Structural basis of proton translocation and force generation in mitochondrial ATP synthase.

ATP synthases produce ATP by rotary catalysis, powered by the electrochemical proton gradient across the membrane. Understanding this fundamental process requires an atomic model of the proton pathway. We determined the structure of an intact mitochondrial ATP synthase dimer by electron cryo-microscopy at near-atomic resolution. Charged and polar residues of the a-subunit stator define two aqueous channels, each spanning one half of the membrane. Passing through a conserved membrane-intrinsic helix hairpin, the lumenal channel protonates an acidic glutamate in the c-ring rotor. Upon ring rotation, the protonated glutamate encounters the matrix channel and deprotonates. An arginine between the two channels prevents proton leakage. The steep potential gradient over the sub-nm inter-channel distance exerts a force on the deprotonated glutamate, resulting in net directional rotation.

Alleviation of metabolic bottleneck by combinatorial engineering enhanced astaxanthin synthesis in Saccharomyces cerevisiae.

Highly efficient biosynthesis of the commercially valuable carotenoid astaxanthin by microbial cells is an attractive alternative to chemical synthesis and microalgae extraction. With the goal of enhancing heterologous astaxanthin production in Saccharomyces cerevisiae, metabolic engineering and protein engineering were integrated to improve both the expression and activity of rate-limiting enzymes. Firstly, to increase the supply of ß-carotene as a key precursor for astaxanthin, a positive mutant of GGPP synthase (CrtE03M) was overexpressed together with three other rate-limiting enzymes tHMG1, CrtI and CrtYB. Subsequently, to accelerate the conversion of ß-carotene to astaxanthin, a color screening system was developed and adopted for directed evolution of ß-carotene ketolase (OBKT), generating a triple mutant OBKTM (H165R/V264D/F298Y) with 2.4-fold improved activity. After adjusting copy numbers of the above-mentioned rate-limiting enzymes to further balance the metabolic flux, a diploid strain YastD-01 was generated by mating two astaxanthin-producing haploid strains carrying the same carotenogenic pathway. Finally, further overexpression of OCrtZ and OBKTM in YastD-01 resulted in accumulation of 8.10mg/g DCW (47.18mg/l) of (3S, 3'S)-astaxanthin in shake-flask cultures. This combinatorial strategy might be also applicable for alleviation of metabolic bottleneck in biosynthesis of other value-added products, especially colored metabolites.

Structural basis for the recognition of spliceosomal SmN/B/B' proteins by the RBM5 OCRE domain in splicing regulation.

The multi-domain splicing factor RBM5 regulates the balance between antagonistic isoforms of the apoptosis-control genes FAS/CD95, Caspase-2 and AID. An OCRE (OCtamer REpeat of aromatic residues) domain found in RBM5 is important for alternative splicing regulation and mediates interactions with components of the U4/U6.U5 tri-snRNP. We show that the RBM5 OCRE domain adopts a unique ß-sheet fold. NMR and biochemical experiments demonstrate that the OCRE domain directly binds to the proline-rich C-terminal tail of the essential snRNP core proteins SmN/B/B'. The NMR structure of an OCRE-SmN peptide complex reveals a specific recognition of poly-proline helical motifs in SmN/B/B'. Mutation of conserved aromatic residues impairs binding to the Sm proteins in vitro and compromises RBM5-mediated alternative splicing regulation of FAS/CD95. Thus, RBM5 OCRE represents a poly-proline recognition domain that mediates critical interactions with the C-terminal tail of the spliceosomal SmN/B/B' proteins in FAS/CD95 alternative splicing regulation.

Salinity stress increases lipid, secondary metabolites and enzyme activity in Amphora subtropica and Dunaliella sp. for biodiesel production.

