The intracellular localisation of malate dehydrogenase in Anacystis nidulans

Abstract Malate dehydrogenase has been reported to be active as a Krebs cycle enzyme in Anabaena cylindrica and Anacystis nidulans [1,2] and as an enzyme of the glycollate pathway in Anabaena cylindrica [1,3]. This enzyme was also reported in Oscillatoria spp. [4] and in Nostoc muscorum [5]. The isoenzyme of eukaryotic organisms was known to participate in various metabolic pathways and to be localized in different subcellular organelles [6–9]. Kovatcheva and Bergman [5] have purified the enzyme from the reddish-brown 20 000 × g × 20 min supernatant. We have determined the intracellular distribution of malate dehydrogenese of Anacystis nidulans and present evidence that it is largely associated with the thylakoids. The significance of this study is discussed in terms of the dual role of cyanobacterial thylakoids in photosynthesis and respiration.     

Crystal structure of NAD-dependent malate dehydrogenase complexed with NADP(H)

For better understanding of the coenzyme specificity in NAD-dependent MDH (tMDH) from Thermus flavus AT-62, we determined the crystal structures of tMDH-NADP(H) complex at maximally 1.65 A resolution. The overall structure is almost the same as that of the tMDH-NADH complex. However, NADP(H) binds to tMDH in the reverse orientation, where adenine occupies the position near the catalytic center and nicotinamide is positioned at the adenine binding site of the tMDH-NADH complex. Consistent with this, kinetic analysis of the malate-oxidizing reaction revealed that NADP(+) inhibited tMDH at high concentrations. This has provided the first evidence for the alternative binding mode of the nicotinamide coenzyme, that has pseudo-symmetry in its structure, in a single enzyme.     

Glyoxysomal malate dehydrogenase and malate synthase from soybean cotyledons (Glycine max L.): enzyme association,antibody production and cDNA cloning

In order to investigate a possible association between soybean malate synthase (MS; l-malate glyoxylate-lyase, CoA-acetylating, EC 4.1.3.2) and glyoxysomal malate dehydrogenase (gMDH; (S)-malate: NAD⁺ oxidoreductase, EC 1.1.1.37), two consecutive enzymes in the glyoxylate cycle, their elution profiles were analyzed on Superdex 200 HR fast protein liquid chromatography columns equilibrated in low- and high-ionicstrength buffers. Starting with soluble proteins extracted from the cotyledons of 5-d-old soybean seedlings and a 45% ammonium sulfate precipitation, MS and gMDH coeluted on Superdex 200 HR (low-ionic-strength buffer) as a complex with an approximate relative molecular mass (M_r) of 670000. Dissociation was achieved in the presence of 50 mM KCl and 5 mM MgCl₂, with the elution of MS as an octamer of M_r 510000 and of gMDH as a dimer of M_r 73 000. Polyclonal antibodies raised to the native copurified enzymes recognized both denatured MS and gMDH on immunoblots, and their native forms after gel filtration. When these antibodies were used to screen a ZAP II expression library containing cDNA from 3-d-old soybean cotyledons, they identified seven clones encoding gMDH, whereas ten clones encoding MS were identified using an antibody to SDS-PAGE-purified MS. Of these cDNA clones a 1.8 kb clone for MS and a 1.3-kb clone for gMDH were fully sequenced. While 88% identity was found between mature soybean gMDH and watermelon gMDH, the N-terminal transit peptides showed only 37% identity. Despite this low identity, the soybean gMDH transit peptide conserves the consensus R(X₆)HL motif also found in plant and mammalian thiolases.The nucleotide sequence data reported in this paper have been submitted to Genbank and assigned the accession numbers LOI628 for gMDH and L01629 for MS.     

