Acetylation of proteins seems a widespread process found in the three domains of life. Several studies have shown that besides histones, acetylation of lysine residues also occurs in non‐nuclear ...proteins. Hence, it has been suggested that this covalent modification is a mechanism that might regulate diverse metabolic pathways by modulating enzyme activity, stability, and/or subcellular localization or interaction with other proteins. However, protein acetylation levels seem to have low correlation with modification of enzyme activity and pathway fluxes. In addition, the results obtained with mutant enzymes that presumably mimic acetylation have frequently been over‐interpreted. Moreover, there is a generalized lack of rigorous enzyme kinetic analysis in parallel to acetylation level determinations. The purpose of this review is to analyze the current findings on the impact of acetylation on metabolic enzymes and its repercussion on metabolic pathways function/regulation.
Aldehyde dehydrogenases (ALDHs) are involved in the detoxification of aldehydes generated as byproducts of lipid peroxidation. In this work, it was determined that, among the three most studied human ...ALDH isoforms, ALDH2 showed the highest catalytic efficiency for oxidation of acrolein, 4-hydroxy-2-nonenal (4-HNE), and malondialdehyde. ALDH1A1 also exhibited significant activity with these substrates, whereas ALDH3A1 only showed activity with 4-HNE. ALDH2 was also the most sensitive isoform to irreversible inactivation by these compounds. Remarkably, ALDH3A1 was insensitive to these aldehydes even at concentrations as high as 20 mM. Formation of adducts of ALDH1A1 and ALDH2 with acrolein increased their K d values for NAD+ by 2- and 3-fold, respectively. NADH exerted a higher protection than propionaldehyde to the inactivation by acrolein, and this protection was additive. These results suggested that both binding sites, those for aldehyde and NAD+ in ALDH2, are targets for the inactivation by lipid peroxidation products. Thus, with the advantage of being relatively inactivation-insensitive, ALDH1A1 and ALDH3A1 may be actively participating in the detoxification of these aldehydes in the cells.
Mitochondrial aldehyde dehydrogenase (ALDH2) has been proposed as a key enzyme in cardioprotection during ischemia–reperfusion processes. This proposal led to the search for activators of ALDH2 with ...the aim to develop cardioprotective drugs. Alda‐1 was the first activator of ALDH2 identified and its cardioprotective effect has been extensively proven in vivo; however, the mechanism of activation is not fully understood. A crystallographic study showed that Alda‐1 binds to the entrance of the aldehyde‐binding site; therefore, Alda‐1 should in essence be an inhibitor. In the present study, kinetic experiments were performed to characterize the effect of Alda‐1 on the properties of ALDH2 (kinetic parameters, determination of the rate‐limiting step, reactivity of the catalytic cysteine) and on the kinetic mechanism (type of kinetics, sequence of substrates entering, and products release). The results showed that Alda‐1 dramatically modifies the properties of ALDH2, the Km for NAD+ decreased by 2.4‐fold, and the catalytic efficiency increased 4.4‐fold; however, the Km for the aldehyde increased 8.6‐fold, thus, diminishing the catalytic efficiency. The alterations in these parameters resulted in a complex behavior, where Alda‐1 acts as inhibitor at low concentrations of aldehyde and as an activator at high concentrations. Additionally, the binding of Alda‐1 to ALDH2 made the deacylation less limiting and diminished the pKa of the catalytic cysteine. Finally, NADH inhibition patterns indicated that Alda‐1 induced a change in the sequence of substrates entry and products release, in agreement with the proposal of both substrates entering ALDH2 by the NAD+ entrance site.
Activator of human ALDH2 Alda‐1 protects cell integrity from diverse stress conditions; however, the activation mechanism is not fully understood. Kinetic characterization showed that binding of Alda‐1 to ALDH2 activates the catalytic cysteine reducing its pKa, which induces the switching of order of substrates binding and products release, additionally, Alda‐1 increases the rate of the limiting step of the reaction.