Amphora subtropica and Dunaliella sp. isolated from Tunisian biotopes were retained for their high lipid contents. Respective optimized parameters for rapid growth were: pH 9 and 10, light period 21 and 24h and temperature 31 and 34°C, respectively. After optimization, Amphora subtropica growth rate increased from 0.2 to 0.5day(-1) and Dunaliella sp. growth rate increased from 0.38 to 0.7day(-1). Amphora subtropica biomass production, productivity and lipid content increased from 0.3 to 0.7gL(-1)(dw), 69-100mgL(-1)d(-1)(dw) and 150-190gkg(-1)(dw), respectively, and Dunaliella sp. from 0.5 to 1.4gL(-1)(dw), 124-200mgL(-1)d(-1) (dw) and 190-280gkg(-1)(dw), respectively. Often to overcome trade-off between microalgae rapid growth and high lipid content which are often conflicting and very difficult to obtain at the same time, separation in a growth stage and a lipid accumulation stage is obvious. Salinity stress in a single stage of culture was studied. Compared to the optimal concentration of growth, excess or deficiency of NaCl engendered the same cellular responses by implication of oxidative stress systems and reactivation of defense and storage systems. Indeed, increasing salinity from 1M to 2M for Amphora subtropica or decreasing salinity from 3M to 2M for Dunaliella sp. have both increased lipids content from (220 and 280) to (350 and 430)gkg(-1), carotenoids from (1.8 and 2.4) to (2.3 and 3.7)pgcell(-1), TBARS amount from (10.4 and 5.3) to (12.1 and 10.7)nmolmg(-1) proteins and SOD activity from of (46.6 and 61.8) to (71.6 and 79.4)Umg(-1) proteins, respectively. With further improved fatty acids profile, the microalgae strains could be potent candidates for biofuel production.

Catalytic improvement and structural analysis of atrazine chlorohydrolase by site-saturation mutagenesis.

To improve the catalytic activity of atrazine chlorohydrolase (AtzA), amino acid residues involved in substrate binding (Gln71) and catalytic efficiency (Val12, Ile393, and Leu395) were targeted to generate site-saturation mutagenesis libraries. Seventeen variants were obtained through Haematococcus pluvialis-based screening, and their specific activities were 1.2-5.2-fold higher than that of the wild type. For these variants, Gln71 tended to be substituted by hydrophobic amino acids, Ile393 and Leu395 by polar ones, especially arginine, and Val12 by alanine, respectively. Q71R and Q71M significantly decreased the Km by enlarging the substrate-entry channel and affecting N-ethyl binding. Mutations at sites 393 and 395 significantly increased the kcat/Km, probably by improving the stability of the dual ß-sheet domain and the whole enzyme, owing to hydrogen bond formation. In addition, the contradictory relationship between the substrate affinity improvement by Gln71 mutation and the catalytic efficiency improvement by the dual ß-sheet domain modification was discussed.

Dissecting the peripheral stalk of the mitochondrial ATP synthase of chlorophycean algae.

The algae Chlamydomonas reinhardtii and Polytomella sp., a green and a colorless member of the chlorophycean lineage respectively, exhibit a highly-stable dimeric mitochondrial F1Fo-ATP synthase (complex V), with a molecular mass of 1600 kDa. Polytomella, lacking both chloroplasts and a cell wall, has greatly facilitated the purification of the algal ATP-synthase. Each monomer of the enzyme has 17 polypeptides, eight of which are the conserved, main functional components, and nine polypeptides (Asa1 to Asa9) unique to chlorophycean algae. These atypical subunits form the two robust peripheral stalks observed in the highly-stable dimer of the algal ATP synthase in several electron-microscopy studies. The topological disposition of the components of the enzyme has been addressed with cross-linking experiments in the isolated complex; generation of subcomplexes by limited dissociation of complex V; detection of subunit-subunit interactions using recombinant subunits; in vitro reconstitution of subcomplexes; silencing of the expression of Asa subunits; and modeling of the overall structural features of the complex by EM image reconstruction. Here, we report that the amphipathic polymer Amphipol A8-35 partially dissociates the enzyme, giving rise to two discrete dimeric subcomplexes, whose compositions were characterized. An updated model for the topological disposition of the 17 polypeptides that constitute the algal enzyme is suggested. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Highly efficient biosynthesis of astaxanthin in Saccharomyces cerevisiae by integration and tuning of algal crtZ and bkt.

