Structural basis for the light regulation of chloroplast NADP malate dehydrogenase

The activity of chloroplast NADP-malate dehydrogenase (NADP-MDH; EC 1.1.1.82) in both C3 and C4 plants is regulated by light intensity. In darkness, the activity of the enzyme can be less than 1% of the maximal activity found at high light intensities. The extent of activation in the light is dynamic, responding rapidly to changes in light intensity and adapting to changes in photosynthetic rate. Enzyme activation is caused by thioredoxin-catalyzed reduction of two regulatory disulfide bonds, while inactivation is accomplished by thioredoxin-catalyzed re-oxidation. In the case of NADP-MDH, the coenzyme substrates NADP+ and NADPH modify the rate of this interconversion and seem to be important to the extent of activation in vivo. The recent determination of the X-ray structure of the oxidized, dark form of NADP-MDH from the C4 plants Flaveria bidentis and Sorghum shows how oxidation of a disulfide bond can inactivate the enzyme. This review discusses the various structural features of NADP-MDH that seem to be responsible for the regulatory properties of the enzyme and emphasizes that large changes of activity can be accomplished by multiple, small, reinforcing changes rather than a single large change in a signal molecule concentration.     

Structural and catalytic properties of oxidized and reduced chloroplast NADP-malate dehydrogenase upon denaturation and renaturation

Chloroplast NADP-dependent malate dehydrogenase exists in two interconvertible forms: the inactive disulfide-containing form and the active dithiol form. No major difference in secondary structure or conformation was found between the oxidized and the reduced enzyme as determined by circular dichroism and intrinsic protein fluorescence. The guanidine/HCl-dependent unfolding of the enzyme is characterized by two transition midpoints: those of the reduced enzyme are lower by about 0.2 M guanidine/HCl compared to the oxidized enzyme. As shown by analytical ultracentrifugation, there was no effect of guanidine/HCl concentrations up to 0.25 M on the quaternary structure of the enzyme in its oxidized and reduced forms: both sedimentation coefficient (S20,w = 4.9 +/- 0.1 S) and sedimentation equilibrium (75 +/- 3 kDa) yield the dimer. In the oxidized state the enzyme undergoes guanidine-dependent dissociation to the monomer with a midpoint of transition at 0.5 M. The kinetics of unfolding were found to be significantly faster for the reduced than for the oxidized enzyme. Renaturation and reactivation of reduced enzyme was more rapid and occurred with higher yields (100%) than for the oxidized enzyme (60-80% yield). Furthermore, the effect of denaturants on catalytic activity, and reductive activation of the oxidized form, were studied. Both increase in protein fluorescence and a stimulatory effect on the activities at low guanidine/HCl concentrations were observed for the oxidized and the reduced form of the enzyme. Denaturants increase the rate of reductive activation of NADP-malate dehydrogenase.     

Energetic aspects of the light activation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase

The light energy requirements for photoactivation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were studied in a reconstituted chloroplast system. This system comprised isolated pea thylakoids, ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR) thioredoxin_m and _f (Td_m, Td_f) and the photoactivatable enzyme. Light-saturation curves of the photoactivation process were established with once washed thylakoids which did not require the addition of Td for light activation. They exhibited a plateau at 10 W·m^–2 under nitrogen and 50 W·m^–2 under air, while NADP photoreduction was saturated at 240 W·m^–2. Cyclic and pseudocyclic phosphorylations saturated at identical levels as enzyme photoactivations. All these observations suggested that the shift of the light saturation plateau towards higher values under air was due to competing oxygen-dependent reactions. With twice washed thylakoids, which required Td for enzyme light-activation, photophosphorylation was stimulated under N₂ by the addition of the components of the photoactivation system. Its rate increased with increasing Td concentrations, just as did the enzyme photoactivation rate, while varying the target enzyme concentration had only a weak effect. Considering that Td concentrations were in a large excess over target enzyme concentrations, it may be assumed that the observed ATP synthesis was essentially dependent on the rate of Td reduction.Under air, Fd-dependent pseudo-cyclic photophosphorylation was not stimulated by the addition of the other enzyme photoactivation components, suggesting that an important site of action of O₂ was located at the level of Fd.Abbreviations Fd ferredoxin - FBPase fructose-1,6-bisphosphatase - FTR ferredoxin-thioredoxin reductase - LEM light effect mediator - NADP-MDH NADP-malate dehydrogenase - Td thioredoxin     

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.     

