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Biomedical subjects

R Douce

Publications and source records attributed to R Douce.

At least 37 records · Page 2Linked to original sources
PubMed1999

Biochemical dissection of photorespiration.

Progress has been made in the understanding of photorespiration and related proteins (Rubisco, glycolate oxidase and glycine decarboxylase) in the context of recent structural information. Numerous shuttles exist to support transamination, ammonia refixation and the supply or export of reductants generated or consumed (via malate-oxaloacetate shuttles) in the photorespiratory pathway. A porin-like channel that is anion selective represents the major permeability pathway of the peroxisomal membrane.

Alcohol Oxidoreductases↗
PubMed1999

Glycine and serine catabolism in non-photosynthetic higher plant cells: their role in C1 metabolism.

Glycine and serine are two interconvertible amino acids that play an important role in C1 metabolism. Using 13C NMR and various 13C-labelled substrates, we studied the catabolism of each of these amino acids in non-photosynthetic sycamore cambial cells. On one hand, we observed a rapid glycine catabolism that involved glycine oxidation by the mitochondrial glycine decarboxylase (GDC) system. The methylenetetra- hydrofolate (CH2-THF) produced during this reaction did not equilibrate with the overall CH2-THF pool, but was almost totally recycled by the mitochondrial serine hydroxymethyltransferase (SHMT) for the synthesis of one serine from a second molecule of glycine. Glycine, in contrast to serine, was a poor source of C1 units for the synthesis of methionine. On the other hand, catabolism of serine was about three times lower than catabolism of glycine. Part of this catabolism presumably involved the glycolytic pathway. However, the largest part (about two-thirds) involved serine-to-glycine conversion by cytosolic SHMT, then glycine oxidation by GDC. The availability of cytosolic THF for the initial SHMT reaction is possibly the limiting factor of this catabolic pathway. These data support the view that serine catabolism in plants is essentially connected to C1 metabolism. The glycine formed during this process is rapidly oxidized by the mitochondrial GDC-SHMT enzymatic system, which is therefore required in all plant tissues.

Journal Article↗
PubMed1999

Biochemical and topological properties of type A MGDG synthase, a spinach chloroplast envelope enzyme catalyzing the synthesis of both prokaryotic and eukaryotic MGDG.

MGDG synthase, the enzyme that catalyzes the synthesis of the major chloroplast membrane lipid monogalactosyldiacylglycerol (MGDG), is encoded by a multigenic family. We have analyzed the biochemical properties, subcellular localization and membrane topology of a spinach chloroplast MGDG synthase, a representative member of the type A family from Spinacia oleracea (soMGD A), using a recombinant protein that was functionally overexpressed in Escherichia coli and specific polyclonal antibodies. We demonstrated that soMGD A could catalyze the synthesis of both 'prokaryotic' and 'eukaryotic' MGDG molecular species in vitro, with a selectivity for diacylglycerol similar to that of purified chloroplast envelope MGDG synthase activity. Furthermore, soMGD A was shown to be sensitive to chemical reagents (dithiothreitol, N-ethylmaleimide and o-phenanthroline) known to affect MGDG synthesis by the partially purified enzyme, as well as in isolated chloroplast envelope membranes. In spinach chloroplasts, soMGD A was localized by Western blot analysis in the inner envelope membrane. Topological studies demonstrated that soMGD A is a monotopic enzyme, embedded within one leaflet of the inner envelope membrane from spinach chloroplasts, a structure which may involve amphipathic alpha helices. We further demonstrated that in vitro, soMGD A precursor is imported and processed to its correct mature form in intact chloroplasts. These results show that soMGD A corresponds to a mature polypeptide of approximately 45 kDa. In addition, inactivation kinetics after gamma-ray irradiation strongly suggest that both native chloroplast envelope MGDG synthase and recombinant soMGD A have a functional molecular mass of 95-100 kDa, indicating that they are probably active as homodimers made of two 45-kDa subunits. This study suggests that, in spite of the growing evidence that MGDG synthesis is catalyzed by a multigenic family of enzymes, in spinach leaves both prokaryotic and eukaryotic MGDG syntheses could be attributable to a unique dimeric enzyme, provided that diacylglycerol is transported from the outer membrane to the inner membrane of the chloroplast envelope.

Amino Acid Sequence↗
PubMed1998

Purification and properties of the chloroplastic form of biotin holocarboxylase synthetase from Arabidopsis thaliana overexpressed in Escherichia coli.