The multisubunit cation/proton antiporter 3 family, also called Mrp, is widely distributed in all three phylogenetic domains (Eukarya, Bacteria, and Archaea). Investigations have focused on Mrp ...complexes from the domain Bacteria to the exclusion of Archaea, with a consensus emerging that all seven subunits are required for Na
/H
antiport activity. The MrpA subunit from the MrpABCDEFG Na
/H
antiporter complex of the archaeon Methanosarcina acetivorans was produced in antiporter-deficient Escherichia coli strains EP432 and KNabc and biochemically characterized to determine the role of MrpA in the complex. Both strains containing MrpA grew in the presence of up to 500 mM NaCl and pH values up to 11.0 with no added NaCl. Everted vesicles from the strains containing MrpA were able to generate a NADH-dependent pH gradient (ΔpH), which was abated by the addition of monovalent cations. The apparent K
values for Na
and Li
were similar and ranged from 31 to 63 mM, whereas activity was too low to determine the apparent K
for K
Optimum activity was obtained between pH 7.0 and 8.0. Homology molecular modeling identified two half-closed symmetry-related ion translocation channels that are linked, forming a continuous path from the cytoplasm to the periplasm, analogous to the NuoL subunit of complex I. Bioinformatics analyses revealed genes encoding homologs of MrpABCDEFG in metabolically diverse methane-producing species. Overall, the results advance the biochemical, evolutionary, and physiological understanding of Mrp complexes that extends to the domain Archaea IMPORTANCE: The work is the first reported characterization of an Mrp complex from the domain Archaea, specifically methanogens, for which Mrp is important for acetotrophic growth. The results show that the MrpA subunit is essential for antiport activity and, importantly, that not all seven subunits are required, which challenges current dogma for Mrp complexes from the domain Bacteria A mechanism is proposed in which an MrpAD subcomplex catalyzes Na
/H
antiport independent of an MrpBCEFG subcomplex, although the activity of the former is modulated by the latter. Properties of MrpA strengthen proposals that the Mrp complex is of ancient origin and that subunits were recruited to evolve the ancestral complex I. Finally, bioinformatics analyses indicate that Mrp complexes function in diverse methanogenic pathways.
Biogenesis and recycling of iron-sulfur (Fe-S) clusters play important roles in the iron homeostasis mechanisms involved in mitochondrial function. In Saccharomyces cerevisiae, the Fe-S clusters are ...assembled into apoproteins by the iron-sulfur cluster machinery (ISC). The aim of the present study was to determine the effects of ISC gene deletion and consequent iron release under oxidative stress conditions on mitochondrial functionality in S. cerevisiae. Reactive oxygen species (ROS) generation, caused by H2O2, menadione, or ethanol, was associated with a loss of iron homeostasis and exacerbated by ISC system dysfunction. ISC mutants showed increased free Fe2+ content, exacerbated by ROS-inducers, causing an increase in ROS, which was decreased by the addition of an iron chelator. Our study suggests that the increment in free Fe2+ associated with ROS generation may have originated from mitochondria, probably Fe-S cluster proteins, under both normal and oxidative stress conditions, suggesting that Fe-S cluster anabolism is affected. Raman spectroscopy analysis and immunoblotting indicated that in mitochondria from SSQ1 and ISA1 mutants, the content of Fe-S centers was decreased, as was formation of Rieske protein-dependent supercomplex III2IV2, but this was not observed in the iron-deficient ATX1 and MRS4 mutants. In addition, the activity of complexes II and IV from the electron transport chain (ETC) was impaired or totally abolished in SSQ1 and ISA1 mutants. These results confirm that the ISC system plays important roles in iron homeostasis, ROS stress, and in assembly of supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Ethanol accumulation during fermentation contributes to the toxic effects in Saccharomyces cerevisiae, impairing its viability and fermentative capabilities. The iron–sulfur (Fe–S) cluster ...biogenesis is encoded by the ISC genes. Reactive oxygen species (ROS) generation is associated with iron release from Fe–S-containing enzymes. We evaluated ethanol toxicity, ROS generation, antioxidant response and mitochondrial integrity in S. cerevisiaeISC mutants. These mutants showed an impaired tolerance to ethanol. ROS generation increased substantially when ethanol accumulated at toxic concentrations under the fermentation process. At the cellular and mitochondrial levels, ROS were increased in yeast treated with ethanol and increased to a higher level in the ssq1∆, isa1∆, iba57∆ and grx5∆ mutants – hydrogen peroxide and superoxide were the main molecules detected. Additionally, ethanol treatment decreased GSH/GSSG ratio and increased catalase activity in the ISC mutants. Examination of cytochrome c integrity indicated that mitochondrial apoptosis was triggered following ethanol treatment. The findings indicate that the mechanism of ethanol toxicity occurs via ROS generation dependent on ISC assembly system functionality. In addition, mutations in the ISC genes in S. cerevisiae contribute to the increase in ROS concentration at the mitochondrial and cellular level, leading to depletion of the antioxidant responses and finally to mitochondrial apoptosis.
