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Molecular Evolution of Alternative Oxidase Proteins: A Phylogenetic and Structure Modeling Approach

Authors:
Rosa Pennisi at Candiolo Cancer Institute IRCCs
Rosa Pennisi
  • Candiolo Cancer Institute IRCCs
Daniele Salvi at Università degli Studi dell'Aquila
Daniele Salvi
Valentina Brandi at Università Degli Studi Roma Tre
Valentina Brandi
Riccardo Angelini at Università Degli Studi Roma Tre
Riccardo Angelini
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Abstract and Figures

Alternative oxidases (AOXs) are mitochondrial cyanide-resistant membrane-bound metallo-proteins catalyzing the oxidation of ubiquinol and the reduction of oxygen to water bypassing two sites of proton pumping, thus dissipating a major part of redox energy into heat. Here, the structure of Arabidopsis thaliana AOX 1A has been modeled using the crystal structure of Trypanosoma brucei AOX as a template. Analysis of this model and multiple sequence alignment of members of the AOX family from all kingdoms of Life indicate that AOXs display a high degree of conservation of the catalytic core, which is formed by a four-α-helix bundle, hosting the di-iron catalytic site, and is flanked by two additional α-helices anchoring the protein to the membrane. Plant AOXs display a peculiar covalent dimerization mode due to the conservation in the N-terminal region of a Cys residue forming the inter-monomer disulfide bond. The multiple sequence alignment has also been used to infer a phylogenetic tree of AOXs whose analysis shows a polyphyletic origin for the AOXs found in Fungi and a monophyletic origin of the AOXs of Eubacteria, Mycetozoa, Euglenozoa, Metazoa, and Land Plants. This suggests that AOXs evolved from a common ancestral protein in each of these kingdoms. Within the Plant AOX clade, the AOXs of monocotyledon plants form two distinct clades which have unresolved relationships relative to the monophyletic clade of the AOXs of dicotyledonous plants. This reflects the sequence divergence of the N-terminal region, probably due to a low selective pressure for sequence conservation linked to the covalent homo-dimerization mode.
Schematic representation of a the three-dimensional structure of the TAO dimer (PDB code 3VV9; [36]) and b of the molecular model of the A. thaliana AOX 1A dimer. Iron atoms are represented by orange spheres (Color figure online)
Schematic representation of a the three-dimensional structure of the TAO dimer (PDB code 3VV9; [36]) and b of the molecular model of the A. thaliana AOX 1A dimer. Iron atoms are represented by orange spheres (Color figure online)
… 
Structural details of the AOXs catalytic center. a TAO di-iron center. bA. thaliana AOX 1A di-iron center. Iron atoms are represented by orange spheres (Color figure online)
Structural details of the AOXs catalytic center. a TAO di-iron center. bA. thaliana AOX 1A di-iron center. Iron atoms are represented by orange spheres (Color figure online)
… 
Conservation of residues important for the structure/function of AOXs mapped onto the AtAOX structural model. a Active site cavity. b Second hydrophobic cavity. Strictly conserved residues are colored in blue, residues not strictly conserved in red. Residues forming the catalytic site, shown as a reference, are colored by atom type (Color figure online)
Conservation of residues important for the structure/function of AOXs mapped onto the AtAOX structural model. a Active site cavity. b Second hydrophobic cavity. Strictly conserved residues are colored in blue, residues not strictly conserved in red. Residues forming the catalytic site, shown as a reference, are colored by atom type (Color figure online)
… 
Maximum likelihood (ML) phylogenetic tree based on the full-length sequence of AOX proteins from member of Eubacteria and Eukaryote kingdoms. Multiple sequence alignment was calculated using Clustal W. The ML tree search implemented the LG + G + I substitution model selected by ProtTest under the AIC criterion. Bootstrap values over 100 replicates are reported in correspondence to the nodes
Maximum likelihood (ML) phylogenetic tree based on the full-length sequence of AOX proteins from member of Eubacteria and Eukaryote kingdoms. Multiple sequence alignment was calculated using Clustal W. The ML tree search implemented the LG + G + I substitution model selected by ProtTest under the AIC criterion. Bootstrap values over 100 replicates are reported in correspondence to the nodes
… 
Maximum likelihood (ML) phylogenetic tree based on the catalytic core sequence of AOX proteins from member of Eubacteria and Eukaryotes kingdoms. Multiple sequence alignment was calculated using Clustal W. The ML tree search implemented the LG + G substitution model selected by ProtTest under the AIC criterion. Bootstrap values over 100 replicates are reported in correspondence to the nodes
