Abstract: Around the world, the frequency and intensity of droughts is increasing as a result of global climate change, with important consequences for the growth and survival of agricultural and native plant species. Understanding how plants respond to water stress is thus crucial for predicting the impacts of climate change on the crop productivity and ecosystem functioning. In contrast to the large number of studies assessing drought impacts on photosynthesis, relatively little attention has been devoted to understanding how mitochondrial respiratory metabolism is altered under water stress conditions. This review provides an overview of the impacts of water stress on mitochondrial respiration (R), combining studies at the whole-plant, individual organ, cellular and organelle levels. To establish whether there are clear patterns in the response of in vivo R to water stress, a wide range of root, leaf and whole-plant studies are reviewed. It is shown that water stress almost always inhibits R in actively growing roots and whole plants. However, in fully expanded, mature leaves the response is more variable, with water stress reducing R in near two-thirds of reported studies, with most of the remainder showing no change. Only a few studies reported increases in leaf R under severe water stress conditions. The mechanisms responsible for these variable responses are discussed. Importantly, the fact is highlighted that irrespective of whether drought increases or decreases respiration, overall the changes in R are minor compared with the large decreases in photosynthetic carbon gain in response to drought. Based on recent work highlighting the link between chloroplast and mitochondrial functions in leaves, we propose a model by which mitochondrial R enables survival and rapid recovery of productivity under water stress conditions. Finally, the effects of water stress on mitochondrial function, protein abundance and overall metabolism are reviewed.
Abstract: Actively respiring animal and plant tissues experience hypoxia because of mitochondrial O-2 consumption. Controlling oxygen balance is a critical issue that involves in mammals hypoxia-inducible factor (HIF) mediated transcriptional regulation, cytochrome oxidase (COX) subunit adjustment and nitric oxide (NO) as a mediator in vasodilatation and oxygen homeostasis. In plants, NO, mainly derived from nitrite, is also an important signalling molecule. We describe here a mechanism by which mitochondrial respiration is adjusted to prevent a tissue to reach anoxia. During pea seed germination, the internal atmosphere was strongly hypoxic due to very active mitochondrial respiration. There was no sign of fermentation, suggesting a down-regulation of O-2 consumption near anoxia. Mitochondria were found to finely regulate their surrounding O-2 level through a nitrite-dependent NO production, which was ascertained using electron paramagnetic resonance (EPR) spin trapping of NO within membranes. At low O-2, nitrite is reduced into NO, likely at complex III, and in turn reversibly inhibits COX, provoking a rise to a higher steady state level of oxygen. Since NO can be re-oxidized into nitrite chemically or by COX, a nitrite-NO pool is maintained, preventing mitochondrial anoxia. Such an evolutionarily conserved mechanism should have an important role for oxygen homeostasis in tissues undergoing hypoxia. (C) 2008 Elsevier B.V. All rights reserved.
Abstract: Seeds of higher plant are desiccation tolerant, which suggests that their mitochondria exhibit particular properties. Insight into the function of seed mitochondria, especially in legume and model plants, has been fostered by the development of proteomics. Seed mitochondria are functional at the onset of imbibition, and their integrity and performance systematically improves during germination. This suggests that repair and biogenesis mechanisms exist, and this is supported by morphological and biochemical evidence. Seed mitochondria generate and operate in a hypoxic environment. They accumulate stress proteins, such as a small heat-shock protein and a late embryogenesis abundant protein. The mitochondria of pea (Pisum sativum L.) seed also display a biased phospholipid composition likely to favour desiccation tolerance. These specific biochemical properties surely contribute to the remarkable tolerance of seed mitochondria to extreme temperatures. Recent progress towards the resolution of the seed mitochondrial proteome is discussed in light of the growing body of genomic data.
Abstract: Few organisms are able to withstand desiccation stress; however, desiccation tolerance is widespread among plant seeds. Survival without water relies on an array of mechanisms, including the accumulation of stress proteins such as the late embryogenesis abundant (LEA) proteins. These hydrophilic proteins are prominent in plant seeds but also found in desiccation-tolerant organisms. In spite of many theories and observations, LEA protein function remains unclear. Here, we show that LEAM, a mitochondrial LEA protein expressed in seeds, is a natively unfolded protein, which reversibly folds into a-helices upon desiccation. Structural modeling revealed an analogy with class A amphipathic helices of apolipoproteins that coat low-density lipoprotein particles in mammals. LEAM appears spontaneously modified by deamidation and oxidation of several residues that contribute to its structural features. LEAM interacts with membranes in the dry state and protects liposomes subjected to drying. The overall results provide strong evidence that LEAM protects the inner mitochondrial membrane during desiccation. According to sequence analyses of several homologous proteins from various desiccation-tolerant organisms, a similar protection mechanism likely acts with other types of cellular membranes.
