COQ4 is required for the oxidative decarboxylation of the C1 carbon of coenzyme Q in eukaryotic cells.

Coenzyme Q (CoQ) is a redox lipid that fulfills critical functions in cellular bioenergetics and homeostasis. CoQ is synthesized by a multi-step pathway that involves several COQ proteins. Two steps of the eukaryotic pathway, the decarboxylation and hydroxylation of position C1, have remained uncharacterized. Here, we provide evidence that these two reactions occur in a single oxidative decarboxylation step catalyzed by COQ4. We demonstrate that COQ4 complements an Escherichia coli strain deficient for C1 decarboxylation and hydroxylation and that COQ4 displays oxidative decarboxylation activity in the non-CoQ producer Corynebacterium glutamicum. Overall, our results substantiate that COQ4 contributes to CoQ biosynthesis, not only via its previously proposed structural role but also via the oxidative decarboxylation of CoQ precursors. These findings fill a major gap in the knowledge of eukaryotic CoQ biosynthesis and shed light on the pathophysiology of human primary CoQ deficiency due to COQ4 mutations.

An organic O donor for biological hydroxylation reactions.

All biological hydroxylation reactions are thought to derive the oxygen atom from one of three inorganic oxygen donors, O2, H2O2, or H2O. Here, we have identified the organic compound prephenate as the oxygen donor for the three hydroxylation steps of the O2-independent biosynthetic pathway of ubiquinone, a widely distributed lipid coenzyme. Prephenate is an intermediate in the aromatic amino acid pathway and genetic experiments showed that it is essential for ubiquinone biosynthesis in Escherichia coli under anaerobic conditions. Metabolic labeling experiments with 18O-shikimate, a precursor of prephenate, demonstrated the incorporation of 18O atoms into ubiquinone. The role of specific iron-sulfur enzymes belonging to the widespread U32 protein family is discussed. Prephenate-dependent hydroxylation reactions represent a unique biochemical strategy for adaptation to anaerobic environments.

Diversification of Ubiquinone Biosynthesis via Gene Duplications, Transfers, Losses, and Parallel Evolution.

The availability of an ever-increasing diversity of prokaryotic genomes and metagenomes represents a major opportunity to understand and decipher the mechanisms behind the functional diversification of microbial biosynthetic pathways. However, it remains unclear to what extent a pathway producing a specific molecule from a specific precursor can diversify. In this study, we focus on the biosynthesis of ubiquinone (UQ), a crucial coenzyme that is central to the bioenergetics and to the functioning of a wide variety of enzymes in Eukarya and Pseudomonadota (a subgroup of the formerly named Proteobacteria). UQ biosynthesis involves three hydroxylation reactions on contiguous carbon atoms. We and others have previously shown that these reactions are catalyzed by different sets of UQ-hydroxylases that belong either to the iron-dependent Coq7 family or to the more widespread flavin monooxygenase (FMO) family. Here, we combine an experimental approach with comparative genomics and phylogenetics to reveal how UQ-hydroxylases evolved different selectivities within the constrained framework of the UQ pathway. It is shown that the UQ-FMOs diversified via at least three duplication events associated with two cases of neofunctionalization and one case of subfunctionalization, leading to six subfamilies with distinct hydroxylation selectivity. We also demonstrate multiple transfers of the UbiM enzyme and the convergent evolution of UQ-FMOs toward the same function, which resulted in two independent losses of the Coq7 ancestral enzyme. Diversification of this crucial biosynthetic pathway has therefore occurred via a combination of parallel evolution, gene duplications, transfers, and losses.

Recent advances in the metabolic pathways and microbial production of coenzyme Q.