+1
Maximum likelihood (ML) phylogenetic tree based on the catalytic core sequence of AOX proteins from member of Eubacteria and Eukaryotes kingdoms. Multiple sequence alignment was calculated using Clustal W. The ML tree search implemented the LG + G substitution model selected by ProtTest under the AIC criterion. Bootstrap values over 100 replicates are reported in correspondence to the nodes
… 
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ORIGINAL ARTICLE
Molecular Evolution of Alternative Oxidase Proteins:
A Phylogenetic and Structure Modeling Approach
Rosa Pennisi
1
•Daniele Salvi
2
•Valentina Brandi
1
•Riccardo Angelini
1
•
Paolo Ascenzi
1
•Fabio Polticelli
1,3
Received: 8 January 2016 / Accepted: 6 April 2016 / Published online: 18 April 2016
ÓSpringer Science+Business Media New York 2016
Abstract Alternative oxidases (AOXs) are mitochondrial
cyanide-resistant membrane-bound metallo-proteins cat-
alyzing the oxidation of ubiquinol and the reduction of
oxygen to water bypassing two sites of proton pumping, thus
dissipating a major part of redox energy into heat. Here, the
structure of Arabidopsis thaliana AOX 1A has been modeled
using the crystal structure of Trypanosoma brucei AOX as a
template. Analysis of this model and multiple sequence
alignment of members of the AOX family from all kingdoms
of Life indicate that AOXs display a high degree of conser-
vation of the catalytic core, which is formed by a four-a-helix
bundle, hosting the di-iron catalytic site, and is flanked by
two additional a-helices anchoring the protein to the mem-
brane. Plant AOXs display a peculiar covalent dimerization
mode due to the conservation in the N-terminal region of a
Cys residue forming the inter-monomer disulfide bond. The
multiple sequence alignment has also been used to infer a
phylogenetic tree of AOXs whose analysis shows a
polyphyletic origin for the AOXs found in Fungi and a
monophyletic origin of the AOXs of Eubacteria, Mycetozoa,
Euglenozoa, Metazoa, and Land Plants. This suggests that
AOXs evolved from a common ancestral protein in each of
these kingdoms. Within the Plant AOX clade, the AOXs of
monocotyledon plants form two distinct clades which have
unresolved relationships relative to the monophyletic clade
of the AOXs of dicotyledonous plants. This reflects the
sequence divergence of the N-terminal region, probably due
to a low selective pressure for sequence conservation linked
to the covalent homo-dimerization mode.
Keywords Alternative oxidase Phylogenetic analysis
Molecular modeling Molecular evolution
Abbreviations
D9 Desaturase
Stearoyl Acyl carrier desaturase
AOX Alternative oxidase
ML Maximum Likelihood
MMO Methane–monooxygenase
RNR R2 subunit from ribonucleotide reductase
ROS Reactive oxygen species
TAO Trypanosomal alternative oxidase
AtAOX Arabidopsis thaliana AOX 1A
Introduction
Alternative oxidases (AOXs) are mitochondrial cyanide—
insensitive membrane-bound proteins involved in redox
reactions. These metallo-proteins, recalling the structural
organization of members of the ‘‘ferritin’’ family, display a
Rosa Pennisi and Daniele Salvi contributed equally to this work.
Database: AtAOX model data are available in the PMDB database
under the accession number PM0080189.
Electronic supplementary material The online version of this
article (doi:10.1007/s00239-016-9738-8) contains supplementary
material, which is available to authorized users.
&Fabio Polticelli
fabio.polticelli@uniroma3.it
1
Department of Sciences, Roma Tre University,
Viale Guglielmo Marconi 446, 00146 Rome, Italy
2
CIBIO-InBIO, Centro de Investigac¸a
˜o em Biodiversidade e
Recursos Gene
´ticos, Universidade do Porto, Campus Agra
´rio
de Vaira
˜o, 4485–661 Vaira
˜o, Portugal
3
National Institute of Nuclear Physics, Roma Tre Section,
00146 Rome, Italy
123
J Mol Evol (2016) 82:207–218
DOI 10.1007/s00239-016-9738-8
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
... Contrary to that view, it has been proposed that O 2 reductases evolved from a more ancient family of NO reductases, such that oxygen-utilizing terminal oxidases were already present when O 2 first appeared [31]. However, that proposal is not supported by current evidence [32][33][34][35][36][37][38], nor have similar proposals been put forward for other O 2 -utilizing enzymes. There are three large and evolutionarily unrelated (independently arisen) superfamilies of oxygen reductases in respiratory chains: (a) the heme copper oxidases (HCO) [32,33] that include cytochrome c oxidases, (b) the heme-containing cytochrome bd oxidases that oxidize membrane quinols [34][35][36], and (c) the alternative oxidase superfamily (AOX) of non-heme diiron proteins that oxidize quinols [37,38]. ...