Holocarboxylase synthetases (HCSs) are key enzymes in biotin utilisation in both prokaryotes and eukaryotes. In a previous study, we demonstrated that, in plants, HCS activity is localised in cytosol, chloroplasts and mitochondria. We also described the cloning and sequencing of a full-length cDNA encoding an Arabidopsis thaliana HCS isoform with a putative organelle-transit peptide. In the study reported here, this cDNA was used to construct an overproducing Escherichia coli strain. The recombinant enzyme was isolated using an efficient three-step purification procedure. Polyclonal antibodies raised against pure HCS were produced to elucidate the subcellular localisation of this protein. Immunodetection carried out by Western blotting of isolated pea leaf subcellular compartments specifically revealed a single polypeptide that was ascribed to the chloroplast compartment. Immunocytochemistry of thin-cut sections from tobacco leaves, transformed by the complete coding sequence of A. thaliana HCS cDNA via Agrobacterium tumefaciens, confirmed that the enzyme encoded by this cDNA is the chloroplastic isoform. Moreover, physicochemical, biochemical and kinetic properties of the pure recombinant HCS were determined. The native recombinant enzyme is a 37-kDa monomer. In contrast to the major part of HCS activity measured in leaf extracts, the recombinant chloroplastic enzyme did not require addition of Mg2+ to be fully active, but was substantially inhibited by EDTA. This suggested that the chloroplastic HCS may contain a tightly-bound divalent cation required for enzyme activity. The recombinant enzyme was able to biotinylate efficiently apo-biotin carboxyl carrier protein (BCCP) from E. coli and apo-methylcrotonoyl-CoA carboxylase (MCCase) from A. thaliana. Apparent Km values for the enzyme substrates D-biotin, ATP and apo-MCCase were found to be 130 nM, 4.4 microM and 32 microM, respectively. Steady-state kinetic analyses of the HCS-catalysed reaction were investigated with respect to reaction mechanism and inhibition by AMP, one of the end-products of the enzyme-catalysed reaction. Substrate interaction and product inhibition patterns indicated that ATP and D-biotin bind sequentially, in an ordered manner, to the enzyme and that ATP or D-biotin and apo-BCCP bind in ping-pong fashion.

Arabidopsis↗
PubMed1998

Allosteric activation of Arabidopsis threonine synthase by S-adenosylmethionine.

Plant threonine synthase, in contrast to its bacterial counterpart, is strongly stimulated by S-adenosylmethionine via a noncovalent interaction [Giovanelli et al. (1984) Plant. Physiol. 76, 285-292]. The mechanism of activation remained, however, largely unknown. To further characterize this unusual role for S-adenosylmethionine, the Arabidopsis thaliana threonine synthase was overexpressed in Escherichia coli, purified to homogeneity, and then used for kinetic and enzyme-bound pyridoxal 5'-phosphate fluorescence equilibrium-binding experiments. We observed that the activating effect of S-adenosylmethionine results from an 8-fold increase in the rate of catalysis and from a 25-fold decrease in the Km value for the O-phosphohomoserine substrate. The data can be well fitted by a kinetic model assuming binding of two S-adenosylmethionine molecules on the native enzyme. We suggest that the dramatic modifications of the enzyme kinetic properties originate most presumably from an allosteric and cooperative transition induced by S-adenosylmethionine. This transition occurs at a much faster rate in the presence of the substrate than in its absence.

Allosteric Site↗
PubMed1998

A loop deletion in the plant acetohydroxy acid isomeroreductase homodimer generates an active monomer with reduced stability and altered magnesium affinity.

Plant acetohydroxy acid isomeroreductase is a stable homodimer which catalyzes in the presence of magnesium an alkyl migration followed by a NADPH-dependent reduction. Since the enzyme exhibits no kinetic cooperativity either for its cofactor (NADPH and magnesium) or for its substrates, the reason for dimerization of this enzyme was not obvious. Recently, crystallographic studies [Biou, V., et al. (1997) EMBO J. 16, 3405-3415] revealed that the loop of residues 422-431 plays a major part in the dimer interface. To understand the role of the quaternary structure of the enzyme, we have deleted residues 423-430 and substituted Phe 431 for serine. This mutant was further overproduced in Escherichia coli, purified to homogeneity, and characterized. Gel filtration and thermodynamic experiments disclosed that this mutant behaves as an active monomer with reduced thermal stability. Furthermore, kinetic and fluorescence experiments showed that the behavior of the monomer with respect to magnesium was greatly altered. These results demonstrate the function of the quaternary structure of plant acetohydroxy acid isomeroreductase in the stabilization of the tertiary structure but also in the stabilization of a high-affinity magnesium binding site.

