibodies raised against the aerobic enzyme do not inhibit the new activity, and hydroxyurea, a potent scavenger of the tyrosyl radical of the aerobic enzyme, only weakly inhibits the anaerobic enzyme. The anaerobic enzyme has interesting evolutionary aspects since it might reflect on an activity that in the absence of oxygen made possible the transition from an "RNA world" into a "DNA world."

Metabolic consequences of resistive-type exercise.

This brief review concerns acute and chronic metabolic responses to resistive-type exercise (RTE) (i.e., Olympic/power weight lifting and bodybuilding). Performance of RTE presents power output substantially greater (10-15-fold) than that evident with endurance-type exercise. Accordingly, RTE relies heavily on the anaerobic enzyme machinery of skeletal muscle for energy supply, with alterations in the rate of aerobic metabolism being modest. Hydrolysis of high energy phosphate compounds (PC, ATP), glycogenolysis, and glycolysis are evident during an acute bout of RTE as indicated by metabolic markers in mixed fiber type skeletal muscle samples. The type of RTE probably influences the magnitude of these responses since the increase in blood lactate is much greater during a typical "bodybuilding" than "power lifting" session. The influence of RTE training on acute metabolic responses to RTE has received little attention. An individual's inherent metabolic characteristics are apparently sufficient to meet the energy demands of RTE as training of this type does not increase VO2max or substantially alter the content of marker enzymes in mixed fiber type skeletal muscle. Analyses of pools of fast- vs slow-twitch fibers, however, indicate that RTE-induced changes may be fiber type specific. Future studies should better delineate the metabolic responses to RTE and determine whether these are related to the enhanced performance associated with such training.

The aerobic capacity of locomotory muscles in the tufted duck, Aythya fuligula.

The locomotory muscles of the tufted duck, Aythya fuligula (L.), were analysed for mass, aerobic and anaerobic enzyme activities, fibre-type proportions, capillarity, mitochondrial and myoglobin content. The estimated aerobic capacity of the muscles correlated well with the muscles' maximal oxygen uptake both when measured during swimming and when predicted for steady-state flight. The results suggest that exercise performance in birds cannot be predicted purely on the basis of muscle mass (see Butler & Woakes, 1985); the specific enzyme complement of each muscle must also be taken into account. The delivery of oxygen to mitochondria is facilitated by the dense capillarity and high myoglobin content of the muscles.

Isolation and characterization of a selenium metabolism mutant of Salmonella typhimurium

Selenium is a constituent in Escherichia coli of the anaerobic enzyme formate dehydrogenase in the form of selenocysteine. Selenium is also present in the tRNA of E. coli in the modified base 5-methylaminomethyl-2-selenouracil (mnm5Se2U). The pathways of bacterial selenium metabolism are largely uncharacterized, and it is unclear whether nonspecific reactions in the sulfur metabolic pathways may be involved. We demonstrated that sulfur metabolic pathway mutants retain a wild-type pattern of selenium incorporation, indicating that selenite (SeO32-) is metabolized entirely via selenium-specific pathways. To investigate the function of mnm5Se2U, we isolated a mutant which is unable to incorporate selenium into tRNA. This strain was obtained by isolating mutants lacking formate dehydrogenase activity and then screening for the inability to metabolize selenium. This phenotype is the result of a recessive mutation which appears to map in the general region of 21 min on the Salmonella typhimurium chromosome. A mutation in this gene, selA, thus has a pleiotropic effect of eliminating selenium incorporation into both protein and tRNA. The selA mutant appears to be blocked in a step of selenium metabolism after reduction, such as in the actual selenium insertion process. We showed that the absence of selenium incorporation into suppressor tRNA reduces the efficiency of suppression of nonsense codons in certain contexts and when wobble base pairing is required. Thus, one function of mnm5Se2U in tRNA may be in codon-anticodon interactions.

