Deciphering the Translation Initiation Factor 5A Modification Pathway in Halophilic Archaea

Translation initiation factor 5A (IF5A) is essential and highly conserved in Eukarya (eIF5A) and Archaea (aIF5A). The activity of IF5A requires hypusine, a posttranslational modification synthesized in Eukarya from the polyamine precursor spermidine. Intracellular polyamine analyses revealed that agmatine and cadaverine were the main polyamines produced in Haloferax volcanii in minimal medium, raising the question of how hypusine is synthesized in this halophilic Archaea. Metabolic reconstruction led to a tentative picture of polyamine metabolism and aIF5A modification in Hfx. volcanii that was experimentally tested. Analysis of aIF5A from Hfx. volcanii by LC-MS/MS revealed it was exclusively deoxyhypusinylated. Genetic studies confirmed the role of the predicted arginine decarboxylase gene (HVO 1958) in agmatine synthesis. The agmatinase-like gene (HVO 2299) was found to be essential, consistent with a role in aIF5A modification predicted by physical clustering evidence. Recombinant deoxyhypusine synthase (DHS) fromS. cerevisiae was shown to transfer 4-aminobutyl moiety from spermidine to aIF5A from Hfx. volcanii in vitro. However, at least under conditions tested, this transfer was not observed with the Hfx. volcanii DHS. Furthermore, the growth of Hfx. volcanii was not inhibited by the classical DHS inhibitor GC7. We propose a model of deoxyhypusine synthesis in Hfx. volcanii that differs from the canonical eukaryotic pathway, paving the way for further studies

Mutations in PROSC Disrupt Cellular Pyridoxal Phosphate Homeostasis and Cause Vitamin B6-Dependent Epilepsy

Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, functions as a cofactor in humans for more than 140 enzymes, many of which are involved in neurotransmitter synthesis and degradation. A deficiency of PLP can present, therefore, as seizures and other symptoms which are treatable with PLP and/or pyridoxine. Deficiency of PLP in the brain can be caused by inborn errors affecting B6 vitamer metabolism or by inactivation of PLP; by compounds accumulating as a result of inborn errors of other pathways or by ingested small molecules. Whole exome sequencing of 2 children from a consanguineous family with pyridoxine-dependent epilepsy revealed a homozygous nonsense mutation in proline synthetase co-transcribed homolog (bacterial) (PROSC), a PLPbinding protein of hitherto unknown function. Subsequent sequencing of 29 unrelated indivduals with pyridoxine-responsive epilepsy identified 4 additional children with biallelic PROSC mutations. Pretreatment cerebrospinal fluid samples showed low PLP concentrations and evidence of reduced activity of PLP-dependent enzymes. However, cultured fibroblasts showed excessive PLP accumulation. An E.coli mutant, lacking the PROSC homologue (ΔYggS) is pyridoxine-sensitive; complementation with human PROSC restored growth whilst hPROSC bearing p.Leu175Pro, p.Arg241Gln and p.Ser78Ter did not. PLP, a highly reactive aldehyde, poses a problem for cells - how to supply enough PLP for apoenzymes while maintaining free PLP concentrations low enough to avoid unwanted reactions with other important cellular nucleophiles. Whilst the mechanism involved is not fully understood our studies suggest that PROSC is involved in intracellular homeostatic regulation of PLP, supplying this cofactor to apoenzymes while minimizing any toxic side reactions

The Elusive Third Subunit IIa of the Bacterial B-Type Oxidases: The Enzyme from the Hyperthermophile Aquifex aeolicus

The reduction of molecular oxygen to water is catalyzed by complicated membrane-bound metallo-enzymes containing variable numbers of subunits, called cytochrome c oxidases or quinol oxidases. We previously described the cytochrome c oxidase II from the hyperthermophilic bacterium Aquifex aeolicus as a ba3-type two-subunit (subunits I and II) enzyme and showed that it is included in a supercomplex involved in the sulfide-oxygen respiration pathway. It belongs to the B-family of the heme-copper oxidases, enzymes that are far less studied than the ones from family A. Here, we describe the presence in this enzyme of an additional transmembrane helix “subunit IIa”, which is composed of 41 amino acid residues with a measured molecular mass of 5105 Da. Moreover, we show that subunit II, as expected, is in fact longer than the originally annotated protein (from the genome) and contains a transmembrane domain. Using Aquifex aeolicus genomic sequence analyses, N-terminal sequencing, peptide mass fingerprinting and mass spectrometry analysis on entire subunits, we conclude that the B-type enzyme from this bacterium is a three-subunit complex. It is composed of subunit I (encoded by coxA2) of 59000 Da, subunit II (encoded by coxB2) of 16700 Da and subunit IIa which contain 12, 1 and 1 transmembrane helices respectively. A structural model indicates that the structural organization of the complex strongly resembles that of the ba3 cytochrome c oxidase from the bacterium Thermus thermophilus, the IIa helical subunit being structurally the lacking N-terminal transmembrane helix of subunit II present in the A-type oxidases. Analysis of the genomic context of genes encoding oxidases indicates that this third subunit is present in many of the bacterial oxidases from B-family, enzymes that have been described as two-subunit complexes