... However, that proposal is not supported by current evidence [32][33][34][35][36][37][38], nor have similar proposals been put forward for other O 2 -utilizing enzymes. There are three large and evolutionarily unrelated (independently arisen) superfamilies of oxygen reductases in respiratory chains: (a) the heme copper oxidases (HCO) [32,33] that include cytochrome c oxidases, (b) the heme-containing cytochrome bd oxidases that oxidize membrane quinols [34][35][36], and (c) the alternative oxidase superfamily (AOX) of non-heme diiron proteins that oxidize quinols [37,38]. Because members of the bd oxidase and AOX superfamilies only have one known electron acceptor substrate, O 2 can be directly inferred as the original substrate for the founding members of those enzyme families following the GOE. ...
... As O 2 first diffused into the environment, it led to oxidation of one-electron donors and enzyme inhibition, but it also introduced a novel, energy-rich oxidant into the trajectory of biochemical evolution. Once the HCO, bd, and AOX families of terminal oxidases arose, they rapidly diversified into new subfamilies [33,35,38]. Moreover, prokaryotes that possessed terminal oxidases came to flourish in heterotrophic settings, amplifying both the gene copy number and protein abundance of terminal oxidases in the environment over evolutionary time. ...
The radical impact of oxygen on prokaryotic evolution-enzyme inhibition first, uninhibited essential biosyntheses second, aerobic respiration third
Article
Molecular oxygen is a stable diradical. All O 2 ‐dependent enzymes employ a radical mechanism. Generated by cyanobacteria, O 2 started accumulating on Earth 2.4 billion years ago. Its evolutionary impact is traditionally sought in respiration and energy yield. We mapped 365 O 2 ‐dependent enzymatic reactions of prokaryotes to phylogenies for the corresponding 792 protein families. The main physiological adaptations imparted by O 2 ‐dependent enzymes were not energy conservation, but novel organic substrate oxidations and O 2 ‐dependent, hence O 2 ‐tolerant, alternative pathways for O 2 ‐inhibited reactions. Oxygen‐dependent enzymes evolved in ancestrally anaerobic pathways for essential cofactor biosynthesis including NAD ⁺ , pyridoxal, thiamine, ubiquinone, cobalamin, heme, and chlorophyll. These innovations allowed prokaryotes to synthesize essential cofactors in O 2 ‐containing environments, a prerequisite for the later emergence of aerobic respiratory chains.
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... The homology model of the AtAOX1A monomer (PMDB Accession number: PM0080189) was chosen as the target protein for the docking study (Pennisi et al., 2016). The 3-dimensional models of the ligands ubiquinol-1 (Q 1 H 2 ), ubiquinone-1 (UQ 1 ), duroquinol (DQH 2 ), duroquinone (DQ), SHAM, n-PG, and pyruvate were generated using PubChem (Kim et al., 2021) and CHARMM-GUI (Jo et al., 2008) programs. ...
... In the case of plants, the crystal structure of AOX is not yet available, so far. Therefore, in the docking studies, we used the homology model generated for AtAOX1A (PMDB Accession number: PM0080189), using the crystal structure of TAO (PDB ID: 3VV9) as a template (Pennisi et al., 2016). The studies of Pennisi et al. (2016) also predicted the protein structure for N-terminal 31 residues (residues 63-93) of AtAOX1A by ab initio/threading program. ...
... Therefore, in the docking studies, we used the homology model generated for AtAOX1A (PMDB Accession number: PM0080189), using the crystal structure of TAO (PDB ID: 3VV9) as a template (Pennisi et al., 2016). The studies of Pennisi et al. (2016) also predicted the protein structure for N-terminal 31 residues (residues 63-93) of AtAOX1A by ab initio/threading program. Thus, irrespective of the origin, AOX possessed a common structural trend of forming a four-α-helix bundle with a diiron catalytic center. ...