Dark modulation of NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase in the chloroplast

The properties of the system which reverses light modulation of NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase activity in pea chloroplasts were examined. A factor catalyzing dark modulation of these enzymes was found. This factor cochromatographed with thioredoxin in all systems used (Sephacryl S-200, Sephadex G-75, DEAE-cellulose). Inhibition of dithiothreitol-dependent modulation and of dark reversal by antibody against Escherichia coli thioredoxin further suggest that the dark factor is in fact thioredoxin. It appears that the reaction is the reverse of the previously described dithiothreitol-dependent thioredoxin-catalyzed modulation of enzymes. The limiting step in vitro seems to be the oxidation of thioredoxin during the dark period.     

Glyoxysomal and mitochondrial malate dehydrogenase of watermelon (Citrullus vulgaris) cotyledons

Molecular properties of the glyoxysomal and mitochondrial isoenzyme of malate dehydrogenase (EC 1.1.1.37; L-malate: NAD⁺ oxidoreductase) from watermelon cotyledons (Citrullus vulgaris Schrad.) were investigated, using completely purified enzyme preparations. The apparent molecular weights of the glyoxysomal and mitochondrial isoenzymes were found to be 67,000 and 74,000 respectively. Aggregation at high enzyme concentrations was observed with the glyoxysomal but not with the mitochondrial isoenzyme. Using sodium dodecyl sulfate electrophoresis each isoenzyme was found to be composed of two polypeptide chains of identical size (33,500 and 37,000, respectively). The isoenzymes differed in their isoelectric points (gMDH: 8,92, mMDH: 5.39), rate of heat inactivation (gMDH: _1/2 at 40°C=3.0 min; mMDH: stable at 40°C; _1/2 at 60°C=4.5 min), adsorption to dextran gels at low ionic strenght, stability against alkaline conditions and their pH optima for oxaloacetate reduction (gMDH: pH 6.6, mMDH: pH 7.5). Very similar pH optima, however, were observed for L-malate oxidation (pH 9.3–9.5). The results indicate that the glyoxysomal and mitochondrial MDH of watermelon cotyledons are distinct proteins of different structural composition.Abbreviations EDTA ethylene diamine tetraacetic acid - gMDH and mMDH glyoxysomal and mitochondrial malate dehydrogenase, respectively     

The malate dehydrogenase isoforms from Trypanosoma brucei: subcellular localization and differential expression in bloodstream and procyclic forms

Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate.     

NAD-dependent malate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase isoenzymes play an important role in dark metabolism of various plastid types

Chloroplasts isolated from spinach (Spinacia oleracea L.) leaves and green sweet-pepper (Capsicum annuum L. var. grossum (L.) Sendt.) fruits contain NADP-dependent malate dehydrogenase (MDH; EC 1.1.1.82) and the bispecific NAD(P)-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; EC 1.2.1.13). The NADP-dependent MDH and GAPDH are activated in the light, and inactive in the dark. We found that chloroplasts possess additional NAD-dependent MDH activity which is, like the NAD-dependent GAPDH activity, not influenced by light. In heterotrophic chromoplasts from red sweet-pepper fruits, the NADP-dependent MDH and the NAD(P)-GAPDH isoenzymes disappear during the developmental transition and only NAD-specific isoforms are found. Spinach chloroplasts contain both NAD/H and NADP/H at significant concentrations. Measurements of the pyridine dinucleotide redox states, performed under dark and various light conditions, indicate that NAD(H) is not involved in electron flow in the light. To analyze the contribution of NAD(H)-dependent reactions during dark metabolism, plastids from spinach leaves or green and red sweet-pepper fruits were incubated with dihydroxyacetone phosphate (DHAP). Exogenously added DHAP was oxidized into 3-phosphoglycerate by all types of plastids only in the presence of oxaloacetate, but not with nitrite or in the absence of added electron acceptors. We conclude that the NAD-dependent activity of GAPDH is essential in the dark to produce the ATP required for starch metabolism; excess electrons produced during triose-phosphate oxidation can selectively be used by NAD-MDH to form malate. Thus NADPH produced independently in the oxidative pentose-phosphate pathway will remain available for reductive processes inside the plastids. Received: 2 July 1997 / Accepted: 20 October 1997     

Purification and characterization of the malate dehydrogenase from Streptomyces aureofaciens