Coenzyme Q (CoQ) serves as an electron carrier in aerobic respiration and has become an interesting target for biotechnological production due to its antioxidative effect and benefits in supplementation to patients with various diseases. Here, we review discovery of the pathway with a particular focus on its superstructuration and regulation, and we summarize the metabolic engineering strategies for overproduction of CoQ by microorganisms. Studies in model microorganisms elucidated the details of CoQ biosynthesis and revealed the existence of multiprotein complexes composed of several enzymes that catalyze consecutive reactions in the CoQ pathways of Saccharomyces cerevisiae and Escherichia coli. Recent findings indicate that the identity and the total number of proteins involved in CoQ biosynthesis vary between species, which raises interesting questions about the evolution of the pathway and could provide opportunities for easier engineering of CoQ production. For the biotechnological production, so far only microorganisms have been used that naturally synthesize CoQ10 or a related CoQ species. CoQ biosynthesis requires the aromatic precursor 4-hydroxybenzoic acid and the prenyl side chain that defines the CoQ species. Up to now, metabolic engineering strategies concentrated on the overproduction of the prenyl side chain as well as fine-tuning the expression of ubi genes from the ubiquinone modification pathway, resulting in high CoQ yields. With expanding knowledge about CoQ biosynthesis and exploration of new strategies for strain engineering, microbial CoQ production is expected to improve.

The Biosynthetic Pathway of Ubiquinone Contributes to Pathogenicity of Francisella novicida.

Francisella tularensis is the causative agent of tularemia. Because of its extreme infectivity and high mortality rate, this pathogen was classified as a biothreat agent. Francisella spp. are strict aerobes, and ubiquinone (UQ) has been previously identified in these bacteria. While the UQ biosynthetic pathways were extensively studied in Escherichia coli, allowing the identification of 15 Ubi proteins to date, little is known about Francisella spp. In this study, and using Francisella novicida as a surrogate organism, we first identified ubiquinone 8 (UQ8) as the major quinone found in the membranes of this bacterium. Next, we characterized the UQ biosynthetic pathway in F. novicida using a combination of bioinformatics, genetics, and biochemical approaches. Our analysis disclosed the presence in Francisella of 10 putative Ubi proteins, and we confirmed 8 of them by heterologous complementation in E. coli. The UQ biosynthetic pathways from F. novicida and E. coli share similar patterns. However, differences were highlighted: the decarboxylase remains unidentified in Francisella spp., and homologs of the Ubi proteins involved in the O2-independent UQ pathway are not present. This is in agreement with the strictly aerobic niche of this bacterium. Next, via two approaches, i.e., the use of an inhibitor (3-amino-4-hydroxybenzoic acid) and a transposon mutant, both of which strongly impair the synthesis of UQ, we demonstrated that UQ is essential for the growth of F. novicida in respiratory medium and contributes to its pathogenicity in Galleria mellonella used as an alternative animal model. IMPORTANCE Francisella tularensis is the causative bacterium of tularemia and is classified as a biothreat agent. Using multidisciplinary approaches, we investigated the ubiquinone (UQ) biosynthetic pathway that operates in F. novicida used as a surrogate. We show that UQ8 is the major quinone identified in the membranes of Francisella novicida. We identified a new competitive inhibitor that strongly decreased the biosynthesis of UQ. Our demonstration of the crucial roles of UQ for the respiratory metabolism of F. novicida and for the involvement in its pathogenicity in the Galleria mellonella model should stimulate the search for selective inhibitors of bacterial UQ biosynthesis.

The O2-independent pathway of ubiquinone biosynthesis is essential for denitrification in Pseudomonas aeruginosa.

Many proteobacteria, such as Escherichia coli, contain two main types of quinones: benzoquinones, represented by ubiquinone (UQ) and naphthoquinones, such as menaquinone (MK), and dimethyl-menaquinone (DMK). MK and DMK function predominantly in anaerobic respiratory chains, whereas UQ is the major electron carrier in the reduction of dioxygen. However, this division of labor is probably not very strict. Indeed, a pathway that produces UQ under anaerobic conditions in an UbiU-, UbiV-, and UbiT-dependent manner has been discovered recently in E. coli Its physiological relevance is not yet understood, because MK and DMK are also present in E. coli Here, we established that UQ9 is the major quinone of Pseudomonas aeruginosa and is required for growth under anaerobic respiration (i.e. denitrification). We demonstrate that the ORFs PA3911, PA3912, and PA3913, which are homologs of the E. coli ubiT, ubiV, and ubiU genes, respectively, are essential for UQ9 biosynthesis and, thus, for denitrification in P. aeruginosa These three genes here are called ubiTPa , ubiVPa , and ubiUPa We show that UbiVPa accommodates an iron-sulfur [4Fe-4S] cluster. Moreover, we report that UbiUPa and UbiTPa can bind UQ and that the isoprenoid tail of UQ is the structural determinant required for recognition by these two Ubi proteins. Since the denitrification metabolism of P. aeruginosa is believed to be important for the pathogenicity of this bacterium in individuals with cystic fibrosis, our results highlight that the O2-independent UQ biosynthetic pathway may represent a target for antibiotics development to manage P. aeruginosa infections.