The internal Cys-207 of sorghum leaf NADP-malate dehydrogenase can form mixed disulphides with thioredoxin

The role of the internal Cys-207 of sorghum NADP-malate dehydrogenase (NADP-MDH) in the activation of the enzyme has been investigated through the examination of the ability of this residue to form mixed disulphides with thioredoxin mutated at either of its two active-site cysteines. The h-type Chlamydomonas thioredoxin was used, because it has no additional cysteines in the primary sequence besides the active-site cysteines. Both thioredoxin mutants proved equally efficient in forming mixed disulphides with an NADP-MDH devoid of its N-terminal bridge either by truncation, or by mutation of its N-terminal cysteines. They were poorly efficient with the more compact WT oxidised NADP-MDH. Upon mutation of Cys-207, no mixed disulphide could be formed, showing that this cysteine is the only one, among the four internal cysteines, which can form mixed disulphides with thioredoxin. These experiments confirm that the opening of the N-terminal disulphide loosens the interaction between subunits, making Cys-207, located at the dimer contact area, more accessible.     

The role of active site arginines of sorghum NADP-malate dehydrogenase in thioredoxin-dependent activation and activity

The activation of sorghum NADP-malate dehydrogenase is initiated by thiol/disulfide interchanges with reduced thioredoxin followed by the release of the C-terminal autoinhibitory extension and a structural modification shaping the active site into a high efficiency and high affinity for oxaloacetate conformation. In the present study, the role of the active site arginines in the activation and catalysis was investigated by site-directed mutagenesis and arginyl-specific chemical derivatization using butanedione. Sequence and mass spectrometry analysis were used to identify the chemically modified groups. Taken together, our data reveal the involvement of Arg-134 and Arg-204 in oxaloacetate coordination, suggest an indirect role for Arg-140 in substrate binding and catalysis, and clearly confirm that Arg-87 is implicated in cofactor binding. In contrast with NAD-malate dehydrogenase, no lactate dehydrogenase activity could be promoted by the R134Q mutation. The decreased susceptibility of the activation of the R204K mutant to NADP and its increased sensitivity to the histidine-specific reagent diethylpyrocarbonate indicated that Arg-204 is involved in the locking of the active site. These results are discussed in relation with the recently published NADP-MDH three-dimensional structures and the previously established three-dimensional structures of NAD-malate dehydrogenase and lactate dehydrogenase.     

Direct NMR observation of the thioredoxin-mediated reduction of the chloroplast NADP-malate dehydrogenase provides a structural basis for the relief of autoinhibition

The chloroplastic NADP-dependent malate dehydrogenase (NADP-MDH) catalyzing the reduction of oxaloacetate into L-malate is regulated by light. Its activation results from the thioredoxin-mediated reduction of two disulfides, located, respectively, in N- and C-terminal sequence extensions typical of all NADP-dependent light-regulated forms. Site-directed mutagenesis studies and the resolution of the three-dimensional structure of the oxidized (inactive) Sorghum vulgare enzyme showed that the C-terminal Cys(365)-Cys(377) disulfide constrains the C-terminal extension to fold into the active site where it acts as an internal inhibitor. In the present study, two-dimensional proton NMR spectra of an engineered NADP-MDH rendered monomeric by a 33-amino acid deletion at the N terminus (38 kDa) revealed that a 15-amino acid-long C-terminal peptide (Ala(375) to C-terminal Val(389)) acquired an increased mobility upon reduction, allowing its direct sequence-specific NMR assignment. The location of the flexible peptide in the sequence suggests that the first part of the C-terminal peptide is still folded near the core of the enzyme, so that cysteines 365 and 377 remain in proximity to allow for an efficient reoxidation/inactivation of the enzyme.     