Alcohol Oxidoreductases↗
PubMed1998

Interactions between serine acetyltransferase and O-acetylserine (thiol) lyase in higher plants--structural and kinetic properties of the free and bound enzymes.

The last steps of cysteine synthesis in plants involve two consecutive enzymes. The first enzyme, serine acetyltransferase, catalyses the acetylation of L-serine in the presence of acetyl-CoA to form O-acetylserine. The second enzyme, O-acetylserine (thiol) lyase, converts O-acetylserine to L-cysteine in the presence of sulfide. We have, in the present work, over-produced in Escherichia coli harboring various type of plasmids, either a plant serine acetyltransferase or this enzyme with a plant O-acetylserine (thiol) lyase. The free recombinant serine acetyltransferase (subunit mass of 34 kDa) exhibited a high propensity to form high-molecular-mass aggregates and was found to be highly unstable in solution. However, these aggregates were prevented in the presence of O-acetylserine (thiol) lyase (subunit mass of 36 kDa). Under these conditions homotetrameric serine acetyltransferase associated with two molecules of homodimeric O-acetylserine (thiol) lyase to form a bienzyme complex (molecular mass approximately 300 kDa) called cysteine synthase containing 4 mol pyridoxal 5'-phosphate/mol complex. O-Acetylserine triggered the dissociation of the bienzyme complex, whereas sulfide counteracted the action of O-acetylserine. Protein-protein interactions within the bienzyme complex strongly modified the kinetic properties of plant serine acetyltransferase: there was a transition from a typical Michaelis-Menten model to a model displaying positive kinetic co-operativity with respect to serine and acetyl-CoA. On the other hand, the formation of the bienzyme complex resulted in a very dramatic decrease in the catalytic efficiency of bound O-acetylserine (thiol) lyase. The latter enzyme behaved as if it were a structural and/or regulatory subunit of serine acetyltransferase. Our results also indicated that bound serine acetyltransferase produces a build-up of O-acetylserine along the reaction path and that the full capacity for cysteine synthesis can only be achieved in the presence of a large excess of free O-acetylserine (thiol) lyase. These findings contradict the widely held belief that such a bienzyme complex is required to channel the metabolite intermediate O-acetylserine.

Acetyltransferases↗
PubMed1998

The specific features of methionine biosynthesis and metabolism in plants.

Plants, unlike other higher eukaryotes, possess all the necessary enzymatic equipment for de novo synthesis of methionine, an amino acid that supports additional roles than simply serving as a building block for protein synthesis. This is because methionine is the immediate precursor of S-adenosylmethionine (AdoMet), which plays numerous roles of being the major methyl-group donor in transmethylation reactions and an intermediate in the biosynthesis of polyamines and of the phytohormone ethylene. In addition, AdoMet has regulatory function in plants behaving as an allosteric activator of threonine synthase. Among the AdoMet-dependent reactions occurring in plants, methylation of cytosine residues in DNA has raised recent interest because impediment of this function alters plant morphology and induces homeotic alterations in flower organs. Also, AdoMet metabolism seems somehow implicated in plant growth via an as yet fully understood link with plant-growth hormones such as cytokinins and auxin and in plant pathogen interactions. Because of this central role in cellular metabolism, a precise knowledge of the biosynthetic pathways that are responsible for homeostatic regulation of methionine and AdoMet in plants has practical implications, particularly in herbicide design.

Cystathionine↗
PubMed1998

Cystathionine gamma-synthase from Arabidopsis thaliana: purification and biochemical characterization of the recombinant enzyme overexpressed in Escherichia coli.

