Intrasteric inhibition in redox signalling: light activation of NADP-malate dehydrogenase

Chloroplast NADP-dependent malate dehydrogenase (NADP-MDH, EC 1.1.1.82) is inactive in the dark and activated in the light via a reduction of specific disulfides by thiol-disulfide interchange with thioredoxin, reduced by the photosynthetic electron transfer. Compared to the constitutively active NAD-dependent forms, NADP-MDH exhibits two regulatory disulfides per subunit, one located in an N-terminal extension and the other in a C-terminal extension. Convergent information gathered from biochemical, site-directed mutagenesis and structural approaches allowed to solve almost completely the activation mechanism. In the oxidized enzyme, the C-terminal extension is pulled back by the disulfide bridge toward the active-site cleft where the penultimate C-terminal glutamate interacts with one of the arginines involved in substrate binding, thus acting as an internal inhibitor obstructing the access of oxaloacetate. The N-terminal extensions are located at the subunit interface area and rigidify the overall structure of the dimer. Their reduction by reduced thioredoxin triggers a conformational change of the active site towards high-activity conformation, whereas the reduction of the C-terminal bridge expells the C-terminal end from the active site, thus opening the way for the substrate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sites of interaction of thioredoxin with sorghum NADP-malate dehydrogenase

The activation pathway of the chloroplastic NADP-dependent malate dehydrogenase (MDH) by reduced thioredoxin has been examined using a method based on the mechanism of thiol/disulfide interchanges, i.e. the transient formation of a mixed disulfide between the target and the reductant. This disulfide can be stabilized when each of the partners is mutated in the less reactive cysteine of the disulfide/dithiol pair. As NADP-MDH has two regulatory disulfides per monomer, four different single cysteine mutants were examined, two for the C-terminal bridge and two for the N-terminal bridge. The results clearly show that the nucleophilic attack of thioredoxin on the C-terminal bridge proceeds through the formation of a disulfide with the most external Cys377. The results are less clear-cut for the N-terminal cysteines and suggest that the Cys24-Cys207 disulfide bridge previously proposed to be an intermediary step in MDH activation can form only when the C-terminal disulfide is reduced.     

Primary structure of sorghum malate dehydrogenase (NADP) deduced from cDNA sequence. Homology with malate dehydrogenase (NAD)

Malate dehydrogenase (NADP) (NADP-MDH) is an important enzyme of the photosynthetic CO2 fixation pathway of C4 plants. We have isolated two clones from a sorghum lambda gt11 cDNA library (CM3, 932 bp, and CM7, 1441 bp). Nucleotide sequence analysis of the cDNAs CM3 and CM7 showed the existence of two NADP-MDH mRNA species encoding different enzyme subunits. Microsequencing of the N-terminus of the mature protein indicated that a specific cleavage of 13 amino acids occurred during the purification steps of the enzyme. The full-length cDNA CM7 contains a large open reading frame encoding an NH2-terminal transit peptide of 40 amino acids and a mature protein of 389 amino acids (42.207 kDa). Alignment of the NADP-MDH sequence with those of several malate dehydrogenases revealed some similarities with NAD-MDHs.     

Cloning and sequencing of the ornithine decarboxylase gene from Trypanosoma brucei. Implications for enzyme turnover and selective difluoromethylornithine inhibition