Structural and Biophysical Characterization of Purified Recombinant Arabidopsis thaliana's Alternative Oxidase 1A (rAtAOX1A): Interaction With Inhibitor(s) and Activator
Article
Full-text available
  • Jun 2022
In higher plants, alternative oxidase (AOX) participates in a cyanide resistant and non-proton motive electron transport pathway of mitochondria, diverging from the ubiquinone pool. The physiological significance of AOX in biotic/abiotic stress tolerance is well-documented. However, its structural and biophysical properties are poorly understood as its crystal structure is not yet revealed in plants. Also, most of the AOX purification processes resulted in a low yield/inactive/unstable form of native AOX protein. The present study aims to characterize the purified rAtAOX1A protein and its interaction with inhibitors, such as salicylhydroxamic acid (SHAM) and n-propyl gallate (n-PG), as well as pyruvate (activator), using biophysical/in silico studies. The rAtAOX1A expressed in E. coli BL21(DE3) cells was functionally characterized by monitoring the respiratory and growth sensitivity of E. coli/pAtAOX1A and E. coli/pET28a to classical mitochondrial electron transport chain (mETC) inhibitors. The rAtAOX1A, which is purified through affinity chromatography and confirmed by western blotting and MALDI-TOF-TOF studies, showed an oxygen uptake activity of 3.86 μmol min⁻¹ mg⁻¹ protein, which is acceptable in non-thermogenic plants. Circular dichroism (CD) studies of purified rAtAOX1A revealed that >50% of the protein content was α-helical and retained its helical absorbance signal (ellipticity) at a wide range of temperature and pH conditions. Further, interaction with SHAM, n-PG, or pyruvate caused significant changes in its secondary structural elements while retaining its ellipticity. Surface plasmon resonance (SPR) studies revealed that both SHAM and n-PG bind reversibly to rAtAOX1A, while docking studies revealed that they bind to the same hydrophobic groove (Met191, Val192, Met195, Leu196, Phe251, and Phe255), to which Duroquinone (DQ) bind in the AtAOX1A. In contrast, pyruvate binds to a pocket consisting of Cys II (Arg174, Tyr175, Gly176, Cys177, Val232, Ala233, Asn294, and Leu313). Further, the mutational docking studies suggest that (i) the Met195 and Phe255 of AtAOX1A are the potential candidates to bind the inhibitor. Hence, this binding pocket could be a ‘potential gateway' for the oxidation-reduction process in AtAOX1A, and (ii) Arg174, Gly176, and Cys177 play an important role in binding to the organic acids like pyruvate.
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... The elucidation of the crystal structures of the trypanosomal and plant (A. thaliana) enzymes have provided structural insights into the nature of the active site, catalytic mechanism and supramolecular organization of the enzyme [83][84][85]. Both in plants and protists, the active site, buried in a hydrophobic cavity, is composed of the diiron centre with four glutamate and two histidine residues. ...
... Both in plants and protists, the active site, buried in a hydrophobic cavity, is composed of the diiron centre with four glutamate and two histidine residues. The Glu-X-X-His motifs stabilize the di-iron center and participate in catalysis [78,[82][83][84]86,87], while other conserved residues are essential for ubiquinol/ubiquinone and oxygen binding [83][84][85][86]88]. Because of the recent interest in AOX as a potential drug target, structural studies of the enzyme from different taxa will undoubtedly intensify. ...
... Both in plants and protists, the active site, buried in a hydrophobic cavity, is composed of the diiron centre with four glutamate and two histidine residues. The Glu-X-X-His motifs stabilize the di-iron center and participate in catalysis [78,[82][83][84]86,87], while other conserved residues are essential for ubiquinol/ubiquinone and oxygen binding [83][84][85][86]88]. Because of the recent interest in AOX as a potential drug target, structural studies of the enzyme from different taxa will undoubtedly intensify. ...
Cyanide resistant respiration and the alternative oxidase pathway: A journey from plants to mammals
Article
Full-text available
  • Apr 2022
  • BBA-BIOENERGETICS
In a large number of organisms covering all phyla, the mitochondrial respiratory chain harbors, in addition to the conventional elements, auxiliary proteins that confer adaptive metabolic plasticity. The alternative oxidase (AOX) represents one of the most studied auxiliary proteins, initially identified in plants. In contrast to the standard respiratory chain, the AOX mediates a thermogenic cyanide-resistant respiration; a phenomenon that has been of great interest for over 2 centuries in that energy is not conserved when electrons flow through it. Here we summarize centuries of studies starting from the early observations of thermogenicity in plants and the identification of cyanide resistant respiration, to the fascinating discovery of the AOX and its current applications in animals under normal and pathological conditions.
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... The tertiary structure of TbAOX was used as the template to build the homology model, in which OhAOX, BgAOX, BaAOX, and BsAOX showed sequence identity of 49 Similar to TbAOX, each snail AOX was identified as a homodimer, with a diiron core in each monomer surrounded by a four-helix bundle and ligated by four glutamic acid and two histidine residues (Additional file 6: Fig. S3), which were considered the active sites of the AOX. ...