Abstract: Theory suggests it should be difficult for asexual organisms to adapt to a changing environment because genetic diversity can only arise from mutations accumulating within direct antecedents and not through sexual exchange. In an asexual microinvertebrate, the bdelloid rotifer, we have observed a mechanism by which such organisms could acquire the diversity needed for adaptation. Gene copies most likely representing former alleles have diverged in function so that the proteins they encode play complementary roles in survival of dry conditions. One protein prevents desiccation-sensitive enzymes from aggregating during drying, whereas its counterpart does not have this activity, but is able to associate with phospholipid bilayers and is potentially involved in maintenance of membrane integrity. The functional divergence of former alleles observed here suggests that adoption of asexual reproduction could itself be an evolutionary mechanism for the generation of diversity.
Abstract: center dot Storage product accumulation in seeds of major crop species is limited by their low internal oxygen concentration. Adjustment of energy and storage metabolism to oxygen deficiency (hypoxia) in seeds is highly relevant for agriculture and biotechnology. However, the mechanisms of low-oxygen sensing and balancing remain a mystery. center dot Here, it is shown that normal hypoxia in seeds of soybean (Glycine max) and pea (Pisum sativum) triggers a nitrite-dependent increase in endogenous nitric oxide (NO) concentrations. NO, in turn, reduces the oxygen consumption of seeds, generating a localized decrease in both ATP availability and biosynthetic activity. Increasing oxygen availability reduces endogenous NO concentrations, thereby abolishing mitochondrial and metabolic inhibition. center dot This auto-regulatory and reversible oxygen balancing, via NO, avoids seed anoxia and suggests a key role for NO in regulating storage activity. This hypothesis is reinforced by changes in energy status (ATP: ADP ratio), steady-state metabolite concentrations and biosynthetic fluxes under NO treatment. center dot The proposed mechanism of low-oxygen sensing and balancing in plants offers the prospect of a new field of study in crop biotechnology.
Abstract: Most seeds are anhydrobiotes, relying on an array of protective and repair mechanisms, and seed mitochondria have previously been shown to harbor stress proteins probably involved in desiccation tolerance. Since temperature stress is a major issue for germinating seeds, the temperature response of pea ( Pisum sativum) seed mitochondria was examined in comparison with that of mitochondria from etiolated epicotyl, a desiccation-sensitive tissue. The functional analysis illustrated the remarkable temperature tolerance of seed mitochondria in response to both cold and heat stress. The mitochondria maintained a well-coupled respiration between -3.5 degrees C and 40 degrees C, while epicotyl mitochondria were not efficient below 0 degrees C and collapsed above 30 degrees C. Both mitochondria exhibited a similar Arrhenius break temperature at 7 degrees C, although they differed in phospholipid composition. Seed mitochondria had a lower phosphatidylethanolamine-to-phosphatidylcholine ratio, fewer unsaturated fatty acids, and appeared less susceptible to lipid peroxidation. They also accumulated large amounts of heat shock protein HSP22 and late-embryogenesis abundant protein PsLEAm. The combination of membrane composition and stress protein accumulation required for desiccation tolerance is expected to lead to an unusually wide temperature tolerance, contributing to the fitness of germinating seeds in adverse conditions. The unique oxidation of external NADH at low temperatures found with several types of mitochondria may play a central role in maintaining energy homeostasis during cold shock, a situation often encountered by sessile and ectothermic higher plants.