The UbiK protein is an accessory factor necessary for bacterial ubiquinone (UQ) biosynthesis and forms a complex with the UQ biogenesis factor UbiJ.

Ubiquinone (UQ), also referred to as coenzyme Q, is a widespread lipophilic molecule in both prokaryotes and eukaryotes in which it primarily acts as an electron carrier. Eleven proteins are known to participate in UQ biosynthesis in Escherichia coli, and we recently demonstrated that UQ biosynthesis requires additional, nonenzymatic factors, some of which are still unknown. Here, we report on the identification of a bacterial gene, yqiC, which is required for efficient UQ biosynthesis, and which we have renamed ubiK Using several methods, we demonstrated that the UbiK protein forms a complex with the C-terminal part of UbiJ, another UQ biogenesis factor we previously identified. We found that both proteins are likely to contribute to global UQ biosynthesis rather than to a specific biosynthetic step, because both ubiK and ubiJ mutants accumulated octaprenylphenol, an early intermediate of the UQ biosynthetic pathway. Interestingly, we found that both proteins are dispensable for UQ biosynthesis under anaerobiosis, even though they were expressed in the absence of oxygen. We also provide evidence that the UbiK-UbiJ complex interacts with palmitoleic acid, a major lipid in E. coli Last, in Salmonella enterica, ubiK was required for proliferation in macrophages and virulence in mice. We conclude that although the role of the UbiK-UbiJ complex remains unknown, our results support the hypothesis that UbiK is an accessory factor of Ubi enzymes and facilitates UQ biosynthesis by acting as an assembly factor, a targeting factor, or both.

Coq6 is responsible for the C4-deamination reaction in coenzyme Q biosynthesis in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is able to use para-aminobenzoic acid (pABA) in addition to 4-hydroxybenzoic acid as a precursor of coenzyme Q, a redox lipid essential to the function of the mitochondrial respiratory chain. The biosynthesis of coenzyme Q from pABA requires a deamination reaction at position C4 of the benzene ring to substitute the amino group with an hydroxyl group. We show here that the FAD-dependent monooxygenase Coq6, which is known to hydroxylate position C5, also deaminates position C4 in a reaction implicating molecular oxygen, as demonstrated with labeling experiments. We identify mutations in Coq6 that abrogate the C4-deamination activity, whereas preserving the C5-hydroxylation activity. Several results support that the deletion of Coq9 impacts Coq6, thus explaining the C4-deamination defect observed in Δcoq9 cells. The vast majority of flavin monooxygenases catalyze hydroxylation reactions on a single position of their substrate. Coq6 is thus a rare example of a flavin monooxygenase that is able to act on two different carbon atoms of its C4-aminated substrate, allowing its deamination and ultimately its conversion into coenzyme Q by the other proteins constituting the coenzyme Q biosynthetic pathway.

Three conserved histidine residues contribute to mitochondrial iron transport through mitoferrins.

Iron is an essential element for almost all organisms. In eukaryotes, it is mainly used in mitochondria for the biosynthesis of iron-sulfur clusters and haem group maturation. Iron is delivered into the mitochondrion by mitoferrins, members of the MCF (mitochondrial carrier family), through an unknown mechanism. In the present study, the yeast homologues of these proteins, Mrs3p (mitochondrial RNA splicing 3) and Mrs4p, were studied by inserting them into liposomes. In this context, they could transport Fe2+ across the proteoliposome membrane, as shown using the iron chelator bathophenanthroline. A series of amino acid-modifying reagents were screened for their effects on Mrs3p-mediated iron transport. The results of the present study suggest that carboxy and imidazole groups are essential for iron transport. This was confirmed by in vivo complementation assays, which demonstrated that three highly conserved histidine residues are important for Mrs3p function. These histidine residues are not conserved in other MCF members and thus they are likely to play a specific role in iron transport. A model describing how these residues help iron to transit smoothly across the carrier cavity is proposed and compared with the structural and biochemical data available for other carriers in this family.