... The AOX of land plants (monocots and eudicots) and the Euglenozoa formed a separate monophyletic clade with very high bootstrap values, which indicated that the AOXs of these species may have evolved from a homologous AOX carried by their common ancestors in each kingdom. This is consistent with the results of a phylogenetic analysis of AOXs from land plants and the phylum Euglenozoa carried out by Pennisi et al. [49]. Other kingdoms showed certain cross-relationships: metazoans in this study clustered into a large clade, except L. anatina in the phylum Brachiopoda. ...
The identification of alternative oxidase in intermediate host snails of Schistosoma and its potential role in protecting Oncomelania hupensis against niclosamide-induced stress
Article
Full-text available
  • Mar 2022
Background Snail intermediate hosts are mandatory for the transmission of schistosomiasis, which has to date infected more than 200 million people worldwide. Our previous studies showed that niclosamide treatment caused the inhibition of aerobic respiration and oxidative phosphorylation, and the disruption of energy supply, in one of the intermediate hosts of schistosomiasis, Oncomelania hupensis , which eventually led to the death of the snails. Meanwhile, the terminal oxidase in the mitochondrial respiratory chain, alternative oxidase (AOX), was significantly up-regulated, which was thought to counterbalance the oxidative stress and maintain metabolic homeostasis in the snails. The aims of the present study are to identify the AOXs in several species of snails and investigate the potential activation of O. hupensis AOX ( OhAOX ) under niclosamide-induced stress, leading to enhanced survival of the snail when exposed to this molluscicide. Methods The complete complementary DNA was amplified from the AOXs of O. hupensis and three species of Biomphalaria ; the sequence characteristics were analysed and the phylogenetics investigated. The dynamic expression and localisation of the AOX gene and protein in O. hupensis under niclosamide-induced stress were examined. In addition, the expression pattern of genes in the mitochondrial respiratory complex was determined and the production of reactive oxygen species (ROS) calculated. Finally, the molluscicidal effect of niclosamide was compared between snails with and without inhibition of AOX activity. Results AOXs containing the invertebrate AOX-specific motif NP-[YF]-XPG-[KQE] were identified from four species of snail, which phylogenetically clustered together into Gastropoda AOXs and further into Mollusca AOXs. After niclosamide treatment, the levels of OhAOX messenger RNA (mRNA) and OhAOX protein in the whole snail were 14.8 and 2.6 times those in untreated snails, respectively, but varied widely among tissues. Meanwhile, the level of cytochrome C reductase mRNA showed a significant decrease in the whole snail, and ROS production showed a significant decrease in the liver plus gonad (liver-gonad) of the snails. At 24 h post-treatment, the mortality of snails treated with 0.06–0.1 mg/L niclosamide and AOX inhibitor was 56.31–76.12% higher than that of snails treated with 0.1 mg/L niclosamide alone. Conclusions AOX was found in all the snail intermediate hosts of Schistosoma examined here. AOX was significantly activated in O. hupensis under niclosamide-induced stress, which led to a reduction in oxidative stress in the snail. The inhibition of AOX activity in snails can dramatically enhance the molluscicidal effect of niclosamide. A potential target for the development of an environmentally safe snail control method, which acts by inhibiting the activity of AOX, was identified in this study. Graphical abstract
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... In green parts of land plants, photosynthesis produces a considerably higher amount of ROS compared to the mitochondrial respiration 6,9 . However, this is partly because, in plant mitochondria, the alternative oxidase, which is absent in vertebrates 48 , partially uncouples electron transfer from proton pumping and ATP generation, thereby reducing ROS generation 6 . In addition, land plants have evolved to produce more ATP in their chloroplasts than in their mitochondria in the light 49 . ...
Taming the perils of photosynthesis by eukaryotes: constraints on endosymbiotic evolution in aquatic ecosystems
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Full-text available
  • Nov 2023
An ancestral eukaryote acquired photosynthesis by genetically integrating a cyanobacterial endosymbiont as the chloroplast. The chloroplast was then further integrated into many other eukaryotic lineages through secondary endosymbiotic events of unicellular eukaryotic algae. While photosynthesis enables autotrophy, it also generates reactive oxygen species that can cause oxidative stress. To mitigate the stress, photosynthetic eukaryotes employ various mechanisms, including regulating chloroplast light absorption and repairing or removing damaged chloroplasts by sensing light and photosynthetic status. Recent studies have shown that, besides algae and plants with innate chloroplasts, several lineages of numerous unicellular eukaryotes engage in acquired phototrophy by hosting algal endosymbionts or by transiently utilizing chloroplasts sequestrated from algal prey in aquatic ecosystems. In addition, it has become evident that unicellular organisms engaged in acquired phototrophy, as well as those that feed on algae, have also developed mechanisms to cope with photosynthetic oxidative stress. These mechanisms are limited but similar to those employed by algae and plants. Thus, there appear to be constraints on the evolution of those mechanisms, which likely began by incorporating photosynthetic cells before the establishment of chloroplasts by extending preexisting mechanisms to cope with oxidative stress originating from mitochondrial respiration and acquiring new mechanisms.