The malate dehydrogenase (MDH) from Streptomyces aureofaciens was purified to homogeneity and its physical and biochemical properties were studied. Its amino-terminal sequence perfectly matched the amino-terminal sequence of the MDH from Streptomyces atratus whose biochemical characteristics have never been determined. The molecular mass of the native enzyme, estimated by size-exclusion chromatography, was 70 kDa. The protein was a homodimer, with a 38-kDa subunit molecular mass. It showed a strong specificity for NADH and was much more efficient for the reduction of oxaloacetate than for the oxidation of malate, with a pH optimum of 8. Unlike MDHs from other sources, it was not inhibited by excess oxaloacetate. This first complete functional characterization of an MDH from Streptomyces shows that the enzyme is very similar in many respects to other bacterial MDHs with the notable exception of a lack of inhibition by excess substrate.     

Binding of malate dehydrogenase and NADH channelling to complex I

As previously reported, mitochondrial malate dehydrogenase (MDH) binds to purified complex I of the electron transport system. With conditions used in previous reports, MDH binds even more extensively, but probably predominantly non-specifically, to the matrix side of the inner mitochondrial membrane of submitochodrial particles (SMP). Herein we report experimental conditions for highly specific binding of malate dehydrogenase to complex I within SMP. These conditions permit us to demonstrate NADH channelling from malate dehydrogenase to complex I using the completing reaction test. This test, though not ideal for all situations, has several advantages over the enzyme buffering test previously used. These advantages should facilitate further studies elucidating NADH channeling to complex I from MDH and other dehydrogenases. Independent evidence of NADH channelling to the electron transport chain and the potential advantages of substrate channelling in general are also discussed. Substrate channelling from MDH in particular may be especially beneficial because of the unfavourable equilibrium and kinetics of this enzyme reaction.     

Purification and characterization of malate dehydrogenase from the syntrophic propionate-oxidizing bacterium strain MPOB

Abstract Malate dehydrogenase from the syntrophic propionate-oxidizing bacterium strain MPOB was purified 42-fold. The native enzyme had an apparent molecular mass of 68 kDa and consisted of two subunits of 35 kDa. The enzyme exhibited maximum activity with oxaloacetate at pH 8.5 and 60 °C. The K _m for oxaloacetate was 50 μM and for NADH 30 μM. The K _m values for l-malate and NAD were 4 and 1.1 mM, respectively. Substrate inhibition was found at oxaloacetate concentrations higher than 250 μM. The N-terminal amino acid sequence of the enzyme was similar to the sequences of a variety of other malate dehydrogenases from plants, animals and micro-organisms.     

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.     

Regulation of malate dehydrogenases from neonatal,adolescent, and mature rat brain

Since the malate-aspartate shuttle in brain has been shown to be closely linked to brain energy metabolism and neurotransmitter synthesis, the activity of MDH, one of the enzymes of the malateaspartate shuttle, was studied in cortical non-synaptic mitochondria (mMDH) and cytosol (cMDH) in 1–4 day, 18–20 day and 7–8 week old rats. The mean mMDH activity (nmol/min/mg protein) was 10,517±734 (mean±SEM), 8,882±241 and 10,323±561 and cMDH activity was 2,453±99, 4,673±152 and 6,821±205 in 1–4 day, 18–20 day and 7–8 week old rats, respectively. While cMDH activity increased with age (p<0.0001), mMDH activity showed no change. This study also determined if endogenous compounds, previously shown to alter malate metabolism, affected MDH activities. Lactate inhibited only cMDH activity, by a competitive mechanism. Oxaloacetate inhibited mMDH by partial non-competitive inhibition and cMDH by competitive inhibition. Alpha-ketoglutarate competitively inhibited both enzymes; however, the inhibition of mMDH activity was more pronounced than that of cMDH activity. Citrate inhibited mMDH via an uncompetitive mechanism and cMDH via a noncompetitive mechanism. The mechanisms of inhibition of mMDH and cMDH by each of the effectors were the same over the three ages. The results suggest mMDH and cMDH activities show a dissimilar developmental pattern and may be regulated differently by endogenous effectors. The greater sensitivity of mMDH, compared to cMDH, to certain effectors may be related to the dual role of mMDH in the tricarboxylic acid cycle and the malate-aspartate shuttle.These data were presented in part at the meeting of the Federation of American Societies for Experimental Biology in Atlanta, Georgia, April 1991. This work was performed in partial fulfillment of the requirements for the M.S. Degree in Nutritional Sciences (P.M.)     