Abstract: Late-embryogenesis abundant (LEA) proteins are hydrophilic proteins that accumulate to a high level in desiccation-tolerant tissues an are thus prominent in seeds. They are expected to play a protective role during dehydration; however, functional evidence is scarce. We identified a LEA protein of group 3 (PsLEAm) that was localized within the matrix space of pea (Pisum sativum) seed mitochondria. PsLEAm revealed typical LEA features such as high hydrophilicity and repeated motifs, except for the N-terminal transit peptide. Most of the highly charged protein was predicted to fold into amphiphilic alpha-helixes. PsLEAm was expressed during late seed development and remained in the dry seed and throughout germination. Application of the stress hormone abscisic acid was found to reinduce the expression of PsLEAm transcripts during germination. PsLEAm could not be detected in vegetative tissues; however, its expression could be reinduced in leaves by severe water stress. The recombinant PsLEAm was shown to protect two mitochondrial matrix enzymes, fumarase and rhodanese, during drying in an in vitro assays The overall results constitute, to our knowledge, the first characterization of a LEA protein in mitochondria and experimental evidence for a beneficial role of a LEA protein with respect to proteins during desiccation.
Abstract: Germination is an energy-demanding process that requires the operation of mitochondria, which must survive desiccation in the quiescent seed and become rapidly functional after imbibition to meet the ATP demand. The relationship between germination and mitochondrial performance was addressed by analysing the properties of mitochondria isolated from control, primed and aged pea (Pisum sativum L.) seeds. Mitochondria were isolated and purified at early stages of germination (before radicle protrusion), and their oxidative properties, membrane integrity and ultrastructure were examined. Mitochondria isolated after 12 h of imbibition readily oxidized exogenous NADH and Krebs cycle substrates at high rates. However, their phosphorylation efficiency was restricted by poor membrane integrity. After 22 h from the beginning of imbibition, purified seed mitochondria had intact outer membranes and oxidized the substrates at slightly lower rates, but with higher respiratory control (improved capacity for phosphorylation). Purified seed mitochondria were always found to be deficient in endogenous NAD, although the organelles were capable of importing and retaining the cofactor. While the priming treatment appeared to slightly increase the performance of mitochondria, seed deterioration by accelerated ageing strongly affected the oxidative properties of mitochondria, which were badly impaired in ATP production. Outer and inner membrane integrity was identified as the primary target for desiccation and ageing stress. A link between mitochondrial function and seed quality was also corroborated by respiration measurements of seed fragments at the onset of imbibition.
Abstract: Dimethyl disulfide exerts insecticidal neurotoxicity through mitochondrial dysfunction and activation of insect K-ATP channels. J Neurophysiol 90: 259-270, 2003; 10.1152/ jn. 01096.2002. The plant-derived insecticides have introduced a new concept in insecticide research. In response to insect attacks, some plants can release volatile sulfur compounds such as dimethyl disulfide (DMDS) in the atmosphere, which are lethal for the generalist insects. We demonstrate that DMDS induced an uncommon complex neurotoxic activity. The studies of in vivo toxicity of DMDS in three insect species and mice indicated a highest bioactivity for insects. Although DMDS did not alter the electrophysiological properties of the cockroach Periplaneta americana giant axon, it affected the synaptic transmission at the presynaptic level resulting in an inhibition of the neurotransmitter release. Whole cell patch-clamp experiments performed on cockroach cultured dorsal unpaired median ( DUM) neurons revealed a dose-dependent hyperpolarization induced by DMDS associated with a decrease in the input resistance and the disappearance of action potentials. The hyperpolarization was inhibited by glibenclamide and tolbutamide, and was dependent on intracellular ATP concentration, demonstrating a neurotoxicity via the activation of K ATP channels. Finally, the same effects observed with oligomycin, 2,4-dinitrophenol, and KCN together with the studies of DMDS toxicity on isolated mitochondria confirmed an unusual action occurring through an inhibition of the mitochondrial respiratory chain complex IV (cytochrome oxydase). This DMDS-induced inhibition of complex IV subsequently decreased the intracellular ATP concentration, which thereby activated neuronal K ATP channels mediating membrane hyperpolarization and reduction of neuronal activity.
Abstract: Two new xanthones, caledonixanthone M 1 and caloxanthone L 2, and one new acid, caledonic acid 6 were isolated from the hexane-soluble extract of the stem bark of Calophyllum caledonicum. In the course of this phytochemical study, seven other known compounds - calothwaitesixanthone, calozeyloxanthone, allanxanthone, isoapetalic acid 3, calolongic acid 4, apetalic acid 5 and isocalolongic acid 7 - were isolated. Their antifungal activity against the growth of the human pathogenic fungus Aspergillus fumigatus was then investigated. The results indicated that the crude extract, calolongic acid 4 and isocalolongic acid 7 exhibited strong inhibitory effects with MIC80 values of 8, 4, 2 mug/mL, respectively. Besides, calolongic acid 4, its lactone derivative 4a and isocalolongic acid 7 markedly reduced the respiration of pea seed mitochondria.