Ornithine decarboxylase of the African trypanosome Trypanosoma brucei brucei had an estimated native molecular weight of 100,000 by gel filtration and a subunit molecular weight of 45,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gene encoding this enzyme, present in a single copy in T. brucei, was identified by mouse ornithine decarboxylase cDNA under relatively stringent conditions of hybridization and subcloned in a 5.9-kilobase (kb) SstI fragment from a cosmid clone into the plasmid pUC 19. This clone encompassed a 2.8-kb SstII fragment that contained the entire T. brucei ornithine decarboxylase gene. The 2.8-kb SstII fragment hybridized to a 2.4-kb mRNA that presumably encodes the parasite enzyme. The 2.8-kb SstII fragment was partially sequenced and found to contain an open reading frame of 445 amino acids that has 61.5% homology with the corresponding sequence of the mouse enzyme. The only major discrepancies between the two enzymes are the addition of a 20-amino acid N-terminal peptide and the deletion of a 36-amino acid C-terminal peptide and the T. brucei ornithine decarboxylase. The C terminus has been postulated to be one of the structural factors associated with rapid in vivo turnover of mammalian ornithine decarboxylase. The absence of this C-terminal peptide in T. brucei ornithine decarboxylase predicts a slow turnover for the parasite enzyme in vivo, and this is supported by our experimental data. The lack of turnover of ornithine decarboxylase in trypanosomes may constitute the basis of selective antitrypanosomal action of the irreversible enzyme inhibitor DL-alpha-difluoromethylornithine.     

Purification and characterization of pea thioredoxin f expressed in Escherichia coli

The recently cloned cDNA for pea chloroplast thioredoxin f was used to produce, by PCR, a fragment coding for a protein lacking the transit peptide. This cDNA fragment was subcloned into a pET expression vector and used to transform E. coli cells. After induction with IPTG the transformed cells produce the protein, mainly in the soluble fraction of the broken cells. The recombinant thioredoxin f has been purified and used to raise antibodies and analysed for activity. The antibodies appear to be specific towards thioredoxin f and do not recognize other types of thioredoxin. The recombinant protein could activate two chloroplastic enzymes, namely NADP-dependent malate dehydrogenase (NADP-MDH) and fructose 1,6-bisphosphatase (FBPase), both using dithiothreitol as a chemical reductant and in a light-reconstituted/thylakoid assay. Recombinant pea thioredoxin f turned out to be an excellent catalyst for NADP-MDH activation, being the more efficient than a recombinant m-type thioredoxin of Chlamydomonas reinhardtii and the thioredoxin of E. coli. At the concentrations of thioredoxin used in the target enzyme activation assays only the recombinant thioredoxin f activated the FBPase.     

Primary structure of the light-dependent regulatory site of corn NADP-malate dehydrogenase

The light-activated NADP-malate dehydrogenase (NADP-MDH) catalyzes the reduction of oxaloacetate to malate in higher plant chloroplasts. This enzyme is regulated in vivo by the ferredoxin-thioredoxin system through redox reactions. NADP-MDH has been photoactivated in vitro in a chloroplast system reconstituted from the pure protein components and thylakoid membranes. Photoactivation was accompanied by the appearance of new thiol groups (followed by [14C]iodoacetate incorporation). 14C-Carboxymethylated NADP-MDH has been purified from the incubation mixture and its amino-terminal sequence analyzed. Two [14C]carboxymethylcysteines were identified at positions 10 and 15 after light activation, while they were not detected in the dark-treated protein. In addition, the analysis of the tryptic digest of light-activated [14C]carboxymethylated NADP-MDH revealed that the radioactive label was mostly incorporated in Cys10 and Cys15, indicating that these 2 residues play a major role in the light activation mechanism. Moreover, an activation model, in which photoreduced thio-redoxin was replaced by the dithiol reductant dithio-threitol, has been developed. When NADP-MDH was activated in this way, the same sulfhydryls were found to be labeled, and alternatively, they did not incorporate any radioactivity when dithiothreitol reduction was performed after carboxymethylation in denaturating conditions. These results indicate that activation (by light or by dithiothreitol) proceeds on each subunit by reduction of a disulfide bridge located at the amino terminus of the enzyme between Cys10 and Cys15.     