The transmembrane prolines of the mitochondrial ADP/ATP carrier are involved in nucleotide binding and transport and its biogenesis.

The mitochondrial ADP/ATP carrier (Ancp) is a paradigm of the mitochondrial carrier family, which allows cross-talk between mitochondria, where cell energy is mainly produced, and cytosol, where cell energy is mainly consumed. The members of this family share numerous structural and functional characteristics. Resolution of the atomic structure of the bovine Ancp, in a complex with one of its specific inhibitors, revealed interesting features and suggested the involvement of some particular residues in the movements of the protein to perform translocation of nucleotides from one side of the membrane to the other. They correspond to three prolines located in the odd-numbered transmembrane helices (TMH), Pro-27, Pro-132, and Pro-229. The corresponding residues of the yeast Ancp (Pro-43, Ser-147, and Pro-247) were mutated into alanine or leucine, one at a time and analysis of the various mutants evidenced a crucial role of Pro-43 and Pro-247 during nucleotide transport. Beside, replacement of Ser-147 with proline does not inactivate Ancp and this is discussed in view of the conservation of the three prolines at equivalent positions in the Ancp sequences. These prolines belong to the signature sequences of the mitochondrial carriers and we propose they play a dual role in the mitochondrial ADP/ATP carrier function and biogenesis. Unexpectedly their mutations cause more general effects on mitochondrial biogenesis and morphology, as evidenced by measurements of respiratory rates, cytochrome contents, and also clearly highlighted by fluorescence microscopy.

Functional characterization of TbMCP5, a conserved and essential ADP/ATP carrier present in the mitochondrion of the human pathogen Trypanosoma brucei.

Trypanosoma brucei is a kinetoplastid parasite of medical and veterinary importance. Its digenetic life cycle alternates between the bloodstream form in the mammalian host and the procyclic form (PCF) in the bloodsucking insect vector, the tsetse fly. PCF trypanosomes rely in the glucose-depleted environment of the insect vector primarily on the mitochondrial oxidative phosphorylation of proline for their cellular ATP provision. We previously identified two T. brucei mitochondrial carrier family proteins, TbMCP5 and TbMCP15, with significant sequence similarity to functionally characterized ADP/ATP carriers from other eukaryotes. Comprehensive sequence analysis confirmed that TbMCP5 contains canonical ADP/ATP carrier sequence features, whereas they are not conserved in TbMCP15. Heterologous expression in the ANC-deficient yeast strain JL1Δ2Δ3u(-) revealed that only TbMCP5 was able to restore its growth on the non-fermentable carbon source lactate. Transport studies in yeast mitochondria showed that TbMCP5 has biochemical properties and ADP/ATP exchange kinetics similar to those of Anc2p, the prototypical ADP/ATP carrier of S. cerevisiae. Immunofluorescence microscopy and Western blot analysis confirmed that TbMCP5 is exclusively mitochondrial and is differentially expressed with 4.5-fold more TbMCP5 in the procyclic form of the parasite. Silencing of TbMCP5 expression in PCF T. brucei revealed that this ADP/ATP carrier is essential for parasite growth, particularly when depending on proline for energy generation. Moreover, ADP/ATP exchange in isolated T. brucei mitochondria was eliminated upon TbMCP5 depletion. These results confirmed that TbMCP5 functions as the main ADP/ATP carrier in the trypanosome mitochondrion. The important role of TbMCP5 in the T. brucei energy metabolism is further discussed.

Role of the Escherichia coli ubiquinone-synthesizing UbiUVT pathway in adaptation to changing respiratory conditions.