Differential roles of the Escherichia coli fumarases and fnr-dependent expression of fumarase B and aspartase

The physiological functions of the Escherichia coli fumarases A, B, and C (FUMA, B and C) were investigated using strains containing multicopy plasmids expressing each of the fum genes, and with single-copy fum-lacZ fusions. The results showed that FUMA is the critic acid cycle enzyme because it was strongly expressed under aerobic conditions but repressed by glucose and anaerobiosis. FUMB and FUMC were less susceptible to repression by glucose and anaerobiosis, and FUMB was identified as an anaerobic enzyme because its anaerobic expression was controlled by the anaerobic transcriptional activator, FNR. The expression of an aspA-lacZ fusion was also shown to be fnr-dependent.

Differential roles of theEscherichia colifumarases andfnr-dependent expression of fumarase B and aspartase

The physiological functions of the Escherichia coli fumarases A, B, and C (FUMA, B and C) were investigated using strains containing multicopy plasmids expressing each of the fum genes, and with single-copy fum-lacZ fusions. The results showed that FUMA is the critic acid cycle enzyme because it was strongly expressed under aerobic conditions but repressed by glucose and anaerobiosis. FUMB and FUMC were less susceptible to repression by glucose and anaerobiosis, and FUMB was identified as an anaerobic enzyme because its anaerobic expression was controlled by the anaerobic transcriptional activator, FNR. The expression of an aspA-lacZ fusion was also shown to be fnr-dependent.

Anaerobic metabolism enzymes as markers of flooding stress in maize seeds

Maize (Zea mays L.) seeds differ in their relative tolerance to the anaerobic environment caused by flooding. Seed tolerance to flooding stress depends on cellular and metabolic processes since gross anatomical responses have not developed at the pre-emergence stage. The study reported here characterizes the activities of four anaerobic respiratory enzymes: pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), and malic enzyme (ME) in the flood-tolerant A632 and floodsusceptible Mo 17 inbred maize seeds during flooding at 10 and 25°C. Each inbred consisted of two seed lots possessing 95% and 75% germination levels. Flooding increased the activities of all four enzymes. However, no consistent correlation between anaerobic enzyme activity and flood tolerance was observed across genotype, seed quality and flooding temperature. The results indicate that it may not be feasible to use whole-seed anaerobic enzyme activities to predict maize seed performance under flooding stress.

Aerobic and anaerobic enzymes in the healthy human heart

Aerobic and anaerobic enzymes in the healthy human heart

Shift to fetal and anaerobic enzyme activity of myocytes in renal hypertension

Shift to fetal and anaerobic enzyme activity of myocytes in renal hypertension

Temporary low oxygen conditions for the formation of nitrate reductase and nitrous oxide reductase by denitrifying Pseudomonas sp. G59.

Formation of nitrate reductase (NaR) and nitrous oxide reductase (N2OR) by a Pseudomonas sp. G59 did not occur in aerobic or anaerobic conditions, but was observed in a microaerobic incubation in which an anaerobically grown culture was agitated in a sealed vessel initially containing 20 kPa oxygen in the headspace. During the microaerobic incubation, the oxygen concentration in the headspace decreased and dissolved oxygen reached 0.1-0.2 kPa. NaR activity was detected immediately and N2OR activity after 3 h of incubation irrespective of the presence or absence of NO3- or N2O. In the presence of NO3-, NO2- was accumulated as a major product, but N2O was observed in low concentrations only after N2OR appeared. After microaerobic incubation for 3 h, N2OR formation continued even anaerobically in an atmosphere of N2O. In contrast, Escherichia coli formed NaR not only microaerobically but also anaerobically. However, NaR formation by E. coli was inhibited by sodium fluoride under anaerobic, but not under microaerobic conditions. The Pseudomonas culture did not possess fermentative activity. It is suggested that the dependence on microaerobiosis for the formation of these reductases by the Pseudomonas culture was due to an inability to produce energy anaerobically until these anaerobic respiratory enzymes were formed.