Abstract: The NADPH/NADP-thioredoxin (Trx) reductase (NTR)/Trx system (NTS) is a redox system that plays a posttranslational regulatory role by reducing protein targets involved in crucial cellular processes in microorganisms and animals. In plants, the system includes several h type Trx isoforms and has been shown to intervene in reserve mobilization during early seedling growth of cereals. To determine whether NTS was operational during germination of legume seeds and which Trx h isoforms could be implicated, Trx h isoforms expression was monitored in germinating pea (Pisum sativum cv Baccara) seeds, together with the amount of NTR and NADPH. Two new isoforms were identified: Trx h3, similar to the two isoforms already described in pea but not expressed in seeds; and the more divergent isoform, Trx h4. Active recombinant proteins were produced in Escherichia coli and used to raise specific antibodies. The expression of new isoforms was analyzed at both mRNA and protein levels. The lack of correlation between mRNA and protein abundances suggests the occurrence of posttranscriptional regulation. Trx h3 protein amount remained constant in both axes and cotyledons of dry and imbibed seeds but then decreased 2 d after radicle protrusion. In contrast, Trx h4 was only expressed in axes of dry and imbibed seeds but not in germinated seeds or in seedlings, therefore appearing as closely linked to germination. The presence of NTR and NADPH in seeds suggests that NTS could be functional during germination. The possible role of Trx h3 and h4 in this context is discussed.
Abstract: To expand the functional analysis of plant mitochondria, we have undertaken the building of the proteome of pea mitochondria purified from leaves (green and etiolated), roots and seeds. In the first stage, we focused our proteomic exploration on the soluble protein complement of the green leaf mitochondria. We used traditional two-dimensional polyacrylamide gel electrophoresis, in combination with size exclusion chromatography as a third dimension, to identify the major proteins and further resolve their macromolecular complexity. The two-dimensional map of soluble proteins of green leaf mitochondria revealed 433 spots (with Coomassie blue staining) and around 73% of the proteins (in mass) were identified using three different approaches: Edman degradation, matrix-assisted laser desorption/ionization mass spectrometry and electrospray ionization tandem mass spectrometry. Quite a lot of the polypeptides were present in multiforms which indicated the presence of isoforms or the occurrence of post-translational modifications. Among these proteins, we uncovered an abundant family that was identified as aldehyde dehydrogenases, representing approximately 7.5% of the soluble proteins. The comparative analysis of soluble mitochondrial proteomes led to the identification of a number of proteins which were specifically present in root or in seed mitochondria, thus revealing the impact of tissue differentiation at the mitochondrial level.
Abstract: Implication of the ubiquitous, highly conserved, Ca2+ sensor calmodulin (CaM) in pea seed germination has been investigated. Mass spectrometry analysis of purified CaM revealed the coexistence in seeds of three protein isoforms, diverging from each other by single amino acid substitution in the N-terminal alpha-helix. CaM was shown to be encoded by a small multigenic family, and full-length cDNAs of the three isoforms (PsCaM1, 2 and 3) were isolated to allow the design of specific primers in more divergent 5 and 3 untranslated regions. Expression studies, performed by semiquantitative RT-PCR, demonstrated differential expression patterns of the three transcripts during germination. PsCaM1 and 2 were detected at different levels in dry axes and cotyledons, and they accumulated during imbibition and prior to radicle protrusion. In contrast, PsCaM3 appeared only upon radicle protrusion, then gradually increased in both tissues. To characterise the biochemical properties of the CaM isoforms, functional analyses were conducted in vitro using recombinant Strep-tagged proteins (CaM1-ST, CaM2-ST and CaM3-ST) expressed in Escherichia coli. Gel mobility shift assays revealed that CaM1-ST exhibited a stoichiometric binding of a synthetic amphiphilic CaM kinase II peptide while CaM2-ST and CaM3-ST affinities for the same peptide were reduced. Affinity differences were also observed for CaM isoform binding to Trp-3, an idealised helical CaM-binding peptide. However, the three proteins activated in the same way the CaM-dependent pea NAD kinase. Finally, the significance of the single substitutions upon CaM interaction with its targets is discussed in a structural context.