Astaxanthin is a highly valued carotenoid with strong antioxidant activity and has wide applications in aquaculture, food, cosmetic, and pharmaceutical industries. The market demand for natural astaxanthin promotes research in metabolic engineering of heterologous hosts for astaxanthin production. In this study, an astaxanthin-producing Saccharomyces cerevisiae strain was created by successively introducing the Haematococcus pluvialis ß-carotenoid hydroxylase (crtZ) and ketolase (bkt) genes into a previously constructed ß-carotene hyperproducer. Further integration of strategies including codon optimization, gene copy number adjustment, and iron cofactor supplementation led to significant increase in the astaxanthin production, reaching up to 4.7 mg/g DCW in the shake-flask cultures which is the highest astaxanthin content in S. cerevisiae reported to date. Besides, the substrate specificity of H. pluvialis CrtZ and BKT and the probable formation route of astaxanthin from ß-carotene in S. cerevisiae were figured out by expressing the genes separately and in combination. The yeast strains engineered in this work provide a basis for further improving biotechnological production of astaxanthin and might offer a useful general approach to the construction of heterologous biosynthetic pathways for other natural products.

Kinetic and hysteretic behavior of ATP hydrolysis of the highly stable dimeric ATP synthase of Polytomella sp.

The F1FO-ATP synthase of the colorless alga Polytomella sp. exhibits a robust peripheral arm constituted by nine atypical subunits only present in chlorophycean algae. The isolated dimeric enzyme exhibits a latent ATP hydrolytic activity which can be activated by some detergents. To date, the kinetic behavior of the algal ATPase has not been studied. Here we show that while the soluble F1 sector exhibits Michaelis-Menten kinetics, the dimer exhibits a more complex behavior. The kinetic parameters (Vmax and Km) were obtained for both the F1 sector and the dimeric enzyme as isolated or activated by detergent, and this activation was also seen on the enzyme reconstituted in liposomes. Unlike other ATP synthases, the algal dimer hydrolyzes ATP on a wide range of pH and temperature. The enzyme was inhibited by oligomycin, DCCD and Mg-ADP, although oligomycin induced a peculiar inhibition pattern that can be attributed to structural differences in the algal subunit-c. The hydrolytic activity was temperature-dependent and exhibited activation energy of 4 kcal/mol. The enzyme also exhibited a hysteretic behavior with a lag phase strongly dependent on temperature but not on pH, that may be related to a possible regulatory role in vivo.

Characterization and expression patterns of nitrate reductase from Dunaliella bardawil under osmotic stress and dilution shock.

A complementary DNA (cDNA) of nitrate reductase (NR) from Dunaliella bardawil was isolated using RT-PCR and RACEs techniques. The full-length D. bardawil NR (DbNR) cDNA is 3,107 bp containing a putative open reading frame of 2,670 bp in length which encodes 889 amino acids with a calculated molecular weight (MW) of 98.37 kDa, a 34-bp 5'-untranslated region, and a 3'-untranslated region of 403 bp with a poly (A) tail. BLAST search showed that the nucleotide and putative protein sequence exhibit sequence identities of 92 and 79% with the corresponding gene from Dunaliella tertiolecta, respectively. Protein structural analysis showed a typical NR structure of DbNR with five structural distinctive domains which form three common subparts of eukaryotic NR (Euk-NR). Phylogenetic analysis based on the holo-DbNR and sulfite oxidase (SO) and cytochrome b reductase (CbR) subparts manifested that (1) DbNR has a closer relationship with those counterparts from algae and higher plants than from other species and (2) DbNR might have evolved from ancient SO and CbR in a "domain shuffling" pattern. The glycerol contents and transcriptional expression patterns of DbNR under salt stress and dilution shock treatments were also traced. The results implied an indirect role of NaCl on the induction of DbNR through an osmoregulation pathway.

In vitro import and assembly of the nucleus-encoded mitochondrial subunit III of cytochrome c oxidase (Cox3).