Isoprenoid quinones are essential for cellular physiology. They act as electron and proton shuttles in respiratory chains and various biological processes. Escherichia coli and many α-, ß-, and γ-proteobacteria possess two types of isoprenoid quinones: ubiquinone (UQ) is mainly used under aerobiosis, while demethylmenaquinones (DMK) are mostly used under anaerobiosis. Yet, we recently established the existence of an anaerobic O2-independent UQ biosynthesis pathway controlled by ubiT, ubiU, and ubiV genes. Here, we characterize the regulation of ubiTUV genes in E. coli. We show that the three genes are transcribed as two divergent operons that are both under the control of the O2-sensing Fnr transcriptional regulator. Phenotypic analyses using a menA mutant devoid of DMK revealed that UbiUV-dependent UQ synthesis is essential for nitrate respiration and uracil biosynthesis under anaerobiosis, while it contributes, though modestly, to bacterial multiplication in the mouse gut. Moreover, we showed by genetic study and 18O2 labeling that UbiUV contributes to the hydroxylation of ubiquinone precursors through a unique O2-independent process. Last, we report the crucial role of ubiT in allowing E. coli to shift efficiently from anaerobic to aerobic conditions. Overall, this study uncovers a new facet of the strategy used by E. coli to adjust its metabolism on changing O2 levels and respiratory conditions. This work links respiratory mechanisms to phenotypic adaptation, a major driver in the capacity of E. coli to multiply in gut microbiota and of facultative anaerobic pathogens to multiply in their host. IMPORTANCE Enterobacteria multiplication in the gastrointestinal tract is linked to microaerobic respiration and associated with various inflammatory bowel diseases. Our study focuses on the biosynthesis of ubiquinone, a key player in respiratory chains, under anaerobiosis. The importance of this study stems from the fact that UQ usage was for long considered to be restricted to aerobic conditions. Here we investigated the molecular mechanism allowing UQ synthesis in the absence of O2 and searched for the anaerobic processes that UQ is fueling in such conditions. We found that UQ biosynthesis involves anaerobic hydroxylases, that is, enzymes able to insert an O atom in the absence of O2. We also found that anaerobically synthesized UQ can be used for respiration on nitrate and the synthesis of pyrimidine. Our findings are likely to be applicable to most facultative anaerobes, which count many pathogens (Salmonella, Shigella, and Vibrio) and will help in unraveling microbiota dynamics.

COQ4 is required for the oxidative decarboxylation of the C1 carbon of Coenzyme Q in eukaryotic cells.

Coenzyme Q (CoQ) is a redox lipid that fulfills critical functions in cellular bioenergetics and homeostasis. CoQ is synthesized by a multi-step pathway that involves several COQ proteins. Two steps of the eukaryotic pathway, the decarboxylation and hydroxylation of position C1, have remained uncharacterized. Here, we provide evidence that these two reactions occur in a single oxidative decarboxylation step catalyzed by COQ4. We demonstrate that COQ4 complements an Escherichia coli strain deficient for C1 decarboxylation and hydroxylation and that COQ4 displays oxidative decarboxylation activity in the non-CoQ producer Corynebacterium glutamicum. Overall, our results substantiate that COQ4 contributes to CoQ biosynthesis, not only via its previously proposed structural role, but also via oxidative decarboxylation of CoQ precursors. These findings fill a major gap in the knowledge of eukaryotic CoQ biosynthesis, and shed new light on the pathophysiology of human primary CoQ deficiency due to COQ4 mutations.

Exploring the conformational dynamics of the bovine ADP/ATP carrier in mitochondria.