The in vitro Entamoeba histolytica pyruvate:ferredoxin oxidoreductase (EhPFOR) kinetic properties and the effect of oxidative stress on glycolytic pathway enzymes and fluxes in live trophozoites were ...evaluated. EhPFOR showed a strong preference for pyruvate as substrate over other oxoacids. The enzyme was irreversibly inactivated by a long period of saturating O2 exposure (IC50 0.034 mm), whereas short‐term exposure (< 30 min) leading to > 90% inhibition allowed for partial restoration by addition of Fe2+. CoA and acetyl‐CoA prevented, whereas pyruvate exacerbated, inactivation induced by short‐term saturating O2 exposure. Superoxide dismutase was more effective than catalase in preventing the inactivation, indicating that reactive oxygen species (ROS) were involved. Hydrogen peroxide caused inactivation in an Fe2+‐reversible fashion that was not prevented by the coenzymes, suggesting different mechanisms of enzyme inactivation by ROS. Structural analysis on an EhPFOR 3D model suggested that the protection against ROS provided by coenzymes could be attributable to their proximity to the Fe–S clusters. After O2 exposure, live parasites displayed decreased enzyme activities only for PFOR (90%) and aldehyde dehydrogenase (ALDH; 68%) of the bifunctional aldehyde–alcohol dehydrogenase (EhADH2), whereas acetyl‐CoA synthetase remained unchanged, explaining the increased acetate and lowered ethanol fluxes. Remarkably, PFOR and ALDH activities were restored after return of the parasites to normoxic conditions, which correlated with higher ethanol and lower acetate fluxes. These results identified amebal PFOR and ALDH of EhADH2 activities as markers of oxidative stress, and outlined their relevance as significant controlling steps of energy metabolism in parasites subjected to oxidative stress.
Oxidative phosphorylation (OxPhos) is functional and sustains tumor proliferation in several cancer cell types. To establish whether mitochondrial β-oxidation of free fatty acids (FFAs) contributes ...to cancer OxPhos functioning, its protein contents and enzyme activities, as well as respiratory rates and electrical membrane potential (ΔΨm) driven by FFA oxidation were assessed in rat AS-30D hepatoma and liver (RLM) mitochondria. Higher protein contents (1.4–3 times) of β-oxidation (CPT1, SCAD) as well as proteins and enzyme activities (1.7–13-times) of Krebs cycle (KC: ICD, 2OGDH, PDH, ME, GA), and respiratory chain (RC: COX) were determined in hepatoma mitochondria vs. RLM. Although increased cholesterol content (9-times vs. RLM) was determined in the hepatoma mitochondrial membranes, FFAs and other NAD-linked substrates were oxidized faster (1.6–6.6 times) by hepatoma mitochondria than RLM, maintaining similar ΔΨm values. The contents of β-oxidation, KC and RC enzymes were also assessed in cells. The mitochondrial enzyme levels in human cervix cancer HeLa and AS-30D cells were higher than those observed in rat hepatocytes whereas in human breast cancer biopsies, CPT1 and SCAD contents were lower than in human breast normal tissue. The presence of CPT1 and SCAD in AS-30D mitochondria and HeLa cells correlated with an active FFA utilization in HeLa cells. Furthermore, the β-oxidation inhibitor perhexiline blocked FFA utilization, OxPhos and proliferation in HeLa and other cancer cells. In conclusion, functional mitochondria supported by FFA β-oxidation are essential for the accelerated cancer cell proliferation and hence anti-β-oxidation therapeutics appears as an alternative promising approach to deter malignant tumor growth.
The marine archaeon Methanosarcina acetivorans contains a putative NAD + -independent d-lactate dehydrogenase (D-iLDH/glycolate oxidase) encoded by the MA4631 gene, belonging to the FAD-oxidase C ...superfamily. Nucleotide sequences similar to MA4631 gene, were identified in other methanogens and Firmicutes with >90 and 35–40% identity, respectively. Therefore, the lactate metabolism in M. acetivorans is reported here. Cells subjected to intermittent pulses of oxygen (air-adapted; AA-Ma cells) consumed lactate only in combination with acetate, increasing methane production and biomass yield. In AA-Ma cells incubated with d-lactate plus 14C-l-lactate, the radioactive label was found in methane, CO2 and glycogen, indicating that lactate metabolism fed both methanogenesis and gluconeogenesis. Moreover, d-lactate oxidation was coupled to O2-consumption which was sensitive to HQNO; also, AA-Ma cells showed high transcript levels of gene dld and those encoding subunits A (MA1006) and B (MA1007) of a putative cytochrome bd quinol oxidase, compared to anaerobic control cells. An E. coli mutant deficient in dld complemented with the MA4631 gene, grew with d-lactate as carbon source and showed membrane-bound d-lactate:quinone oxidoreductase activity. The product of the MA4631 gene is a FAD-containing monomer showing activity of iLDH with preference to d-lactate. The results suggested that air adapted M. acetivorans is able to co-metabolize lactate and acetate with associated oxygen consumption by triggering the transcription and synthesis of the D-iLDH and a putative cytochrome bd: methanophenazine (quinol) oxidoreductase. Biomass generation and O2 consumption, suggest a potentially new oxygen detoxification mechanism coupled to energy conservation in this methanogen.
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•Air-adapted Methanosarcina acetivorans can grow in presence of acetate + lactate.•Lactate was oxidized in the presence of O2, producing methane, glycogen and biomass.•D-iLDH and cyt bd oxidase transcripts increased in cells showing high O2-consumption.•Respiration linked to lactate oxidation was sensitive to HQNO and partially to azide.•Lactate oxidation potentially through cytochromes, consumed the otherwise toxic O2.
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