Chloroplast NADP-malate dehydrogenase: structural basis of light-dependent regulation of activity by thiol oxidation and reduction

BACKGROUND: NADP-dependent malate dehydrogenase (EC 1.1.1.82) is a light-activated chloroplast enzyme that functions in the C4 pathway of photosynthesis. The light regulation is believed to be mediated in vivo by thioredoxin-catalyzed reduction and re-oxidation of cystine residues. The rates of reversible activation and inactivation of the enzyme are strongly influenced by the coenzyme substrates that seem to ultimately determine the steady-state extent of activation in vivo. RESULTS: The X-ray structure of the inactive, oxidized enzyme was determined at 2.8 A resolution. The core structure is homologous to AND-dependent malate dehydrogenases. Two surface-exposed and thioredoxin-accessible disulfide bonds are present, one in the N-terminal extension and the other in the C-terminal extension. The C-terminal peptide of the inactive, oxidized enzyme is constrained by its disulfide bond to fold into the active site over NADP+, hydrogen bonding to the catalytic His225 as well as obstructing access of the C4 acid substrate. Two loops flanking the active site, termed the Arg2 and Trp loops, that contain the C4 acid substrate binding residues are prevented from closing by the C-terminal extension. CONCLUSIONS: The structure explains the role of the C-terminal extension in inhibiting activity. The negative C terminus will interact more strongly with the positively charged nicotinamide of NADP+ than NADPH, explaining why the coenzyme-binding affinities of the enzyme differ so markedly from those of all other homologous alpha-hydroxy acid dehydrogenases. NADP+ may also slow dissociation of the C terminus upon reduction, providing a mechanism for the inhibition of activation by NADP+ but not NADPH.     

Capping protein binding to S100B: implications for the tentacle model for capping the actin filament barbed end

S100B binds tightly to a 12-amino acid peptide derived from heterodimeric capping protein. In native intact capping protein, this sequence is in the C terminus of the alpha-subunit, which is important for capping the actin filament. This C-terminal region is proposed to act as a flexible "tentacle," extending away from the body of capping protein in order to bind actin. To this hypothesis, we analyzed the interaction between S100B and capping protein in solution. The C-terminal 28 amino acids of the alpha-subunit, the proposed tentacle, bound to S100B as a free synthetic peptide or a glutathione S-transferase fusion (K(d) approximately 0.4-1 microm). In contrast, S100B did not bind to whole native capping protein or functionally affect its capping activity. S100B does not bind, with any significant affinity, to the proposed alpha-tentacle sequence of whole native capping protein in solution. In the NMR structure of S100B complexed with the alpha-subunit-derived 12-amino acid peptide, the hydrophobic side of a short alpha-helix in the peptide, containing an important tryptophan residue, contacts S100B. In the x-ray structure of native capping protein, the corresponding sequence of the alpha-subunit C terminus, including Trp(271), interacts closely with the body of the protein. Therefore, our results suggest the alpha-subunit C terminus is not mobile as predicted by the tentacle model. Addition of non-ionic detergent allowed whole capping protein to bind weakly to S100B, indicating that the alpha-subunit C terminus can be mobilized from the surface of the capping protein molecule, presumably by weakening the hydrophobic binding at the contact site.     

The polar region consecutive to the HIV fusion peptide participates in membrane fusion

The fusion peptide of HIV-1 gp41 is formed by the 16 N-terminal residues of the protein. This 16-amino acid peptide, in common with several other viral fusion peptides, caused a reduction in the bilayer to hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine (T(H)), suggesting its ability to promote negative curvature in membranes. Surprisingly, an elongated peptide corresponding to the 33 N-terminal amino acids raised T(H), although it was more potent than the 16-amino acid fusion peptide in inducing lipid mixing with large unilamellar liposomes of 1:1:1 dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine/choleste rol. The 17-amino acid C-terminal fragment of the peptide can induce membrane fusion by itself, if it is anchored to a membrane by palmitoylation of the amino terminus, indicating that the additional 17 hydrophilic amino acids contribute to the fusogenic potency of the peptide. This is not solely a consequence of the palmitoylation, as a random peptide with the same amino acid composition with a palmitoyl anchor was less potent in promoting membrane fusion and palmitic acid itself had no fusogenic activity. The 16-amino acid N-terminal fusion peptide and the longer 33-amino acid peptide were labeled with NBD. Fluorescence binding studies indicate that both peptides bind to the membrane with similar affinities, indicating that the increased fusogenic activity of the longer peptide was not a consequence of a greater extent of membrane partitioning. We also determined the secondary structure of the peptides using FTIR spectroscopy. We find that the amino-terminal fusion peptide is inserted into the membrane as a beta-sheet and the 17 C-terminal amino acids lie on the surface of the membrane, adopting an alpha-helical conformation. It was further demonstrated with the use of rhodamine-labeled peptides that the 33-amino acid peptide self-associated in the membrane while the 16-amino acid N-terminal peptide did not. Thus, the 16-amino acid N-terminal fusion peptide of HIV inserts into the membrane and, like other viral fusion peptides, lowers T(H). In addition, the 17 consecutive amino acids enhance the fusogenic activity of the fusion peptide presumably by promoting its self-association.     