Cystathionine gamma-synthase catalyses the first reaction specific for methionine biosynthesis in plants, the gamma-replacement of the phosphoryl substituent of O-phosphohomoserine by cysteine. A cDNA encoding cystathionine gamma-synthase from Arabidopsis thaliana has been cloned and used to overexpress the enzyme in Escherichia coli. The native recombinant enzyme is a homotetramer composed of 53 kDa subunits, each being tightly associated with one molecule of pyridoxal 5'-phosphate that binds at lysine-379 of the protein precursor. The replacement reaction follows a Ping Pong mechanism with a Vmax of 33.6 units/mg and Km values of 2.5 mM and 460 microM for O-phosphohomoserine and cysteine respectively. The protective effect of O-phosphohomoserine against enzyme inactivation by propargylglycine indicated that the Kd for the substrate is approx. 1/2500 of its Km value. Thus most of these biochemical properties are similar to those previously reported for plant and bacterial cystathionine gamma-synthases. However, the plant enzyme differs markedly from its enterobacterial counterparts because it catalyses a very faint gamma-elimination of O-phosphohomoserine in the absence of cysteine, this process being about 1/2700 as fast as the gamma-replacement reaction and approx. 1/1500 as fast as the gamma-elimination catalysed by the E. coli enzyme. This huge difference could be attributed to the inability of the A. thaliana cystathionine gamma-synthase to accumulate a long-wavelength-absorbing species that is characteristic for the efficient gamma-elimination reaction catalysed by the enterobacterial enzyme.

Alkynes↗
PubMed1998

Kinetic and mass spectrometric analyses of the interactions between plant acetohydroxy acid isomeroreductase and thiadiazole derivatives.

Plant acetohydroxy acid isomeroreductase (EC 1.1.1.86), the second enzyme of the branched chain amino acid biosynthetic pathway, has been submitted to high-throughput screening for herbicide discovery. We report here the discovery of a new class of compounds belonging to the thiadiazole family, which exhibit a strong inhibitory effect on this plant enzyme. Kinetic analyses revealed that these compounds act as either reversible or irreversible noncompetitive inhibitors of the plant enzyme. Reversibility or irreversibility of these compounds can be attributed to the nature of the additional groups of the thiadiazole ring favoring or not favoring the formation of a covalent adduct. Mass spectrometric experiments on the complex between an irreversible compound belonging to the thiadiazole family and the plant enzyme identified Cys498 as the binding site of the inhibitor.

Alcohol Oxidoreductases↗
PubMed1998

Transport, Compartmentation, and Metabolism of Homoserine in Higher Plant Cells. Carbon-13- and phosphorus-31-nuclear magnetic resonance studies Carbon-13- and Phosphorus-31-Nuclear Magnetic Resonance Studies

The transport, compartmentation, and metabolism of homoserine was characterized in two strains of meristematic higher plant cells, the dicotyledonous sycamore (Acer pseudoplatanus) and the monocotyledonous weed Echinochloa colonum. Homoserine is an intermediate in the synthesis of the aspartate-derived amino acids methionine, threonine (Thr), and isoleucine. Using 13C-nuclear magnetic resonance, we showed that homoserine actively entered the cells via a high-affinity proton-symport carrier (Km approximately 50-60 mum) at the maximum rate of 8 +/- 0.5 mumol h-1 g-1 cell wet weight, and in competition with serine or Thr. We could visualize the compartmentation of homoserine, and observed that it accumulated at a concentration 4 to 5 times higher in the cytoplasm than in the large vacuolar compartment. 31P-nuclear magnetic resonance permitted us to analyze the phosphorylation of homoserine. When sycamore cells were incubated with 100 mum homoserine, phosphohomoserine steadily accumulated in the cytoplasmic compartment over 24 h at the constant rate of 0.7 mumol h-1 g-1 cell wet weight, indicating that homoserine kinase was not inhibited in vivo by its product, phosphohomoserine. The rate of metabolism of phosphohomoserine was much lower (0.06 mumol h-1 g-1 cell wet weight) and essentially sustained Thr accumulation. Similarly, homoserine was actively incorporated by E. colonum cells. However, in contrast to what was seen in sycamore cells, large accumulations of Thr were observed, whereas the intracellular concentration of homoserine remained low, and phosphohomoserine did not accumulate. These differences with sycamore cells were attributed to the presence of a higher Thr synthase activity in this strain of monocot cells.

Journal Article↗
PubMed1998

The gene encoding T protein of the glycine decarboxylase complex involved in the mitochondrial step of the photorespiratory pathway in plants exhibits features of light-induced genes.

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.

Amino Acid Oxidoreductases↗
PubMed1998

Molecular characterization of the PEND protein, a novel bZIP protein present in the envelope membrane that is the site of nucleoid replication in developing plastids.