The cox3 gene, encoding subunit III of cytochrome c oxidase (Cox3) is in mitochondrial genomes except in chlorophycean algae, where it is localized in the nucleus. Therefore, algae like Chlamydomonas reinhardtii, Polytomella sp. and Volvox carteri, synthesize the Cox3 polypeptide in the cytosol, import it into mitochondria, and integrate it into the cytochrome c oxidase complex. In this work, we followed the in vitro internalization of the Cox3 precursor by isolated, import-competent mitochondria of Polytomella sp. In this colorless alga, the precursor Cox3 protein is synthesized with a long, cleavable, N-terminal mitochondrial targeting sequence (MTS) of 98 residues. In an import time course, a transient Cox3 intermediate was identified, suggesting that the long MTS is processed more than once. The first processing step is sensitive to the metalo-protease inhibitor 1,10-ortophenantroline, suggesting that it is probably carried out by the matrix-located Mitochondrial Processing Protease. Cox3 is readily imported through an energy-dependent import pathway and integrated into the inner mitochondrial membrane, becoming resistant to carbonate extraction. Furthermore, the imported Cox3 protein was assembled into cytochrome c oxidase, as judged by the presence of a labeled band co-migrating with complex IV in Blue Native Electrophoresis. A model for the biogenesis of Cox3 in chlorophycean algae is proposed. This is the first time that the in vitro mitochondrial import of a cytosol-synthesized Cox3 subunit is described.

Molecular cloning and functional characterization of MnSOD from Dunaliella salina.

Dunaliella salina, a unicellular green alga, has the potential to grow in hypersaline environments via one of its gene products, superoxide dismutase (SOD). The superoxide radicals (O2 (-) ) produced by environmental stresses can cause damage to cells, and SOD catalyzes the turnover of such free radicals to protect cells. In this study, the gene coding for SOD in D. salina was cloned and the product was further identified and characterized. The open reading frame of this gene was 651 bp long, encoding for 217 amino acids. According to the sequence alignment using BLAST, native polyacrylamide electrophoresis for SOD activity analysis, and atomic absorption spectroscopy analysis, this protein belongs to the manganese-containing superoxide dismutase (MnSOD) family. Complementation analysis, performed by introducing plasmids carrying an inducible version of the D. salina gene encoding for MnSOD into an SOD-deficient mutant of E.coli, revealed that this gene could not only complement the defects in SOD activity, but was also capable of providing a stronger tolerance to restrictive growth conditions, such as high salt and prolonged UV exposure, compared to the tolerance of wild-type strains.

Fluorimetric analysis of the binding characteristics of 5-enolpyruvylshikimate-3-phosphate synthase with substrates in Dunaliella salina.

A general model of the catalytic mechanism for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) has already been proposed. But whether shikimate-3-phosphate (S3P) alone can cause EPSPs' conformation changes, and whether the binding site of phosphoenolpyruvate (PEP) and glyphosate is the same are still in debate. In this paper, DsaroA gene amplified and cloned from Dunaliella salina (our laboratory's early study) was used for DsEPSPs expression and purification. Then the DsEPSP conformation changes as it bind with different substrates were detected by fluorimetry. The results show that we obtained the DsEPSPs by prokaryotic expression and purification, and the S3P binding with DsEPSPs alone cannot cause DsEPSPs to form "close" conformation directly. However, when S3P exits, DsEPSPs did have a trend to change to the "close" conformation. Then the "close" conformation can be formed completely with the addition of phosphoenolpyruvate (PEP) or glyphosate. The inorganic phosphorus can help S3P to induce two domains of DsEPSPs to form "close" conformation. Besides, when DsEPSPs binds with S3P, in 295 nm, only the intensity of emission peak decreases, however, in 280 nm, not only the peak intensity reduces but also the blue-shift phenomenon takes place. The reason for blue-shift phenomenon was the distribution of aromatic amino acids in EPSPs. EPSPs is a good target for novel antibiotics and herbicides, because of shikimic acid pathway is only present in plants and microorganisms, completely absent in mammals, fish, birds, reptiles, and insects. The results demonstrate that the binding of substrates to EPSPs causes a conformational change from an open form to a closed form, that might be important for designing of novel antimicrobial and herbicidal agents that block closure of the enzyme.