Malate Dehydrogenases in Guard Cells of Pisum sativum

Guard cell protoplasts of Pisum sativum show considerable NADP-dependent malate dehydrogenase (MDH) activity in darkness which can be enhanced severalfold by illumination or treatment with dithiothreitol (DTT). The question arose whether guard cells possess an NADP-MDH different from that present in the chloroplasts of the mesophyll (which is inactive in darkness or in the absence of DTT). MDH activities were determined in extracts of isolated protoplasts from mesophyll and epidermis, and in mechanically prepared epidermal pieces (with guard cells as the only living cells and no interference from proteases originating from the cell wall digesting enzymes). Guard cells possessed NAD-dependent MDHs of high activity and incomplete exclusion of NADP as a coenzyme. This NADP-dependent activity of the NAD-MDH(s) could not be stimulated by DTT or, inferentially, by light. The DTT- (and light-) dependent NADP-MDH represented 0.05% of the total protein of the guard cells and had a specific activity of 0.1 unit per milligram protein; both values are in the same range as the corresponding ones of the mesophyll cells. Agreement was also found in the extent of light activation, in subunit molecular weight, immunological cross-reactions, and in the behavior on an ion exchange column. The activity of the chloroplastic NADP-MDH in guard cells barely suffices to meet the malate requirement for stomatal opening in the light. It is therefore likely that NAD-MDHs residing in other compartments of the guard cells supplement the activity of the chloroplastic NADP-MDH particularly during stomatal opening in darkness.     

Cloning and nucleotide sequence of the gene encoding the γ-d-glutamyl-l-diamino acid endopeptidase II of Bacillus sphaericus

The gene encoding the Bacillus sphaericus gamma-D-glutamyl-L-diamino acid endopeptidase II, a cytoplasmic enzyme involved in cell sporulation [1], contains the information for a 271-amino acid protein devoid of a signal peptide. The endopeptidase lacks sequence relatedness with other proteins of known primary structure except that its C-terminal region has significant similarity with the C-terminal region of the 54-kDa P54 protein of Enterococcus faecium, of unknown function [2].     

A single C-terminal peptide segment mediates both membrane association and localization of lysyl hydroxylase in the endoplasmic reticulum

Hydroxylation of lysyl residues is crucial for the unique glycosylation pattern found in collagens and for the mechanical strength of fully assembled extracellular collagen fibers. Hydroxylation is catalyzed in the lumen of the endoplasmic reticulum (ER) by a specific enzyme, lysyl hydroxylase (LH). The absence of the known ER-specific retrieval motifs in its primary structure and its association with the ER membranes in vivo have suggested that the enzyme is localized in the ER via a novel retention/retrieval mechanism. We have identified here a 40-amino acid C-terminal peptide segment of LH that is able to convert cathepsin D, normally a soluble lysosomal protease, into a membrane-associated protein. The same segment also markedly slows down the transport of the reporter protein from the ER into post-ER compartments, as assessed by our pulse-chase experiments. The retardation efficiency mediated by this C-terminal peptide segment is comparable with that of the intact LH but lower than that of the KDEL receptor-based retrieval mechanism. Within this 40-amino acid segment, the first 25 amino acids appear to be the most crucial ones in terms of membrane association and ER localization, because the last 15 C-terminal amino acids did not possess substantial retardation activity alone. Our findings thus define a short peptide segment very close to the extreme C terminus of LH as the only necessary determinant both for its membrane association and localization in the ER.     