Abstract: Fatty acid and lipoic acid biosynthesis were investigated in plant mitochondria. Although the mitochondria lack acetyl-CoA carboxylase, our experiments reveal that they contain the enzymatic equipment necessary to transform malonate into the two main building units for fatty acid synthesis: malonyl- and acetyl-acyl carrier protein (ACP), We demonstrated, by a new method based on a complementary use of high performance liquid chromatography and mass spectrometry, that the soluble mitochondrial fatty-acid synthase produces mainly three predominant acyl-ACPs as follows: octanoyl(C8)-, hexadecanoyl(C16)-, and octadecanoyl (C18)-ACP. Octanoate production is of primary interest since it has been postulated long ago to be a precursor of lipoic acid. By using a recombinant H apoprotein mutant as a potential acceptor for newly synthesized lipoic acid, we were able to detect limited amounts of lipoylated H protein in the presence of malonate, several sulfur donors, and cofactors, Finally, we present a scheme outlining the new biochemical pathway of fatty acid and lipoic acid synthesis in plant mitochondria.
Abstract: The glycine decarboxylase complex consists of four different component enzymes (P-, H-, T- and L-proteins). The 14-kDa lipoamide-containing H-protein plays a pivotal role in the complete sequence of reactions as its prosthetic group (lipoic acid) interacts successively with the three other components of the complex and undergoes a cycle of reductive methylamination, methylamine transfer and electron transfer. With the aim to understand the interaction between the H-protein and its different partners, we have previously determined the crystal structure of the oxidized and methylaminated forms of the H-protein. In the present study, we have crystallized the H-protein in its reduced state and the L-protein (lipoamide dehydrogenase or dihydrolipoamide dehydrogenase). The L-protein has been overexpressed in Escherichia coli and refolded from inclusion bodies in an active form. Crystals were obtained from the refolded L-protein and the structure has been determined by X-ray crystallography. This first crystal structure of a plant dihydrolipoamide dehydrogenase is similar to other known dihydrolipoamide dehydrogenase structures. The crystal structure of the H-protein in its reduced form has been determined and compared to the structure of the other forms of the protein. It is isomorphous to the structure of the oxidized form. In contrast with methylaminated H-protein where the loaded lipoamide arm was locked into a cavity of the protein, the reduced lipoamide arm appeared freely exposed to the solvent. Such a freedom is required to allow its targeting inside the hollow active site of L-protein. Our results strongly suggest that a direct interaction between the H- and L-proteins is not necessary for the reoxidation of the reduced lipoamide arm bound to the H-protein. This hypothesis is supported by biochemical data [Neuburger, M., Polidori, A.M., Pietre, E., Faure, M., Jourdain, A., Bourguignon, J., Pucci, B. & Douce, R. (2000) Eur. J. Biochem. 267, 2882-2889] and by small angle X-ray scattering experiments reported herein.
Abstract: The mitochondrial glycine decarboxylase complex (GDC) consists of four component enzymes (P, H, T, and L proteins) involved in the breakdown of glycine. in order to investigate structural interactions involved in the stabilization of the methylamine-loaded H protein (a transient species in the GDC reaction), we designed several mutants of H apoprotein. Structural analysis of the wild-type and mutants of H apoprotein emphasized the necessity to carefully assess, by biophysical techniques, the correct folding of mutated proteins prior to investigate their biochemical properties. The correctly folded wild-type and mutants of H apoprotein were in vitro lipoylated and then characterized in the context of GDC reaction by studying the reconstituted complex and partial reactions. We showed that Val(62) and Ala(64), surrounding the lipoyl-lysine, play an important role in the molecular events that govern the reaction between P and H protein but do not intervene in the recognition of the binding site of Lipoic acid by lipoyl ligase, The biochemical results obtained with the HE14A mutant of H protein pointed out the major role of the Glu(14) amino acid residue in the GDC catalysis and highlighted the importance of the ionic and hydrogen bounds in the hydrophobic cleft of H protein for the stabilization of the methylamine-loaded lipoyl arm.