Interactions of subunits Asa2, Asa4 and Asa7 in the peripheral stalk of the mitochondrial ATP synthase of the chlorophycean alga Polytomella sp.

Mitochondrial F1FO-ATP synthase of chlorophycean algae is a complex partially embedded in the inner mitochondrial membrane that is isolated as a highly stable dimer of 1600kDa. It comprises 17 polypeptides, nine of which (subunits Asa1 to 9) are not present in classical mitochondrial ATP synthases and appear to be exclusive of the chlorophycean lineage. In particular, subunits Asa2, Asa4 and Asa7 seem to constitute a section of the peripheral stalk of the enzyme. Here, we over-expressed and purified subunits Asa2, Asa4 and Asa7 and the corresponding amino-terminal and carboxy-terminal halves of Asa4 and Asa7 in order to explore their interactions in vitro, using immunochemical techniques, blue native electrophoresis and affinity chromatography. Asa4 and Asa7 interact strongly, mainly through their carboxy-terminal halves. Asa2 interacts with both Asa7 and Asa4, and also with subunit α in the F1 sector. The three Asa proteins form an Asa2/Asa4/Asa7 subcomplex. The entire Asa7 and the carboxy-terminal half of Asa4 seem to be instrumental in the interaction with Asa2. Based on these results and on computer-generated structural models of the three subunits, we propose a model for the Asa2/Asa4/Asa7 subcomplex and for its disposition in the peripheral stalk of the algal ATP synthase.

S-adenosyl homocysteine hydrolase (SAHH) accelerates flagellar regeneration in Dunaliella salina.

S-adenosylhomocysteine hydrolase (SAHH) is an enzyme, which catalyzes the hydrolysis of S-adenosylhomocysteine (SAH) which is formed after the donation of the methyl group of S-adenosylmethionine (SAM) to a methyl acceptor in methylation reaction. As a potent regulator of methylation, SAHH plays a critical role in methylation reaction in the cells. Here we cloned the SAHH gene from unicellular green alga Dunaliella salina (dsSAHH) and investigated its effects on flagellar regeneration of D. salina, and found that dsSAHH was upregulated both at the protein and the transcription levels during pH shock-triggered flagellar regeneration of D. salina. The flagellar regeneration was accelerated when dsSAHH was overexpressed, but it was inhibited by SAHH inhibitor 3-deazaadenosine (DZA). Moreover, a receptor for activated C kinase 1 from D. salina (dsRACK1), which was identified to interact with dsSAHH, was increased when dsSAHH was overexpressed in D. salina as shown by real-time PCR. The findings of this study suggest that dsSAHH may participate in the regulation of flagellar regeneration of D. salina.

Role of Lys281 in the Dunaliella salina (6-4) photolyase reaction.

His(354) and His(358), two highly conserved histidines in Xenopus laevis (6-4) photolyase [equivalent to His(401) and His(405), in Dunaliella salina (6-4) photolyase], are critical for photoreactivation. They act as a base and an acid, respectively. However, the remaining high repair activity when the pH value is higher than the pKa of histidine suggests the involvement of other basic amino acids in photoreactivation. According to the results of in vivo enzyme assay and three-dimension structural model of Dunaliella salina (6-4) photolyase we hypothesized that Lys(281) might be involved in the photoreactivation over the pH range from 10.0 to 11.0. To test this, we generated two mutant forms of the (6-4) photolyase, K281G and K281R mutant, by overlap extension polymerase chain reaction, and performed the enzyme assay with these mutants. From these results we conclude that the Lys(281), which is highly conserved in (6-4) photolyases, participates in the photoreactivation and acts as an acid to donate a proton to His(401) when the environmental pH is higher than the pKa value of histidine.

Molecular cloning and characterization of a vacuolar H+₋pyrophosphatase from Dunaliella viridis.