Structural basis for leucine-induced allosteric activation of glutamate dehydrogenase

Glutamate dehydrogenase (GDH) catalyzes reversible conversion between glutamate and 2-oxoglutarate using NAD(P)(H) as a coenzyme. Although mammalian GDH is regulated by GTP through the antenna domain, little is known about the mechanism of allosteric activation by leucine. An extremely thermophilic bacterium, Thermus thermophilus, possesses GDH with a unique subunit configuration composed of two different subunits, GdhA (regulatory subunit) and GdhB (catalytic subunit). T. thermophilus GDH is unique in that the enzyme is subject to allosteric activation by leucine. To elucidate the structural basis for leucine-induced allosteric activation of GDH, we determined the crystal structures of the GdhB-Glu and GdhA-GdhB-Leu complexes at 2.1 and 2.6 Å resolution, respectively. The GdhB-Glu complex is a hexamer that binds 12 glutamate molecules: six molecules are bound at the substrate-binding sites, and the remaining six are bound at subunit interfaces, each composed of three subunits. The GdhA-GdhB-Leu complex is crystallized as a heterohexamer composed of four GdhA subunits and two GdhB subunits. In this complex, six leucine molecules are bound at subunit interfaces identified as glutamate-binding sites in the GdhB-Glu complex. Consistent with the structure, replacement of the amino acid residues of T. thermophilus GDH responsible for leucine binding made T. thermophilus GDH insensitive to leucine. Equivalent amino acid replacement caused a similar loss of sensitivity to leucine in human GDH2, suggesting that human GDH2 also uses the same allosteric site for regulation by leucine.     

Effect of high light intensities on oxygen evolution and the light activation of NADP-malate dehydrogenase in intact spinach chloroplasts

The factors limiting the photosynthetic carbon metabolism of intact spinach (Spinacia oleracea L.) chloroplasts after a high-light pretreatment have been studied. Photosynthetic CO₂ fixation was decreased and became more sensitive to the inhibitory effect of the cyclic-electron-flow inhibitor, antimycin A. Depending on the extent of photoinhibition, changing the balance of linear to cyclic electron flow by adding oxaloacetate and antimycin A either did not relieve, or partially relieved the photoinhibitory effect. The decrease in CO₂ fixation appeared to be the consequence of either a limitation by photosystem-II activity (in the case of moderate inhibition) or, at least partially an unfavourable balance between the linear and cyclic electron flows (in the case of strong inhibition). The light activation of NADP-malate dehydrogenase (EC 1.1.1.82) was decreased only in the presence of CO₂, i.e. when there was strong competition for reducing power; otherwise, it was unaffected by photoinhibitory treatments, in accordance with its low energy requirement.Abbreviations Chl chlorophyll - NADP-MDH NADP-dependent malate dehydrogenase - PFD photon flux density - PSI, II photosystem I, II     

Site-directed mutagenesis reveals the involvement of an additional thioredoxin-dependent regulatory site in the activation of recombinant sorghum leaf NADP-malate dehydrogenase.

The chloroplastic enzyme NADP-malate dehydrogenase is activated by a reversible thiol/disulfide interchange with reduced thioredoxin. Its target disulfide bridge is considered to be located at the amino terminus. To further substantiate the regulatory role of this disulfide, site-directed mutagenesis has been used to replace each or both of the amino-terminal cysteines of the sorghum leaf NADP-malate dehydrogenase, expressed in Escherichia coli, by serines. A truncation mutant lacking the amino terminus has also been produced. Surprisingly, the mutant proteins still required activation by reduced thioredoxin. However, their activation was almost instantaneous, whereas the native enzyme reached full activity after a 10-20 min preincubation. The 8 1/2 for reduced thioredoxin was decreased 2-fold in the mutants, but their Km values for NADPH and oxaloacetate did not change significantly. The inhibition of activation by NADP and inhibition of activity by thiol-derivatizing agents were also retained. These results are interpreted as an indication that two thioredoxin-dependent reduction steps are involved in NADP-dependent malate dehydrogenase light activation, hence that two disulfides per monomer participate in the process. The overall activation rate would depend on a conformational change following the reduction of the amino-terminal disulfide bridge. The amino terminus also plays a role in the dimerization of the protein.     

The dark (oxidized) form of the light-activatable NADP-malate dehydrogenase from pea chloroplasts is catalytically active in the presence of guanidine-HCl

The dark form of NADP-malate dehydrogenase from pea leaves which has been shown to contain one disulfide bridge per subunit does not exhibit any catalytic activity in the absence of thiol reducing agents. Upon reduction of these disulfide bridges the enzyme becomes catalytically active. In this presentation, however, it is shown that the oxidized dark form of NADP-malate dehydrogenase becames catalytically competent when assayed in the presence of 200–250 mM guanidine-HCl. This guanidine-dependent activity of the oxidized enzyme is characterized by higher apparent K_m values for the substratres as compared to the reduced enzyme, but is still specific for NADPH. Up to 25% of the V_maxOf the reduced enzyme was obtained for the oxidized guanidine-activated NADP-malate dehydrogenase. The results suggest that the reduction of the regulatory disulfide is not essential for catalytic activity.     