Plastid nucleoids are known to bind to the envelope membrane in developing chloroplasts. Here, plastid DNA is extensively replicated. We previously detected a DNA binding protein in the inner envelope membranes of developing plastids in pea and named it PEND (for plastid envelope DNA binding) protein. In this study, we report on the structure and molecular characterization of a cDNA for the PEND protein. As a result of screening cDNA libraries in lambdagt11 with one of the target sequences of the PEND protein as a probe, we obtained a clone (PD2) for a novel DNA binding protein consisting of 633 amino acid residues. Analysis of the N-terminal sequence of the purified PEND protein indicated that the transit peptide is just 16 residues long. The PEND protein was detected specifically in the plastid envelope membrane of young unopened leaf buds by immunoblot analysis. The PEND protein consists of a basic region plus zipper region, an unprecedented sextuple repeat region, and a putative membrane-spanning region. The basic region with a zipper region seems to have diverged from that of other plant transcription factors. In addition, the PEND protein could be a distant homolog of the trans-Golgi network integral membrane proteins. The PEND protein is therefore a novel type of DNA binding protein that binds to the membrane as an intrinsic membrane protein.

Amino Acid Sequence↗
PubMed1997

Biotin synthesis in higher plants: purification and characterization of bioB gene product equivalent from Arabidopsis thaliana overexpressed in Escherichia coli and its subcellular localization in pea leaf cells.

Biotin synthase catalyses the final step in the biotin biosynthetic pathway and is encoded by the bioB gene in Escherichia coli. To investigate the conversion of dethiobiotin to biotin in the plant kingdom, the cDNA encoding the bioB gene product equivalent from Arabidopsis thaliana was used to construct an E. coli overexpression strain. The purified A. thaliana bioB gene product is a homodimer (100 kDa) with a reddish color and has an absorbance spectrum characteristic of protein with [2Fe-2S] clusters. Its intracellular compartmentation in pea leaves discloses a unique polypeptide of 39 kDa within the matrix of mitochondria.

Arabidopsis↗
PubMed1997

The crystal structure of plant acetohydroxy acid isomeroreductase complexed with NADPH, two magnesium ions and a herbicidal transition state analog determined at 1.65 A resolution.

Acetohydroxy acid isomeroreductase catalyzes the conversion of acetohydroxy acids into dihydroxy valerates. This reaction is the second in the synthetic pathway of the essential branched side chain amino acids valine and isoleucine. Because this pathway is absent from animals, the enzymes involved in it are good targets for a systematic search for herbicides. The crystal structure of acetohydroxy acid isomeroreductase complexed with cofactor NADPH, Mg2+ ions and a competitive inhibitor with herbicidal activity, N-hydroxy-N-isopropyloxamate, was solved to 1.65 A resolution and refined to an R factor of 18.7% and an R free of 22.9%. The asymmetric unit shows two functional dimers related by non-crystallographic symmetry. The active site, nested at the interface between the NADPH-binding domain and the all-helical C-terminus domain, shows a situation analogous to the transition state. It contains two Mg2+ ions interacting with the inhibitor molecule and bridged by the carboxylate moiety of an aspartate residue. The inhibitor-binding site is well adjusted to it, with a hydrophobic pocket and a polar region. Only 24 amino acids are conserved among known acetohydroxy acid isomeroreductase sequences and all of these are located around the active site. Finally, a 140 amino acid region, present in plants but absent from other species, was found to make up most of the dimerization domain.

Alcohol Oxidoreductases↗
PubMed1997

Purification and characterization of a fusion protein of plant acetohydroxy acid synthase and acetohydroxy acid isomeroreductase.

The nucleotide sequence coding for the Arabidopsis thaliana acetohydroxy acid synthase was genetically fused in frame with the nucleotide sequence coding for the Spinacia oleracea acetohydroxy acid isomeroreductase and expressed in Escherichia coli. This construction allowed the production of large amounts of soluble fusion protein. The pure chimeric enzyme exhibits high acetohydroxy acid synthase and acetohydroxy acid isomeroreductase specific activities. Fusion and native enzymes exhibit similar Km values for their substrates and for most cofactors. Furthermore, whereas native plant acetohydroxy acid synthase is highly unstable, the stability of this enzyme in the fusion has been increased. Thus, the chimeric enzyme appears to be a useful tool for the determination of kinetic and structural properties of plant acetohydroxy acid synthase.

2-Acetolactate Mutase↗