Plasmalemma-associated malate dehydrogenase activity in onion root cells

Summary Plasma membrane vesicles isolated from onion roots showed oxaloacetate reductase activity as well as other oxidoreductase activities. Purification and further sequencing showed that the protein responsible for the activity is a 40 kDa protein which corresponds to the cytosolic soluble malate dehydrogenase. However, the activity remained bound to the membrane after repeated freezing and thawing cycles and further washing, excluding a cytosolic contamination as the source of the activity. Furthermore, a second 28 kDa protein has been copurified together with the 40 kDa protein. The plasmalemma oxaloacetate reductase activity shows both donor and acceptor sites located towards the cytoplasmic side of the plasma membrane. This enzyme catalyzed the oxidation of NADH by oxaloacetate and the reduction of NAD⁺ by malate in the presence of an oxaloacetate-withdrawing system. We conclude that a significant amount of the cytosolic malate dehydrogenase can be specifically attached to the cytosolic face of the plasmalemma. A possible role in a putative malate shuttle associated to the plasma membrane is discussed.Abbreviations AFR ascorbate free radical - DQ duroquinone - OA oxaloacetate - DPIP dichlorophenolindophenol - MDH malate dehydrogenase - PHMB p-hydroxymercuribenzoate     

Influence of an anion-binding site in the stabilization of halophilic malate dehydrogenase from Haloarcula marismortui

Halophilic proteins have evolved to be soluble, stable and active in high salt concentration. Crystallographic studies have shown that surface enrichment by acidic amino acids is a common structural feature of halophilic proteins. In addition, ion-binding sites have also been observed in most of the cases. The role of chloride-binding sites in halophilic adaptation was addressed in a site-directed mutagenesis study of tetrameric malate dehydrogenase from Haloarcula marismortui. The mutation of K 205, which is involved in an inter-subunit chloride-binding site, drastically modified the enzyme stability in the presence of KCl, but not in the presence of KF. The oligomeric state of the [K205A] mutant changes with the nature of the anion. At high salt concentration, the [K205A] mutant is a dimer when the anion is a chloride ion, whereas it is a tetramer when the fluoride ion is used. The results highlight the role of anion-binding sites in protein adaptation to high salt conditions.     

Characterization of the malate dehydrogenase from Thermoleophilum album NM

Malate dehydrogenase (MDH; EC 1.1.1.37) was characterized from Thermoleophilum album NM, a gram-negative aerobic bacterium obligate for thermophily and n-alkane substrates. The enzyme was purified by affinity chromatography and electroelution. The MDH had a mol.wt. of 61,000 and consisted of two subunits, each with a mol.wt. of 32,500. T. album NM MDH migrated further on nondenaturing polyacrylamide gels than did other MDHs. The MDH was active from 30°–95° C with optimum activity occurring at 60° C and pH 7.5. Kinetic data were determined at 60° C and pH 7.5. The K _m values for malate and NAD were 1.41 mM and 0.26 mM, respectively. The K _m for reduction of oxalacetate was 5.43 mM and 0.31 mM for NADH. The amino acid composition of T. album NM MDH differed in the amounts of Arg, Lys, Gly, Pro and His from the MDHs of other thermophilic and mesophilic organism. The N-terminal amino acid sequence had no appreciable homology with MDHs of other species.     