Inhibition of the thioredoxin-dependent activation of the NADP-malate dehydrogenase and cofactor specificity

The chloroplastic NADP-malate dehydrogenase is activated by reduction of its N- and C-terminal disulfides by reduced thioredoxin. The activation is inhibited by NADP(+), the oxidized form of the cofactor. Previous studies suggested that the C-terminal disulfide was involved in this process. Recent structural data pointed toward a possible direct interaction between the C terminus of the oxidized enzyme and the cofactor. In the present study, the relationship between the cofactor specificity for catalysis and for inhibition of activation has been investigated by changing the cofactor specificity of the enzyme by substitution of selected residues of the cofactor-binding site. An NAD-specific thiol-regulated MDH was engineered. Its activation was inhibited by NAD(+) but no longer by NADP(+). These results demonstrate that the oxidized cofactor is bound at the same site as the reduced cofactor and support the idea of a direct interaction between the negatively charged C-terminal end of the enzyme and the positively charged nicotinamide ring of the cofactor, in agreement with the structural data. The structural requirements for cofactor specificity are modeled and discussed.     

The dimer contact area of sorghum NADP-malate dehydrogenase: role of aspartate 101 in dimer stability and catalytic activity

During thioredoxin-mediated activation of chloroplastic NADP-malate dehydrogenase, a homodimeric enzyme, the interaction between subunits is known to be loosened but maintained. A modeling of the 3D structure of the protein identified Asp-101 as being potentially involved in the association between subunits through an electrostatic interaction. Indeed, upon site-directed substitution of Asp-101 by an asparagine, the mutated enzyme behaved mainly as a monomer. The mutation strongly affected the catalytical efficiency of the enzyme. The now available 3D structure of the enzyme shows that Asp-101 is protruding at the dimer interface, interacting with Arg-268 of the neighbouring subunit.     

Structural basis for activation of the thiamin diphosphate-dependent enzyme oxalyl-CoA decarboxylase by adenosine diphosphate

Oxalyl-coenzyme A decarboxylase is a thiamin diphosphate-dependent enzyme that plays an important role in the catabolism of the highly toxic compound oxalate. We have determined the crystal structure of the enzyme from Oxalobacter formigenes from a hemihedrally twinned crystal to 1.73 A resolution and characterized the steady-state kinetic behavior of the decarboxylase. The monomer of the tetrameric enzyme consists of three alpha/beta-type domains, commonly seen in this class of enzymes, and the thiamin diphosphate-binding site is located at the expected subunit-subunit interface between two of the domains with the cofactor bound in the conserved V-conformation. Although oxalyl-CoA decarboxylase is structurally homologous to acetohydroxyacid synthase, a molecule of ADP is bound in a region that is cognate to the FAD-binding site observed in acetohydroxyacid synthase and presumably fulfils a similar role in stabilizing the protein structure. This difference between the two enzymes may have physiological importance since oxalyl-CoA decarboxylation is an essential step in ATP generation in O. formigenes, and the decarboxylase activity is stimulated by exogenous ADP. Despite the significant degree of structural conservation between the two homologous enzymes and the similarity in catalytic mechanism to other thiamin diphosphate-dependent enzymes, the active site residues of oxalyl-CoA decarboxylase are unique. A suggestion for the reaction mechanism of the enzyme is presented.     

Structural basis for catalytic activation of a serine recombinase

Sin resolvase is a site-specific serine recombinase that is normally controlled by a complex regulatory mechanism. A single mutation, Q115R, allows the enzyme to bypass the entire regulatory apparatus, such that no accessory proteins or DNA sites are required. Here, we present a 1.86 ? crystal structure of the Sin Q115R catalytic domain, in a tetrameric arrangement stabilized by an interaction between Arg115 residues on neighboring subunits. The subunits have undergone significant conformational changes from the inactive dimeric state previously reported. The structure provides a new high-resolution view of a serine recombinase active site that is apparently fully assembled, suggesting roles for the conserved active site residues. The structure also suggests how the dimer-tetramer transition is coupled to assembly of the active site. The tetramer is captured in a different rotational substate than that seen in previous hyperactive serine recombinase structures, and unbroken crossover site DNA can be readily modeled into its active sites.     