Métabolisme énergétique d'une bactérie hyperthermophile (organisation en supercomplexes et caractérisation de la voie de réduction de l'oxygène)

Aquifex aeolicus, une des bactéries les plus hyperthermophiles connue à ce jour, se développe à 85C. Cet organisme a été isolé de sources hydrothermales près de l île de Vulcano. Aquifex aeolicus est retrouvée au niveau des branches les plus profondes de l arbre phylogénétique universel basé sur les séquences de gène de l ARNr. C est une bactérie chimiolithoautotrophe qui nécessite pour sa croissance la présence d H2, d O2, de CO2, et d un composé soufré (soufre élémentaire ou thiosulfate). De part son aptitude à vivre dans des environnements extrêmes, ce microorganisme est un objet d études intéressant pour la compréhension de l adaptation des microorganismes, mais également pour la connaissance des voies bioénergétiques ancestrales et leur évolution. L équipe a caractérisé différentes enzymes impliquées dans le métabolisme énergétique d Aquifex aeolicus : des enzymes clés du métabolisme de l hydrogène, (les hydrogénases), et des enzymes du métabolisme du soufre, (une soufre réductase, une Soufre Oxygénase Réductase ainsi que deux soufre transférases). Cependant, les voies bioénergétiques mises en jeu par la bactérie pour se développer sont peu connues, en particulier celle impliquant l oxygène, bien que le complexe bc1 ait été caractérisé. Une approche protéomique intégrée non conventionnelle à partir des membranes d Aquifex aeolicus, nous a permis de mettre en évidence les différents complexes et enzymes respiratoires présents lorsque la bactérie se développe en présence de soufre élémentaire. Des études de transfert d électrons nous ont permis de proposer que plusieurs chaînes de transfert d électrons sont fonctionnelles dans les membranes. (1) la voie hydrogène-oxygène, (2) la voie hydrogène-soufre (H2/S), (3) la voie sulfure d hydrogène-oxygène (H2S/O2). Des travaux précédents de l équipe ont montré que la voie H2/S est organisée en un supercomplexe membranaire impliquant une hydrogénase et une soufre réductase. Une approche pluridisciplinaire nous a permis de mettre en évidence que la voie H2S/O2 s organise en un supercomplexe membranaire de 350 kDa composé d une Sulfure Quinone Réductase (SQR), d un dimère de complexe bc1 et d une cytochrome c oxydase. Nous avons montré que cette dernière est une enzyme de type ba3 et qu elle constituée de 3 sousunités. Ce supercomplexe fonctionnel constitue un respirasome complet très stable, en effet il résiste aux agents dénaturants. Le sous-complexe complexe bc1-cytochrome c oxydase est le coeur stable du supercomplexe, et la SQR est en interaction plus faible avec ce coeur . Par résonance plasmonique de surface une interaction entre la SQR et le sous-complexe a été mise en évidence. La SQR, voie d entrée des électrons au sein du supercomplexe membranaire, a retenu notre attention et a été caractérisée. Cette enzyme ne semble pas avoir les mêmes caractéristiques qu elle soit libre ou associée à ses partenaires au sein du supercomplexe. Nous avons proposé un modèle global du métabolisme énergétique d Aquifex aeolicus.AIX-MARSEILLE1-BU Sci.St Charles (130552104) / SudocSudocFranceF

Insertion and self-diffusion of a monotopic protein, the Aquifex aeolicus sulfide quinone reductase, in supported lipid bilayers