Abstract: The flavoprotein component (SiR-FP) of the sulfite reductase from Escherichia coli is an octamer containing one FAD and one FMN per polypeptide chain. SiR-FP60, a SiR-FP fragment starting with alanine-52, was overexpressed in E. coli and purified as a monomer. The N-terminal part of the native protein contains thus all the determinants required for the polymerization. SiR-FP60 retains both FAD and FMN with comparable contributions of the two flavins and the catalytic properties of SIR-FP. Thus, SiR-FP60 can be considered as a reliable simplified model of the sulfite reductase flavoprotein component. The formation and the stabilization of the neutral FMN semiquinone is thermodynamically favorable in SiR-FP60 upon reduction with photoreduced deazaflavin, dithionite, or NADPH. Generation of FMNH. is explained from a disproportionation of electrons between the reduced and oxidized FMN moieties during an intermolecular reaction, as shown with SiR-FP23, the FMN-binding domain of SiR-FP. The neutral FAD semiquinone can be observed only within SiR-FP43, the isolated FAD-binding domain. NADPH was used as a titrant or in excess to demonstrate that electron transfer is possible only because the FMN cofactor is coupled to FAD as an electron acceptor in the protein. The electron distribution within the various reduced forms of SiR-FP60 has been compared with that of the reduced forms of cytochrome P450 reductase, bacterial cytochrome P450, and nitric-oxide synthase. Despite the conservation of the bi-flavin-domain structure between these proteins over evolutionary time, each of them provides significantly different flavin reactivities.
Abstract: We have isolated and characterized a genomic clone encoding the 41 kDa monomer T-protein. This gene called gdcT spans approximately 3 kbp and is composed of four exons interrupted by three introns (321, 691 and 114 bp). The splice sites for donor and acceptor are in agreement with the canonical GT/AG rule. Primer extension strongly suggests the presence of two major transcription start sites. The first transcription start site around 43 bases downstream of a putative TATA box was assigned the +1 position. The second (+31) is not correlated with a putative TATA box, but revealed a pyrimidine-rich region which is very similar to the initiator element. Sequence analysis of the 5'-upstream region of the gene reveals three consensus regions found in the nuclear genes encoding the chloroplastic proteins of ribulose-1,5-bisphosphate carboxylase (rbcS) and the chlorophyll a/b-binding protein (cab) such as an AT-rich sequence localized at -539 to -530, a box II core sequence GGTTAA (-123 to -118) and between -364 and -354 a tandem GATA motif. These elements are known to be involved respectively in the regulation of light-responsiveness and cell-type specificity expression of plant genes. Gel shift assays indicate that the box II core sequence could bind protein nuclear factors similar to the trans-acting factor which interact with corresponding promoter region of rbcS gene.
Abstract: In pea leaves, the synthesis of 7,8-dihydropteroate a primary step in folate synthesis, was only detected in mitochondria. This reaction is catalyzed by a bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase enzyme, which represented 0.04-0.06% of the matrix proteins. The enzyme had a native mel. wt of 280-300 kDa and was made up of identical subunits of 53 kDa. The reaction catalyzed by the 7,8-dihydropteroate synthase domain of the protein was Mg2+-dependent and behaved like a random bireactant system. The related cDNA contained an open reading frame of 1545 bp and the deduced amino acid sequence corresponded to a polypeptide of 515 residues with a calculated M(r) of 56 454 Da. Comparison of the deduced amino acid sequence with the N-terminal sequence of the purified protein indicated that the plant enzyme is synthesized with a putative mitochondrial transit peptide of 28 amino acids, The calculated M(r) of the mature protein was 53 450 Da. Southern blot experiments suggested that a single-copy gene codes for the enzyme. This result, together with the facts that the protein is synthesized with a mitochondrial transit peptide and that the activity was only detected in mitochondria, strongly supports the view that mitochondria is the major (unique?) site of 7,8-dihydropteroate synthesis in higher plant cells.