The halotolerant alga Dunaliella adapts to exceptionally high salinity and possesses efficient mechanisms for regulating intracellular Na(+). In plants, sequestration of Na(+) into the vacuole is driven by the electrochemical H(+) gradient generated by H(+) pumps, and this Na(+) sequestration is one mechanism that confers salt tolerance to plants. To investigate the role of vacuolar H(+) pumps in the salt tolerance of Dunaliella, we isolated the cDNA of the vacuolar proton-translocating inorganic pyrophosphatase (V-H(+)-PPase) from Dunaliella viridis. The DvVP cDNA is 2,984 bp in length, codes for a polypeptide of 762 amino acids and has 15 transmembrane domains. The DvVP protein is highly similar to V-H(+)-PPases from other green algae and higher plant species, in terms of its amino acid sequence and its transmembrane model. A phylogenetic analysis of V-H(+)-PPases revealed the close relationship of Dunaliella to green algal species of Charophyceae and land plants. The heterologous expression of DvVP in the yeast mutant G19 (Δena1-4) suppressed Na(+) hypersensitivity, and a GFP-fusion of DvVP localized to the vacuole membranes in yeast, indicating that DvVP encodes a functional V-H(+)-PPase. A northern blot analysis showed a decrease in the transcript abundance of DvVP at higher salinity in D. viridis cells, which is in contrast to the salt-induced upregulation of V-H(+)-PPase in some plants, suggesting that the expression of DvVP under salt stress may be regulated by different mechanisms in Dunaliella. This study not only enriched our knowledge about the biological functions of V-H(+)-PPases in different organisms but also improved our understanding of the molecular mechanism of salt tolerance in Dunaliella.

Characterization and alternative splicing of the complex I 19-kD subunit in Dunaliella salina: expression and mutual correlation of splice variants under diverse stresses.

Complex I is the first enzyme in the mitochondrial respiratory chain. It extracts energy from NADH, which is produced by the oxidation of sugars and fats, and traps the energy by virtue of a potential difference or voltage across the mitochondrial inner membrane. Herein, the genomic sequence and four splice variants encoding the complex I 19-kD subunit were isolated from Dunaliella salina. There were four transcripts coding for the complex I 19-kD subunit due to alternative splicing in algae, and the four transcripts were translated to two protein isoforms with varying C-terminals. We report the splicing pattern in the 3'-region of the D. salina 19-kD subunit, in which three of the exons (5, 6, and 7) could be alternatively spliced. Moreover, we found that four alternatively spliced variants were subject to coordinated transcription in response to different stresses by real-time quantitative PCR.

A highly active ubiquinol-cytochrome c reductase (bc1 complex) from the colorless alga Polytomella spp., a close relative of Chlamydomonas. Characterization of the heme binding site of cytochrome c1.

The alga Polytomella spp. offers extraordinary advantages in the preparation of mitochondria since it lacks chloroplasts and a cell wall. In this work the mitochondrial bc1 complex from Polytomella spp. was solubilized and purified by ion exchange chromatography. The complex was found to be composed of 10 polypeptides and exhibited high rates of ubiquinol-cytochrome c oxidoreductase activity (> 300 s-1) sensitive to antimycin and myxothiazol. The molecular mass of the bc1 complex from Polytomella spp. was assayed by gel filtration and estimated to be of 256,300 Da. Therefore, this complex exhibits the unique property of behaving as a monomer. Amino-terminal sequencing of cytochrome c1 identified 7 residues, from which a deoxyoligonucleotide was designed. A second deoxyoligonucleotide was constructed based on a highly conserved region of the c1 type cytochromes. With these probes, a fragment of the cytochrome c1 gene was amplified by polymerase chain reaction and sequenced. The deduced sequence of the apoprotein exhibited a consensus binding site CXXCH. The data suggest that the cytochrome c1 from Polytomella spp. differs from other protoctists like Crithidia and Euglena, i.e. it exhibits a heme binding domain structurally related to the bovine, yeast, and Neurospora c1 type cytochromes.