International audienceMonotopic proteins constitute a class of membrane proteins that bind tightly to cell membranes, but do not span them. We present a FRAPP (Fluorescence Recovery After Patterned Photobleaching) study of the dynamics of a bacterial monotopic protein, SQR (sulfide quinone oxidoreductase) from the thermophilic bacteria Aquifex aeolicus, inserted into two different types of lipid bilayers (EggPC: L-α-phosphatidylcholine (Egg, Chicken) and DMPC: 1,2-dimyristoyl-sn-glycero-3-phosphocholine) supported on two different types of support (mica or glass). It sheds light on the behavior of a monotopic protein inside the bilayer. The insertion of SQR is more efficient when the bilayer is in the fluid phase than in the gel phase. We observed diffusion of the protein, with no immobile fraction, and deduced from the diffusion coefficient measurements that the resulting inserted object is the same whatever the incubation conditions, i.e. homogeneous in terms of oligomerization state. As expected, the diffusion coefficient of the SQR is smaller in the gel phase than in the fluid phase. In the supported lipid bilayer, the diffusion coefficient of the SQR is smaller than the diffusion coefficient of phospholipids in both gel and fluid phase. SQR shows a diffusion behavior different from the transmembrane protein α-hemolysin, and consistent with its monotopic character. Preliminary experiments in the presence of the substrate of SQR, DecylUbiquinone, an analogue of quinone, component of transmembrane electrons transport systems of eukaryotic and prokaryotic organisms, have been carried out. Finally, we studied the behavior of SQR, in terms of insertion and diffusion, in bilayers formed with lipids from Aquifex aeolicus. All the conclusions that we have found in the biomimetic systems applied to the biological syste

Evidence that the metabolite repair enzyme NAD(P)HX epimerase has a moonlighting function

NAD(P)H-hydrate epimerase (EC 5.1.99.6) is known to help repair NAD(P)H hydrates (NAD(P)HX), which are damage products existing as R and S epimers. The S epimer is reconverted to NAD(P)H by a dehydratase; the epimerase facilitates epimer interconversion. Epimerase deficiency in humans causes a lethal disorder attributed to NADHX accumulation. However, bioinformatic evidence suggest caution about this attribution by predicting that the epimerase has a second function connected to vitamin B6 (pyridoxal 5'-phosphate and related compounds). Specifically, (i) the epimerase is fused to a B6 salvage enzyme in plants, (ii) epimerase genes cluster on the chromosome with B6-related genes in bacteria, and (iii) epimerase and B6-related genes are coexpressed in yeast and Arabidopsis The predicted second function was explored in Escherichia coli, whose epimerase and dehydratase are fused and encoded by yjeF The putative NAD(P)HX epimerase active site has a conserved lysine residue (K192 in E. coli YjeF). Changing this residue to alanine cut in vitro epimerase activity by ≥95% but did not affect dehydratase activity. Mutant cells carrying the K192A mutation had essentially normal NAD(P)HX dehydratase activity and NAD(P)HX levels, showing that the mutation had little impact on NAD(P)HX repair in vivo However, these cells showed metabolome changes, particularly in amino acids, which exceeded those in cells lacking the entire yjeF gene. The K192A mutant cells also had reduced levels of 'free' (i.e. weakly bound or unbound) pyridoxal 5'-phosphate. These results provide circumstantial evidence that the epimerase has a metabolic function beyond NAD(P)HX repair and that this function involves vitamin B6

Evidence that the metabolite repair enzyme NAD(P)HX epimerase has a moonlighting function

NAD(P)H-hydrate epimerase (EC 5.1.99.6) is known to help repair NAD(P)H hydrates (NAD(P)HX), which are damage products existing as R and S epimers. The S epimer is reconverted to NAD(P)H by a dehydratase; the epimerase facilitates epimer interconversion. Epimerase deficiency in humans causes a lethal disorder attributed to NADHX accumulation. However, bioinformatic evidence suggest caution about this attribution by predicting that the epimerase has a second function connected to vitamin B6 (pyridoxal 5'-phosphate and related compounds). Specifically, (i) the epimerase is fused to a B6 salvage enzyme in plants, (ii) epimerase genes cluster on the chromosome with B6-related genes in bacteria, and (iii) epimerase and B6-related genes are coexpressed in yeast and Arabidopsis The predicted second function was explored in Escherichia coli, whose epimerase and dehydratase are fused and encoded by yjeF The putative NAD(P)HX epimerase active site has a conserved lysine residue (K192 in E. coli YjeF). Changing this residue to alanine cut in vitro epimerase activity by ≥95% but did not affect dehydratase activity. Mutant cells carrying the K192A mutation had essentially normal NAD(P)HX dehydratase activity and NAD(P)HX levels, showing that the mutation had little impact on NAD(P)HX repair in vivo However, these cells showed metabolome changes, particularly in amino acids, which exceeded those in cells lacking the entire yjeF gene. The K192A mutant cells also had reduced levels of 'free' (i.e. weakly bound or unbound) pyridoxal 5'-phosphate. These results provide circumstantial evidence that the epimerase has a metabolic function beyond NAD(P)HX repair and that this function involves vitamin B6