Abstract: The flavoprotein component (SiR-FP) of the sulfite reductase from Escherichia coli is an octamer containing one FAD and one FMN as cofactors per polypeptide chain. We have constructed an expression vector containing the DNA fragment encoding for the FMN-binding domain of SiR-FP. The overexpressed protein (SiR-FP23) was purified as a partially flavin-depleted polymer. It could incorporate FMN exclusively upon flavin reconstitution to reach a maximum flavin content of 1.2 per polypeptide chain. Moreover, the protein could stabilize a neutral air-stable semiquinone radical over a wide range of pHs. During photoreduction, the flavin radical accumulated first, followed by the fully reduced state. The redox potentials, determined at room temperature [E-1' (FMNH./FMN) = -130 +/- 10 mV and E-2' (FMNH2/FMNH.) = -335 +/- 10 mV], were very close to those previously reported for Salmonella typhimurium SiR-FP [Ostrowski, J., Barber, M. J., Rueger, D. C., Miller, B. E., Siegel, L. M., & Kredich, N. M. (1989) J. Biol. Chem. 264, 15796-15808]. Both the radical and fully reduced forms of SiR-FP23 were able to transfer their electrons to cytochrome c quantitatively. Altogether, the results presented herein demonstrate that the N-terminal end of E. coli SiR-FP forms the FT;IN-binding domain: It folds independently, thus retaining the chemical properties of the bound FMN, and provides a good model of the FAD-depleted form of native SiR-FP. Moreover, the FMN prosthetic group in SiR-FP23 and native SiR-FP is compared to that of cytochrome P450 reductase and bacterial cytochrome P450, which also contain one FAD and one FMN per polypeptide chain.
Abstract: The glycine decarboxylase complex consists of four different component enzymes (P-, H-, T-and L-proteins). The 14-kDa lipoamide-containing H-protein plays a pivotal role in the complete sequence of reactions since its prosthetic group (lipoic acid) interacts successively with the three other components of the complex and undergoes a cycle of reductive methylamination, methylamine transfer and electron transfer. The X-ray crystal structure of different forms of the H-protein has shown a unique conformation of the protein. This leads to the hypothesis of a three-dimensional recognition of the H-protein by the other components of the system and also by the ligase which lipoylates the H-protein. Striking structural similarities are observed between the H-protein and other lipoate domains of 2-oxo acid dehydrogenases and with the biotin carrier protein of acetyl-CoA carboxylase. In the H-protein, the lipoamide arm is free to move in the solvent when oxidized but is pivoted and tightly bound into a cleft at the protein surface when methylamine-loaded. This implies that the H-protein and the T-component form a stable complex during the catalytic transfer of the methylene unit to the tetrahydrofolate cofactor of the T-protein, This complex has been detected by small angle scattering experiments. In conclusion, in the glycine decarboxylase system, the lipoamide arm does not swing freely from one catalytic site to another as was proposed in other systems.
Abstract: A synthetic gene encoding the entire mature H protein of the glycine decarboxylase complex from pea (Pisum sativum L.) was constructed and expressed in Escherichia coli. The recombinant H protein, which after the induction period constituted more than half of the E. coli protein, was found in a soluble form, Activity measurements and mass-spectrometry analysis of the purified protein showed that, in the absence or presence of 5[3-(1,2)-dithiolanyl]pentanoic acid (lipoic acid) in the culture medium, recombinant H protein could be produced as the unlipoylated apoform or as the lipoylated form, respectively. Addition of chloramphenicol to the culture medium after induction increased the proportion of lipoylated H protein. High rates of lipoylation of the H apoprotein were measured in vivo and in vitro, revealing that the recombinant pea H protein was an excellent substrate for the E. coli lipoyl-ligase. The three-dimensional structure of the recombinant H apoprotein was determined at a 0.25-nm resolution. It was almost identical to the structure of the native pea leaf enzyme, which indicates that the recombinant protein folds properly in E. coli and that the lipoyl-ligase recognizes a three-dimensional structure in order to add lipoic acid to its specific lysine residue. It is postulated that the high level of expression and lipoylation of recombinant H protein may be due to the protein retaining the structure of the original enzyme.