The mitochondrial ADP/ATP carrier catalyzes the transport of ADP and ATP across the mitochondrial inner membrane by switching between two different conformations. They can be blocked by two inhibitors: carboxyatractyloside (CATR) and bongkrekic acid (BA). Our understanding of the ADP/ATP transport process is largely based on analysis of structural differences between the individual inhibited states. The X-ray crystallographic three-dimensional structure of bovine ADP/ATP carrier isoform 1 (bAnc1p) complexed with CATR was determined, but the structure of the BA-carrier complex remains unknown. We recently investigated the conformational dynamics of bAnc1p in detergent solution using hydrogen/deuterium exchange and mass spectrometry (HDX-MS). This study shed light on some features of ADP/ATP translocation, but the mechanism itself and the organization of bAnc1p in the membrane required further investigation. This paper describes the first study of bAnc1p in the mitochondria on the whole-protein scale using HDX-MS. Membrane-embedded bAnc1p was deuterated and purified under HDX-MS-compatible conditions. Our results for the carrier in the mitochondrial inner membrane differed from those published for the carrier in a detergent solution. These differences were mainly in the upper half of the cavity that globally showed a limited H/D exchange whatever the complex analyzed and at the level of the matrix loops that were less accessible to the solvent in the BA-carrier complex than in the CATR-carrier complex. They are discussed with respect to published data for bAnc1p and have provided new insights into the conformation of the matrix loops of the bovine carrier in complex with BA in mitochondria.

Yeast ADP/ATP carrier isoform 2: conformational dynamics and role of the RRRMMM signature sequence methionines.

The mitochondrial ADP/ATP carrier, or Ancp, is a member of the mitochondrial carrier family responsible for exchanging ADP and ATP across the mitochondrial inner membrane. ADP/ATP transport involves Ancp switching between two conformational states. These can be analyzed using specific inhibitors, carboxyatractyloside (CATR) and bongkrekic acid (BA). The high resolution three-dimensional structure of bovine Anc1p (bAnc1p), as a CATR-carrier complex, has been solved. However, because the structure of the BA-carrier complex has not yet been determined, the detailed mechanism of transport remains unknown. Recently, sample processing for hydrogen/deuterium exchange experiments coupled to mass spectrometry was improved, providing novel insights into bAnc1p conformational transitions due to inhibitor binding. In this work we performed both hydrogen/deuterium exchange-mass spectrometry experiments and genetic manipulations. Because these are very difficult to apply with bovine Anc1p, we used Saccharomyces cerevisiae Anc isoform 2 (ScAnc2p). Significant differences in solvent accessibility were observed throughout the amino acid sequence for ScAnc2p complexed to either CATR or BA. Interestingly, in detergent solution, the conformational dynamics of ScAnc2p were dissimilar to those of bAnc1p, in particular for the upper half of the cavity, toward the intermembrane space, and the m2 loop, which is thought to be easily accessible to the solvent from the matrix in bAnc1p. Our study then focused on the methionyl residues of the Ancp signature sequence, RRRMMM. All our results indicate that the methionine cluster is involved in the ADP/ATP transport mechanism and confirm that the Ancp cavity is a highly dynamic structure.

Rational Engineering of Non-Ubiquinone Containing Corynebacterium glutamicum for Enhanced Coenzyme Q10 Production.

Coenzyme Q10 (CoQ10) is a lipid-soluble compound with important physiological functions and is sought after in the food and cosmetic industries owing to its antioxidant properties. In our previous proof of concept, we engineered for CoQ10 biosynthesis the industrially relevant Corynebacterium glutamicum, which does not naturally synthesize any CoQ. Here, liquid chromatography-mass spectrometry (LC-MS) analysis identified two metabolic bottlenecks in the CoQ10 production, i.e., low conversion of the intermediate 10-prenylphenol (10P-Ph) to CoQ10 and the accumulation of isoprenologs with prenyl chain lengths of not only 10, but also 8 to 11 isopentenyl units. To overcome these limitations, the strain was engineered for expression of the Ubi complex accessory factors UbiJ and UbiK from Escherichia coli to increase flux towards CoQ10, and by replacement of the native polyprenyl diphosphate synthase IspB with a decaprenyl diphosphate synthase (DdsA) to select for prenyl chains with 10 isopentenyl units. The best strain UBI6-Rs showed a seven-fold increased CoQ10 content and eight-fold increased CoQ10 titer compared to the initial strain UBI4-Pd, while the abundance of CoQ8, CoQ9, and CoQ11 was significantly reduced. This study demonstrates the application of the recent insight into CoQ biosynthesis to improve metabolic engineering of a heterologous CoQ10 production strain.

Manganese-driven CoQ deficiency.