The reversible inactivation of the Clostridium thermoaceticum formate dehydrogenase by butanedione

2,3-Butanedione reacts with the formate dehydrogenase from Clostridium thermoaceticum to inactivate selectively the reduction of the physiological electron acceptor, NADP, without altering the rates of methyl viologen reduction or formate oxidation. Chemical modification of this extremely oxygen-sensitive formate dehydrogenase is reversible and obeys pseudo-first-order kinetics with respect to inactivator concentration. The presence of sodium azide and NADP protects the enzyme from inactivation. In contrast, enzymatic incubation with sodium formate or sodium bicarbonate does not retard the rate of enzymatic activity loss. From these results we conclude that butanedione reacts with a critical arginine residue at a site which is separate from formate oxidation and which is involved in the binding of the pyridinium nucleotide cofactor. The procedures reported for chemical modification of this anaerobic enzyme should also be widely applicable in identifying amino acids participating in the catalytic mechanisms of other oxygen-sensitive proteins.

Experimental-morphological study of the effect of liposomes in CCl4 poisoning

The experiments on white mice have shown CCl4 to induce 24 hours after its injection focal changes in the liver, such as hepatocyte necrotization accompanied by lell organoid destruction, drastic reduction in glycogen content and increased fat content, as well as considerable decline in the activity of aerobic and anaerobic metabolic enzymes. Liposome administration attenuated the lytic effect of poison, promoting the integrity of subcellular hepatocyte structures. Distrophic changes in the latter led to increased glycogen content lower lipid level and intensified bioenergic processes.

Inactivation of glycerol dehydrogenase of Klebsiella pneumoniae and the role of divalent cations.

Anaerobically induced NAD-linked glycerol dehydrogenase of Klebsiella pneumoniae for fermentative glycerol utilization was reported previously to be inactivated in the cell during oxidative metabolism. In vitro inactivation was observed in this study by incubating the purified enzyme in the presence of O2, Fe2+, and ascorbate or dihydroxyfumarate. It appears that O2 and the reducing agent formed H2O2 and that H2O2 reacted with Fe2+ to generate an activated species of oxygen which attacked the enzyme. The in vitro-oxidized enzyme, like the in vivo-inactivated enzyme, showed an increased Km for NAD (but not glycerol) and could no longer be activated by Mn2+ which increased the Vmax of the native enzyme but decreased its apparent affinity for NAD. Ethanol dehydrogenase and 1,3-propanediol oxidoreductase, two enzymes with anaerobic function, also lost activity when the cells were incubated aerobically with glucose. However, glucose 6-phosphate dehydrogenase (NADP-linked), isocitrate dehydrogenase, and malate dehydrogenase, expected to function both aerobically and anaerobically, were not inactivated. Thus, oxidative modification of proteins in vivo might provide a mechanism for regulating the activities of some anaerobic enzymes.

Calf muscle adaptation to peripheral vascular disease.

130 muscle biopsies were taken from the gastrocnemius of 82 patients with different degrees of peripheral vascular disease (PVD) and 19 normal controls for histochemical analysis, and 30 patients and 7 controls for biochemical analysis of "aerobic" and "anaerobic" enzymes. The results showed that the gastrocnemius of patients with PVD did not adapt by increasing its "aerobic" potential as previously suggested. Histochemical studies showed that the cross-sectional area of both Type 1 and Type 2 muscle fibres became smaller than those from age-matched controls in the presence of PVD, and in some cases biopsies from both legs confirmed that this trend was greater in those limbs with increasing evidence of PVD. There is also some evidence to suggest a decrease in absolute capillary numbers per muscle fibre. The biochemical assays confirmed decreased levels of aerobic enzymes with increasing evidence of PVD as judged by ankle systolic pressure, and those patients with intermittent claudication showed some evidence of increased anaerobic enzyme levels in comparison to both the normal controls and those with rest pain. Patients with PVD do not adapt to ischaemia/anoxia by increasing their aerobic capability, but show signs of muscle atrophy probably due to reduced mobility. Training regimes may benefit patients with intermittent claudication by reversing these changes.