Redox regulation of chloroplast enzymes in Galdieria sulphuraria in view of eukaryotic evolution

Redox modulation is a general mechanism for enzyme regulation, particularly for the post-translational regulation of the Calvin cycle in chloroplasts of green plants. Although red algae and photosynthetic protists that harbor plastids of red algal origin contribute greatly to global carbon fixation, relatively little is known about post-translational regulation of chloroplast enzymes in this important group of photosynthetic eukaryotes. To address this question, we used biochemistry, phylogenetics and analysis of recently completed genome sequences. We studied the functionality of the chloroplast enzymes phosphoribulokinase (PRK, EC 2.7.1.19), NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH, GapA, EC 1.2.1.13), fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) and glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), as well as NADP-malate dehydrogenase (NADP-MDH, EC 1.1.1.37) in the unicellular red alga Galdieria sulphuraria (Galdieri) Merola. Despite high sequence similarity of G. sulphuraria proteins to those of other photosynthetic organisms, we found a number of distinct differences. Both PRK and GAPDH co-eluted with CP12 in a high molecular weight complex in the presence of oxidized glutathione, although Galdieria CP12 lacks the two cysteines essential for the formation of the N-terminal peptide loop present in higher plants. However, PRK inactivation upon complex formation turned out to be incomplete. G6PDH was redox modulated, but remained in its tetrameric form; FBPase was poorly redox regulated, despite conservation of the two redox-active cysteines. No indication for the presence of plastidic NADP-MDH (and other components of the malate valve) was found.     

Evidence for a C4 NADP-ME photosynthetic pathway in Vetiveria zizanioides Stapf

ABSTRACT

Leaf anatomy (light and transmission electron microscopy), immunogold localization of Rubisco, photosynthetic enzyme activities, CO₂ assimilation and stomatal conductance were studied in Vetiveria zizanioides Stapf., a graminaceous plant native to tropical and subtropical areas, and cultivated in temperate climates (Northwestern Italy). Leaves possess a NADP-ME Kranz anatomy with bundle sheath cells containing chloroplasts located in a centrifugal position. Dimorphic chloroplasts were also observed; they are agranal and starchy in the bundle sheath and granal starchless in the mesophyll cells. Rubisco immunolocalization studies indicate that this enzyme occurs solely in the bundle sheath chloroplasts. Pyruvate-orthophosphate dikinase, NADP-dependent malate dehydrogenase (NADP-MDH), NADP-dependent malic enzyme (NADP-ME), PEP-carboxykinase and NAD-dependent malic enzyme (NAD-ME) activities were determined. Enzyme activity and some kinetic properties of NADP-ME and NADP-MDH as well as CO₂ compensation point and stomatal conductance values were calculated indicating a NADP-ME C₄ photosynthetic pathway. Biochemical and structural results indicate that V. zizanioides belongs to the C₄ NADP-ME variant. This plant appears to be well adapted to the varying environmental conditions typical of temperate climates, by retaining high enzyme activities and a low CO₂ compensation point.     