Overexposure to manganese disrupts cellular energy metabolism across species, but the molecular mechanism underlying manganese toxicity remains enigmatic. Here, we report that excess cellular manganese selectively disrupts coenzyme Q (CoQ) biosynthesis, resulting in failure of mitochondrial bioenergetics. While respiratory chain complexes remain intact, the lack of CoQ as lipophilic electron carrier precludes oxidative phosphorylation and leads to premature cell and organismal death. At a molecular level, manganese overload causes mismetallation and proteolytic degradation of Coq7, a diiron hydroxylase that catalyzes the penultimate step in CoQ biosynthesis. Coq7 overexpression or supplementation with a CoQ headgroup analog that bypasses Coq7 function fully corrects electron transport, thus restoring respiration and viability. We uncover a unique sensitivity of a diiron enzyme to mismetallation and define the molecular mechanism for manganese-induced bioenergetic failure that is conserved across species.

Conformational dynamics of the bovine mitochondrial ADP/ATP carrier isoform 1 revealed by hydrogen/deuterium exchange coupled to mass spectrometry.

The mitochondrial adenine nucleotide carrier (Ancp) catalyzes the transport of ADP and ATP across the mitochondrial inner membrane, thus playing an essential role in cellular energy metabolism. During the transport mechanism the carrier switches between two different conformations that can be blocked by two toxins: carboxyatractyloside (CATR) and bongkrekic acid. Therefore, our understanding of the nucleotide transport mechanism can be improved by analyzing structural differences of the individual inhibited states. We have solved the three-dimensional structure of bovine carrier isoform 1 (bAnc1p) in a complex with CATR, but the structure of the carrier-bongkrekic acid complex, and thus, the detailed mechanism of transport remains unknown. Improvements in sample processing in the hydrogen/deuterium exchange technique coupled to mass spectrometry (HDX-MS) have allowed us to gain novel insights into the conformational changes undergone by bAnc1p. This paper describes the first study of bAnc1p using HDX-MS. Results obtained with the CATR-bAnc1p complex were fully in agreement with published results, thus, validating our approach. On the other hand, the HDX kinetics of the two complexes displays marked differences. The bongkrekic acid-bAnc1p complex exhibits greater accessibility to the solvent on the matrix side, whereas the CATR-bAnc1p complex is more accessible on the intermembrane side. These results are discussed with respect to the structural and biochemical data available on Ancp.

Effective removal of nonionic detergents in protein mass spectrometry, hydrogen/deuterium exchange, and proteomics.

Detergents are frequently used for protein isolation and solubilization. Their presence is crucial in membrane protein protocols or in lipid raft proteomics. However, they are usually poorly compatible with mass spectrometry. Several different sample preparation protocols are routinely used, but they are either laborious or suffer from sample losses. Here, we describe our alternative method for nonionic detergent removal. It is based on selective detergent extraction after capture of the sample on a reversed phase cartridge. The extraction is performed by chlorinated solvents and works well for polyoxyethylene based nonionic detergents, but also for polymers like polyethylene and propylene glycol. Detergent removal can be also carried out on the protein level but a special care must be taken with hydrophobic proteins. In such cases, it is preferable to perform detergent removal after proteolysis which digests the protein to peptides and reduces the hydrophobicity. The method can easily be automated and is compatible with hydrogen/deuterium exchange coupled to mass spectrometry.

Advances in bacterial pathways for the biosynthesis of ubiquinone.

Ubiquinone is an important component of the electron transfer chains in proteobacteria and eukaryotes. The biosynthesis of ubiquinone requires multiple steps, most of which are common to bacteria and eukaryotes. Whereas the enzymes of the mitochondrial pathway that produces ubiquinone are highly similar across eukaryotes, recent results point to a rather high diversity of pathways in bacteria. This review focuses on ubiquinone in bacteria, highlighting newly discovered functions and detailing the proteins that are known to participate to its biosynthetic pathways. Novel results showing that ubiquinone can be produced by a pathway independent of dioxygen suggest that ubiquinone may participate to anaerobiosis, in addition to its well-established role for aerobiosis. We also discuss the supramolecular organization of ubiquinone biosynthesis proteins and we summarize the current understanding of the evolution of the ubiquinone pathways relative to those of other isoprenoid quinones like menaquinone and plastoquinone.