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... Phylogenetic studies indicate that T. brucei TAO and fungi AOX sequences are phylogenetically related as they cluster in taxonomic distribution analyses (Luévano-Martínez et al 2020; Pennisi et al 2016). Importantly, adenylates were shown to regulate the activity of TAO and fungi AOX (Woyda-Ploszczyca et al 2009; Sakajo et al 1997;Luévano-Martínez et al 2020). ...
The extraordinary energy metabolism of the bloodstream Trypanosoma brucei forms: a critical review and hypothesis
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  • Dec 2022
The parasite Trypanosoma brucei is the causative agent of sleeping sickness and involves an insect vector and a mammalian host through its complex life cycle. T. brucei mammalian bloodstream forms (BSF) exhibits unique metabolic features including: (1) reduced expression and activity of mitochondrial enzymes; (2) respiration mediated by the glycerol phosphate shuttle (GPSh) and the Trypanosome alternative oxidase (TAO) that is intrinsically uncoupled from generation of mitochondrial protonmotive force; (3) maintenance of mitochondrial membrane potential by ATP hydrolysis through the reversal of F1FO-ATP synthase activity; (4) strong reliance on glycolysis to meet their energy demands; (5) high susceptibility to oxidants. Here, we critically review the main metabolic features of BSF and provide a hypothesis to explain the unusual metabolic network and its biological significance for this parasite form. We postulate that intrinsically uncoupled respiration provided by the GPSh-TAOsystem acts as a preventive antioxidant defense by limiting mitochondrial superoxide production and complementing the NADPH-dependent scavenging antioxidant defenses to maintain redox balance. Given the uncoupled nature of the GPSh-TAOsystem, BSF avoidscell death processes by maintaining mitochondrial protonmotiveforce through the reversal of ATP synthase activity using the ATP generated by glycolysis. This unique “metabolic design” in BSF has no biological parallel outside of trypanosomatids and highlights the enormous diversity of the parasite mitochondrial processes to adapt to distinct environments.
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... Regarding the eukaryotic distribution, in addition to previously described groups Alveolata, Euglenozoa, Metazoa, Choanoflagellates, Stramenopiles, Fungi, Rhodophya, Heterolobosea, and Viridiplantae (Pennisi et al., 2016), we identified AOX homologs in some eukaryotes that were not reported: Apusuzoa, Amoebozoa, Filasterea, Haptophyceae, and Rhizaria. No homologs were found in Kipferlia, Metamonada, and Hexamitida ( Figure S1 and Table S1). ...
Localization and Functional Characterization of the Alternative Oxidase in Naegleria
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Full-text available
  • Mar 2022
The alternative oxidase (AOX) is a protein involved in supporting enzymatic reactions of the Krebs cycle in instances when the canonical (cytochrome‐mediated) respiratory chain has been inhibited, while allowing for the maintenance of cell growth and necessary metabolic processes for survival. Among eukaryotes, alternative oxidases have dispersed distribution and are found in plants, fungi, and protists, including Naegleria ssp. Naegleria species are free‐living unicellular amoeboflagellates, and include the pathogenic species of N. fowleri, the so‐called brain eating amoeba. Using a multidisciplinary approach, we aimed to understand the evolution, localization and function of AOX and the role that plays in Naegleria’s biology. Our analyses suggest that AOX was present in last common ancestor of the genus and structure prediction showed that all functional residues are also present in Naegleria species. Using cellular and biochemical techniques, we also functionally characterize N. gruberi’s AOX in its mitochondria and we demonstrate that its inactivation affects its proliferation. Consequently, we discuss the benefits of the presence of this protein in Naegleria species, along with its potential pathogenicity role in N. fowleri. We predict that our findings will spearhead new explorations to understand the cell biology, metabolism and evolution of Naegleria and other free‐living relatives.