Sequence homologies between glyoxysomal and mitochondrial malate dehydrogenase

The comparison of mitochondrial and glyoxysomal malate dehydrogenase (EC 1.1.1.37) from cotyledons of germinating watermelon (Citrullus vulgaris Schrad., cv. Kleckey's Sweet No. 6) by means of serological methods and peptide patterns revealed a high degree of homology. The N-terminal sequence analysis yielded a distinct presequence of eight or nine amino-acid residues, respectively, which is followed by an almost identical stretch of at least 20 amino-acid residues. A very similar domain has been recognized for mitochondrial malate dehydrogenase from porcine heart and yeast, and for Escherichia coli malate dehydrogenase.Abbreviations gMDH glyoxysomal malate dehydrogenase - mMDH mitochondrial malate dehydrogenase - SDS sodium dodecyl sulfate     

Evidence for chloroplastic localization of spinach leaf NADP malate dehydrogenase activating factors

Dithiothreitol activation of spinach leaf NADP malate dehydrogenase is mediated by protein factors that have been partially purified by chromatography on DEAE cellulose. Evidence for their intrachloroplastic localization has been obtained.Abbreviations DTT dithiothreitol - MDH malate dehydrogenase     

Electrostatic interactions across the dimer-dimer interface contribute to the pH-dependent stability of a tetrameric malate dehydrogenase

Malate dehydrogenase (MDH) from the moderately thermophilic bacterium Chloroflexus aurantiacus (CaMDH) is a tetrameric enzyme, while MDHs from mesophilic bacteria usually are dimers. Using site-directed mutagenesis, we show here that a network of electrostatic interactions across the extra dimer-dimer interface in CaMDH is important for thermal stability and oligomeric integrity. Stability effects of single point mutations (E25Q, E25K, D56N, D56K) varied from −1.2°C to −26.8°C, and depended strongly on pH. Gel-filtration experiments indicated that the 26.8°C loss in stability observed for the D56K mutant at low pH was accompanied by a shift towards a lower oligomerization state.     

The chromosomal location of a third set of malate dehydrogenase loci,Mdh-3, in wheat,barley and related species

Summary A third set of malate dehydrogenase loci have been identified and located on the short arms of homoeologous group 5 chromosomes in wheat. Allelic differences have been found at each of the three Mdh-3 loci. However, Mdh-D3 appears to be least variable, with a second allele found only in Sears' Synthetic among a survey of 42 varieties. Homoeoloci were identified on chromosome 7 (5H) of Hordeum vulgare, the short arm of 5E in Agropyron elongatum and 5U in Aegilops umbellulata.     

Plasma-membrane-bound malate dehydrogenase activity in maize roots

Summary Plasma membranes were isolated and purified from 14-day-old maize roots (Zea mays L.) by two-phase partitioning at a 6.5% polymer concentration, and compared to isolated mitochondria, microsomes, and soluble fraction. Marker enzyme analysis demonstrated that the plasma membranes were devoid of cytoplasmic, mitochondrial, tonoplast, and endoplasmic-reticulum contaminations. Isolated plasma membranes exhibited malate dehydrogenase activity, catalyzing NADH-dependent reduction of oxaloacetate as well as NAD⁺-dependent malate oxidation. Malate dehydrogenase activity was resistant to osmotic shock, freeze-thaw treatment, and salt washing and stimulated by solubilization with Triton X-100, indicating that the enzyme is tightly bound to the plasma membrane. Malate dehydrogenase activity was highly specific to NAD⁺ and NADH. The enzyme exhibited a high degree of latency in both right-side-out (80%) and inside-out (70%) vesicle preparations. Kinetic and regulatory properties with ATP and P_i, as well as pH dependence of plasma-membrane-bound malate dehydrogenase were different from mitochondrial and soluble malate dehydrogenases. Starch gel electrophoresis revealed a characteristic isozyme form present in the plasma membrane isolate, but not present in the soluble, mitochondrial, and microsomal fractions. The results presented show that purified plasma membranes isolated from maize roots contain a tightly associated malate dehydrogenase, having properties different from mitochondrial and soluble malate dehydrogenases.Abbreviations FCR ferricyanide reductase - MDH malate dehydrogenase