An enzyme histochemical study of human sinus node, coronary sinus, and mitral valve muscle.

An enzyme histochemical study of the sinoatrial node, the coronary sinus, and the atrial muscle extending into the anterior mitral valve was performed on human hearts. Investigation of the activity and localization of the structurally bound enzymes was performed by conventional histochemical techniques. Determination of the activity of nonstructurally or weakly structurally bound enzymes was performed by histochemical techniques in which leakage of enzymes during the incubation period was reduced by the application of semipermeable membranes. The sinoatrial node is characterized by a high degree of anaerobic enzyme capacity and a relatively low degree of aerobic enzyme capacity. The discriminatory nature of these reactions allows examination of the structure of the sinoatrial node and its approaches. The presence of transitional cells was confirmed; isolated clusters of nodal cells were found in the atrial myocardium around the sinoatrial node, but no evidence of specialized tissue forming the beginning of an internodal pathway was found by this technique. The specific histochemical reactions that characterize the sinoatrial node also occur in the atrial muscle, extending into the anterior mitral valve, the anterior wall of the coronary sinus, and the atrial tissue near the orifice of the coronary sinus. These observations seem to corroborate the hypothesis that arrhythmic ectopic foci can arise in these regions.

Phosphofructokinase from foot muscle of the whelk, Busycotypus canaliculatum: Evidence for covalent modification of the enzyme during anaerobiosis

Phosphofructokinase (PFK) was purified from foot muscle of aerobic and anaerobic (24 h of anoxia) whelks, Busycotypus canaliculatum. Fructose-6-P kinetics were sigmoidal at pH 7.0 with affinity constants, S 0.5, of 2.18 ± 0.10 ( n H = 2.5 ± 0.1) and 2.48 ± 0.13 m m ( n H = 2.7 ± 0.1) for the enzyme from aerobic versus anaerobic muscle. Affinity for ATP, like that for fructose-6-P, did not differ for the two enzymes (0.031 ± 0.003 for the aerobic vs 0.041 ± 0.007 m m for the anaerobic enzyme), but S 0.5 for Mg 2+ was significantly different for the two enzymes (0.060 ± 0.006 vs 0.130 ± 0.020 m m). Whelk muscle PFK was activated by NH 4 +, P i, AMP, ADP, and fructose-2,6-P 2. NH 4 + and fructose-2,6-P 2 were less effective activators of PFK from anoxic muscle, with apparent K a 's 1.6- and 3.5-fold higher for the anaerobic vs aerobic enzyme. Activators decreased S 0.5 for fructose-6-P and reduced n H. With the exception of fructose-2,6-P 2, the effects of activators on S 0.5 were the same for the enzyme from aerobic and anaerobic muscle; fructose-2,6-P 2 at 2.5 μ m reduced S 0.5 by only 3.3-fold for the anaerobic enzyme compared to 5.5-fold for the aerobic enzyme. ATP was a strong substrate inhibitor of PFK; the enzyme from anaerobic muscle showed greater ATP inhibition, with I 50's 1.5- to 2.0-fold lower than those for the aerobic enzyme. The kinetic differences between PFK from anaerobic versus aerobic foot muscle (stronger ATP inhibition and decreased sensitivity to activators for the anaerobic enzyme) were consistent with kinetic differences reported for the phosphorylated versus dephosphorylated forms, respectively, of PFK in other systems. Treatment of PFK from anaerobic muscle with alkaline phosphatase resulted in a decrease in the K a for fructose-2,6-P 2 to a level similar to that of the aerobic enzyme. The physiological stress of anoxia may, therefore, induce a covalent modification of PFK.