Characterization of the C-terminal flanking peptide of human beta-preprotachykinin

The nucleotide sequence of cDNA encoding the common biosynthetic precursor of substance P, neurokinin A and neuropeptide K (beta-preprotachykinin) predicts that, in the human, the precursor contains a C-terminal flanking peptide of 19 amino acid residues [beta-preprotachykinin(111-129)-peptide]. Using an antiserum raised against synthetic human beta-preprotachykinin(117-126)-peptide in radioimmunoassay, we have demonstrated that an extract of a human neuroendocrine tumor of the adrenal medulla contained approximately equimolar concentrations of C-terminal preprotachykinin immunoreactivity (C-PPT-IR), substance P and neurokinin A. The C-terminal preprotachykinin flanking peptide was purified to homogeneity and its primary structure was determined. The amino acid sequence of the peptide, Ala-Leu-Asn-Ser-Val-Ala-Tyr-Glu-Arg-Ser-Ala-Met-Gln-Asn-Tyr-Glu, indicates identity with beta-preprotachykinin(111-126)-peptide. The data suggest that the C-terminal flanking peptide, like the tachykinins, is packed into secretory storage vesicles but the Arg127-Arg128-Arg129 residues in human beta-preprotachykinin are removed from the peptide by the action of endogenous processing enzyme(s).     

Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase

The nuclear-encoded chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme controlling the malate valve, to allow the indirect export of reducing equivalents. Arabidopsis thaliana (L.) Heynh. T-DNA insertion mutants of NADP-MDH were used to assess the role of the light-activated NADP-MDH in a typical C(3) plant. Surprisingly, even when exposed to high-light conditions in short days, nadp-mdh knockout mutants were phenotypically indistinguishable from the wild type. The photosynthetic performance and typical antioxidative systems, such as the Beck-Halliwell-Asada pathway, were barely affected in the mutants in response to high-light treatment. The reactive oxygen species levels remained low, indicating the apparent absence of oxidative stress, in the mutants. Further analysis revealed a novel combination of compensatory mechanisms in order to maintain redox homeostasis in the nadp-mdh plants under high-light conditions, particularly an increase in the NTRC/2-Cys peroxiredoxin (Prx) system in chloroplasts. There were indications of adjustments in extra-chloroplastic components of photorespiration and proline levels, which all could dissipate excess reducing equivalents, sustain photosynthesis, and prevent photoinhibition in nadp-mdh knockout plants. Such metabolic flexibility suggests that the malate valve acts in concert with other NADPH-consuming reactions to maintain a balanced redox state during photosynthesis under high-light stress in wild-type plants.     

Solution structure of the extended neuronal nitric oxide synthase PDZ domain complexed with an associated peptide.

The PDZ domain of neuronal nitric oxide synthase (nNOS) functions as a scaffold for organizing the signal transduction complex of the enzyme. The NMR structure of a complex composed of the nNOS PDZ domain and an associated peptide suggests that a two-stranded beta-sheet C-terminal to the canonical PDZ domain may mediate its interaction with the PDZ domains of postsynaptic density-95 and alpha-syntrophin. The structure also provides the molecular basis of recognition of Asp-X-Val-COOH peptides by the nNOS PDZ domain. The role of the C-terminal extension in Asp-X-Val-COOH peptide binding is investigated. Additionally, NMR studies further show that the Asp-X-Val-COOH peptide and a C-terminal peptide from a novel cytosolic protein named CAPON bind to the same pocket of the nNOS PDZ domain.     

Structural characterization of the entire 1.3S subunit of transcarboxylase from Propionibacterium shermanii.

Transcarboxylase (TC) from Propionibacterium shermanii, a biotin-dependent enzyme, catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate in two partial reactions. Within the multisubunit enzyme complex, the 1.3S subunit functions as the carboxyl group carrier. The 1.3S is a 123-amino acid polypeptide (12.6 kDa), to which biotin is covalently attached at Lys 89. We have expressed 1.3S in Escherichia coli with uniform 15N labeling. The backbone structure and dynamics of the protein have been characterized in aqueous solution by three-dimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy. The secondary structure elements in the protein were identified based on NOE information, secondary chemical shifts, homonuclear 3J(HNHalpha) coupling constants, and amide proton exchange data. The protein contains a predominantly disordered N-terminal half, while the C-terminal half is folded into a compact domain comprising eight beta-strands connected by short loops and turns. The topology of the C-terminal domain is consistent with the fold found in both carboxyl carrier and lipoyl domains, to which this domain has approximately 26-30% sequence similarity.