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Mitochondrial alternative oxidase across the tree of life: Presence, absence, and putative cases of lateral gene transfer
Article
  • Aug 2023
  • BBA-BIOENERGETICS
The alternative oxidase (AOX) is a terminal oxidase in the electron transport system that plays a role in mitochondrial bioenergetics. The past 20 years of research shows AOX has a wide yet patchy distribution across the tree of life. AOX has been suggested to have a role in stress tolerance, growth, and development in plants, but less is known about its function in other groups, including animals. In this study, we analyzed the taxonomic distribution of AOX across >2800 species representatives from prokaryotes and eukaryotes and developed a standardized workflow for finding and verifying the authenticity of AOX sequences. We found that AOX is limited to proteobacteria among prokaryotes, but is widely distributed in eukaryotes, with the highest prevalence in plants, fungi, and protists. AOX is present in many invertebrates, but is absent in others including most arthropods, and is absent from vertebrates. We found aberrant AOX sequences associated with some animal groups. Some of these aberrant AOXs were contaminants, but we also found putative cases of lateral gene transfer of AOX from fungi and protists to nematodes, springtails, fungus gnats, and rotifers. Our findings provide a robust and detailed analysis of the distribution of AOX and a method for identifying and verifying putative AOX sequences, which will be useful as more sequence data becomes available on public repositories.
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Polymorphisms in alternative oxidase (AOX) genes from ecotypes of Arabidopsis and rice revealed an environment‐induced linkage to altitude and rainfall
Article
Full-text available
  • Dec 2022
We investigated SNPs in alternative oxidase (AOX) genes and their connection to ecotype origins (climate, altitude and rainfall) by using genomic data sets of Arabidopsis and rice populations from 1190 and 90 ecotypes, respectively. Parameters were defined to detect non‐synonymous SNPs in the AOX ORF, which revealed amino acid (AA) changes in AOX1c, AOX1d and AOX2 from Arabidopsis and AOX1c from rice in comparison to AOX references from Columbia‐0 and Japonica ecotypes, respectively. Among these AA changes, Arabidopsis AOX1c_A161E&G165R and AOX1c_R242S revealed a link to high rainfall and high altitude, respectively, while all other changes in Arabidopsis and rice AOX were connected to high altitude and rainfall. Comparative 3D modeling showed that all mutant AOX presented structural differences in relation to the respective references. Molecular docking analysis uncovered lower binding affinity values between AOX and the substrate ubiquinol for most of the identified structures compared to their reference, indicating better enzyme‐substrate binding affinities. Thus, our in silico data suggest that the majority of the AA changes found in the available ecotypes will confer better enzyme‐subtract interactions and thus indicate environment‐related, more efficient AOX activity. This article is protected by copyright. All rights reserved.
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Physiological role of AOX1a in photosynthesis and maintenance of cellular redox homeostasis under high light in Arabidopsis thaliana
Article
Full-text available
  • Aug 2014
As plants are sessile, they often face high light (HL) stress that causes damage of the photosynthetic machinery leading to decreased photosynthesis. The importance of alternative oxidase (AOX) in optimizing photosynthesis is well documented. In the present study, the role of AOX in sustaining photosynthesis under HL was studied using AOX1a knockout mutants (aox1a) of Arabidopsis thaliana. Under growth light (GL; 50 μmol photons m-2 s-1) conditions, aox1a plants did not show any changes in photosynthetic parameters, NAD(P)/H redox ratios, or respiratory O2 uptake when compared to wild-type (WT). Upon exposure to HL (700 μmol photons m-2 s-1), respiratory rates did not vary between WT and aox1a. But, photosynthetic parameters related to photosystem II (PSII) and NaHCO3 dependent O2 evolution decreased, while the P700 reduction state increased in aox1a compared to WT. Further, under HL, the redox state of cellular NAD(P)/H pools increased with concomitant rise in reactive oxygen species (ROS) and malondialdehyde (MDA) content in aox1a compared to WT. In presence of HL, the transcript levels of several genes related to antioxidant, malate-oxaloacetate (malate-OAA) shuttle, photorespiratory and respiratory enzymes was higher in aox1a compared to WT. Taken together, these results demonstrate that under HL, in spite of significant increase in transcript levels of several genes mentioned above to maintain cellular redox homeostasis and minimize ROS production, Arabidopsis plants deficient in AOX1a were unable to sustain photosynthesis as is the case in WT plants.