Abstract: The expression of the genes encoding the four proteins (P, H, T, and L) of glycine decarboxylase, a multienzymatic complex involved in the mitochondrial step of the photorespiration pathway, was examined during pea (Pisum sativum) leaf development in comparison with ribulose-1,5-bisphosphate carboxylase/oxygenase. Mitochondria from the primary leaf were isolated at several well-defined stages of development. Their capacity to oxidize glycine was negligible during the earlier stages but increased dramatically once the leaflet opened. This was correlated with the accumulation of the glycine decarboxylase complex (CDC) proteins, which was shown to occur in preexisting mitochondria, producing an increase in their density. The transcription of the CDC genes was coordinated and occurred early, with a peak at 7 d, a stage at which mitochondria are unable to oxidize glycine. This implies the existence of posttranscriptional control of gene expression. The comparison of the expression patterns of the genes encoding specific proteins of CDC with that of rbcS genes suggests a common regulation scheme that is related to light induction. However, ribulose-1,5-bisphosphate carboxylase/oxygenase is present in the chloroplast well before GDC fills the mitochondria, suggesting that the setup of photorespiration occurs in cells already engaged in active photosynthesis.
Abstract: In the cyanobacterium Synechocystis sp, PCC 6803, there are four acyl-lipid desaturases that are, respectively, specific to the Delta 6, Delta 9, Delta 12 and omega 3 positions of fatty acids. The desA gene for the Delta 12 acyl-lipid desaturase was modified by site-directed mutagenesis, such that four of the histidine residues that are conserved in the four desaturases and one histidine residue that is not conserved were replaced by arginine, and the mutated desA genes were overexpressed in Escherichia coli. All of these mutations eliminated the Delta 12 desaturase activity. These results demonstrate that the five histidine residues are essential for the activity of the Delta 12 desaturase, perhaps by providing the ligands for the catalytic Fe center.
Abstract: H-protein is the lipoyl-protein component of the glycine decarboxylase complex, which catalyses, with serine hydroxymethyltransferase, the mitochondrial step of photorespiration in plants. We have isolated and characterized the gene (gdcH) encoding the H-protein in pea (Pisum sativum L.). The H-protein gene is distributed in a stretch of about 1.55 kbp and contains three introns (75, 64 and 185 bp) located in the coding region. No intervening sequences were detected in the 5' and 3' non-coding regions. This intron-exon structure contrasts with the preliminary H-protein gene structures reported for human and chicken, where these genes (dispersed on 13 and 8 kbp genomic fragments respectively) are composed of five highly conserved exons and are interrupted by four long introns. Two main transcription sites were detected by primer extension of RNA. The first transcriptional initiation site was assigned the +1 position and correlated with a putative TATA box located at position -26. The second transcriptional start site was not correlated with a putative TATA box, but may be regulated by an 'initiator' element described by Smale & Baltimore [(1989) Cell (Cambridge, Mass.) 57, 103-113] which contains, within itself, the transcription start site. The presence of two potential promoters may be related to the specialized overexpression pattern of H-protein in leaves, in order to support photorespiration.
Abstract: L-protein is the dihydrolipoamide dehydrogenase component of the glycine decarboxylase complex which catalyses, with serine hydroxymethyltransferase, the mitochondrial step of photorespiration. We have isolated and characterized a cDNA from a gamma gt11 pea library encoding the complete L-protein precursor. The derived amino acid sequence indicates that the protein precursor consists of 501 amino acid residues, including a presequence peptide of 31 amino acid residues. The N-terminal sequence of the first 18 amino acid residues of the purified L-protein confirms the identity of the cDNA. Alignment of the deduced amino acid sequence of L-protein with human, porcine and yeast dihydrolipoamide dehydrogenase sequences reveals high similarity (70% in each case), indicating that this enzyme is highly conserved. Most of the residues located in or near the active sites remain unchanged. The results described in the present paper strongly suggest that, in higher plants, a unique dihydrolipoamide dehydrogenase is a component of different mitochondrial enzyme complexes. Confidence in this conclusion comes from the following considerations. First, after fractionation of a matrix extract of pea-leaf mitochondria by gel-permeation chromatography followed by gel electrophoresis and Western-blot analysis, it was shown that polyclonal antibodies raised against the L-protein of the glycine-cleavage system recognized proteins with an M(r) of about 60 000 in different elution peaks where dihydrolipoamide dehydrogenase activity has been detected. Second, Northern-blot analysis of RNA from different tissues such as leaf, stem, root and seed, using L-protein cDNA as a probe, indicates that the mRNA of the dihydrolipoamide dehydrogenase accumulates to high levels in all tissues. In contrast, the H-protein (a specific protein component of the glycine-cleavage system) is known to be expressed primarily in leaves. Third, Southern-blot analysis indicated that the gene coding for L-protein in pea is most likely to be present in a single copy/haploid genome.