Structural basis for the alteration of coenzyme specificity in a malate dehydrogenase mutant

To elucidate the structural basis for the alteration of coenzyme specificity from NADH toward NADPH in a malate dehydrogenase mutant EX7 from Thermus flavus, we determined the crystal structures at 2.0 A resolution of EX7 complexed with NADPH and NADH, respectively. In the EX7-NADPH complex, Ser42 and Ser45 form hydrogen bonds with the 2'-phosphate group of the adenine ribose of NADPH, although the adenine moiety is not seen in the electron density map. In contrast, although Ser42 and Ser45 occupy a similar position in the EX7-NADH complex structure, both the adenine and adenine ribose moieties of NADH are missing in the map. These results and kinetic analysis of site-directed mutant enzymes indicate (1) that the preference of EX7 for NADPH over NADH is ascribed to the recognition of the 2'-phosphate group by two Ser and Arg44, and (2) that the adenine moiety of NADPH is not recognized in this mutant.     

Extraction of chloroplast light effect mediator(s) and reconstitution of light activation of NADP-linked malate dehydrogenase

Washed thylakoid membranes of pea (Pisum sativum var. Little Marvel), on brief exposure to zwittergent, an amphoteric detergent, lost the property of supporting the light activation of stromal NADP-linked malate dehydrogenase. But, these depleted membranes, on reconstitution with dialyzed, high-speed supernatant of the detergent extract, showed marked light activation of the enzyme when assayed in the presence of 2,6-dichlorophenolindophenol-ascorbate. The component of the high-speed supernatant which is required for light activation is sensitive to sulfite and is heat labile. The analysis of the high-speed supernatant on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two prominent polypeptides at approximately 18,000 and 36,000 daltons. The surface-specific, chloroglycoluril-mediated iodination of the washed thylakoid membranes revealed that zwittergent had extracted these two polypeptides. The results reveal that the light effect mediator (LEM) is a surface-exposed, tightly bound protein existing in the thylakoid membranes, and that it can be removed by zwitterionic detergent and used in reconstitution studies.     

Structural feature of sorghum chloroplast psbA gene and regulation effects of its 5'-noncoding region*

Comparative analysis reveals remarkable homology between the sequences of both psbA gene nucleotides and the inferred amino acids of sorghum, a C_4 plant, and those of rice, a C_3 plant. The 5'-noncoding region of sorghum psbA gene contains the conservative promoter elements, "-35" element and "-10" element, like the prokaryote and the promoter element, TATA box, like the eukaryote. As compared with that of the rice, an extra sequence of 7 bp is found in the leader sequence of the mRNA in the former. Using an in vitro system, it has been demonstrated that protein factor exists in sorghum chloroplast protein extract which specifically binds to the 5'-noncoding region of psbA gene. Measurement of the expression of luciferase shows a 2—5 time greater reaction of the expression plasmids pALqs which contain leader region of sorghum psbA gene than that of the expression plasmids pALqr which contain leader region of rice psbA gene in E. coli.     

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.     

Structural basis for the oxidation of thiosulfate by a sulfur cycle enzyme

Reduced inorganic sulfur compounds are utilized by many bacteria as electron donors to photosynthetic or respiratory electron transport chains. This metabolism is a key component of the biogeochemical sulfur cycle. The SoxAX protein is a heterodimeric c-type cytochrome involved in thiosulfate oxidation. The crystal structures of SoxAX from the photosynthetic bacterium Rhodovulum sulfidophilum have been solved at 1.75 A resolution in the oxidized state and at 1.5 A resolution in the dithionite-reduced state, providing the first structural insights into the enzymatic oxidation of thiosulfate. The SoxAX active site contains a haem with unprecedented cysteine persulfide (cysteine sulfane) coordination. This unusual post-translational modification is also seen in sulfurtransferases such as rhodanese. Intriguingly, this enzyme shares further active site characteristics with SoxAX such as an adjacent conserved arginine residue and a strongly positive electrostatic potential. These similarities have allowed us to suggest a catalytic mechanism for enzymatic thiosulfate oxidation. The atomic coordinates and experimental structure factors have been deposited in the PDB with the accession codes 1H31, 1H32 and 1H33.