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Alternative Mitochondrial Electron Transport Proteins in Higher Plants
Chapter
  • Jan 2004
The so-called “alternative” electron transport protems, the rotenone-insensitive NAD(P)H dehydrogenases and the alternative oxidase, distinguish the inner membrane of plant mitochondria from its animal counterpart. These proteins provide plant tissues possessing them with the potential to modulate the efficiency with which energy is conserved by respiratory electron transport. The activities associated with these enzymes have intrigued scientists from at least 1778, when Lamarck commented on the thermogenesis displayed by some highly specialized flowers, a process in which alternative electron transport proteins are now known to have an important role. In more recent times, many studies have been undertaken to determine the nature of the alternative electron transport proteins and to understand their role in plant physiology. We discuss, within a historical perspective, our current understanding of the biochemistry and molecular biology of the enzymes, including their biochemical regulation. We also discuss the possible roles of the enzymes in the normal and stress physiology of plants, and the physiological interactions of the enzymes with each other and the classical respiratory pathway.
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Increased Respiratory Restriction during Phosphate-Limited Growth in Transgenic Tobacco Cells Lacking Alternative Oxidase
Article
  • Dec 1999
  • PLANT PHYSIOL
We found that mitochondrial alternative oxidase (AOX) protein and the capacity for CN-resistant respiration are dramatically increased in wild-type tobacco (Nicotiana tabacum) suspension-cultured cells in response to growth under P limitation, and antisense (AS8) tobacco cells unable to induce AOX under these conditions have altered growth and metabolism. Specifically, we found that the respiration of AS8 cells was restricted during P-limited growth, when the potential for severe adenylate control of respiration (at the level of C supply to the mitochondrion and/or at the level of oxidative phosphorylation) is high due to the low cellular levels of ADP and/or inorganic P. As a result of this respiratory restriction, AS8 cells had altered growth, morphology, cellular composition, and patterns of respiratory C flow to amino acid synthesis compared with wild-type cells with abundant AOX protein. Also, AS8 cells under P limitation displayed high in vivo rates of generation of active oxygen species compared with wild-type cells. This difference could be abolished by an uncoupler of mitochondrial oxidative phosphorylation. Our results suggest that induction of non-phosphorylating AOX respiration (like induction of adenylate and inorganic P-independent pathways in glycolysis) is an important plant metabolic adaptation to P limitation. By preventing severe respiratory restriction, AOX acts to prevent both redirections in C metabolism and the excessive generation of harmful active oxygen species in the mitochondrion.
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The Alternative Oxidase: in vivo Regulation and Function
Article
  • Jan 2003
  • PLANT BIOLOGY
This review focuses on the biochemical regulation and function of the alternative oxidase in vivo. About 10 years ago, two activation mechanisms were discovered in isolated mitochondria, namely activation by reducing sulfur bonds in the protein and activation by an allosteric effect of pyruvate. It was proposed that plants would have a regulatory mechanism to modify alternative oxidase activity in vivo. However, more recent studies have shown that these two activation mechanisms may not play such an important role in regulation of alternative oxidase activity in vivo after all. Pyruvate and reduction of the sulfide bonds in the protein are definitely required for alternative oxidase activity, but they do not appear to be regulating the activity in vivo. Despite the energy wasting nature of the alternative oxidase, there was no obvious physiological function for the pathway for many years. It is now more clear that the alternative oxidase can prevent the production of excess reactive oxygen species radicals by stabilizing the redox state of the mitochondrial ubiquinone pool, while allowing continued activity of the citric acid cycle. This may be important under conditions when the NADH supply is relatively high (reductant overflow), or when the cytochrome pathway is restrained. The cytochrome pathway might be inhibited by naturally occurring cyanide, nitric oxide, sulfide, high concentrations of CO 2 , low temperatures, or by limited phosphate supply.
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Application of Phylogenetic Networks in Evolutionary Studies.
Article
  • Jan 2006
  • MOL BIOL EVOL
The evolutionary history of a set of taxa is usually represented by a phylogenetic tree, and this model has greatly facilitated the discussion and testing of hypotheses. However, it is well known that more complex evolutionary scenarios are poorly described by such models. Further, ...
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Cyanide tolerance in millipedes: The biochemical basis
Article
  • Apr 1971
  • Comp Biochem Physiol B Biochem Mol Biol
1.1. The millipedes Euryurus leachii and Pleuroloma flavipes butleri produce HCN in their defensive secretion. They have a high resistance to HCN vapors, injected cyanide and azide compared to the cockroaches Blattella germanica and Blaberus discoidalis.2.2. Failure of cyanide to penetrate, ability to tolerate anaerobiosis and detoxication were ruled out as the resistance mechanism in the millipedes.3.3. Mitochondrial respiration in E. leachii was very resistant to cyanide although general mitochondrial properties resembled those of the cockroaches.4.4. Evidence favors the existence of a resistant terminal oxidase rather than an excess of cytochrome oxidase as the reason for mitochondrial insensitivity.
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