Redox regulation of NADP-malate dehydrogenase is vital for land plants under fluctuating light environment.

Many enzymes involved in photosynthesis possess highly conserved cysteine residues that serve as redox switches in chloroplasts. These redox switches function to activate or deactivate enzymes during light-dark transitions and have the function of fine-tuning their activities according to the intensity of light. Accordingly, many studies on chloroplast redox regulation have been conducted under the hypothesis that "fine regulation of the activities of these enzymes is crucial for efficient photosynthesis." However, the impact of the regulatory system on plant metabolism is still unclear. To test this hypothesis, we here studied the impact of the ablation of a redox switch in chloroplast NADP-malate dehydrogenase (MDH). By genome editing, we generated a mutant plant whose MDH lacks one of its redox switches and is active even in dark conditions. Although NADPH consumption by MDH in the dark is expected to be harmful to plant growth, the mutant line did not show any phenotypic differences under standard long-day conditions. In contrast, the mutant line showed severe growth retardation under short-day or fluctuating light conditions. These results indicate that thiol-switch redox regulation of MDH activity is crucial for maintaining NADPH homeostasis in chloroplasts under these conditions.

Thermal adaptation of mRNA secondary structure: stability versus lability.

Macromolecular function commonly involves rapidly reversible alterations in three-dimensional structure (conformation). To allow these essential conformational changes, macromolecules must possess higher order structures that are appropriately balanced between rigidity and flexibility. Because of the low stabilization free energies (marginal stabilities) of macromolecule conformations, temperature changes have strong effects on conformation and, thereby, on function. As is well known for proteins, during evolution, temperature-adaptive changes in sequence foster retention of optimal marginal stability at a species' normal physiological temperatures. Here, we extend this type of analysis to messenger RNAs (mRNAs), a class of macromolecules for which the stability-lability balance has not been elucidated. We employ in silico methods to determine secondary structures and estimate changes in free energy of folding (ΔGfold) for 25 orthologous mRNAs that encode the enzyme cytosolic malate dehydrogenase in marine mollusks with adaptation temperatures spanning an almost 60 °C range. The change in free energy that occurs during formation of the ensemble of mRNA secondary structures is significantly correlated with adaptation temperature: ΔGfold values are all negative and their absolute values increase with adaptation temperature. A principal mechanism underlying these adaptations is a significant increase in synonymous guanine + cytosine substitutions with increasing temperature. These findings open up an avenue of exploration in molecular evolution and raise interesting questions about the interaction between temperature-adaptive changes in mRNA sequence and in the proteins they encode.

Effects of Aerobic Treadmill Training on Oxidative Stress Parameters, Metabolic Enzymes, and Histomorphometric Changes in Colon of Rats with Experimentally Induced Hyperhomocysteinemia.

The aim of this study was to investigate the effects of aerobic treadmill training regimen of four weeks duration on oxidative stress parameters, metabolic enzymes, and histomorphometric changes in the colon of hyperhomocysteinemic rats. Male Wistar albino rats were divided into four groups (n = 10, per group): C, 0.9% NaCl 0.2 mL/day subcutaneous injection (s.c.) 2x/day; H, homocysteine 0.45 µmol/g b.w./day s.c. 2x/day; CPA, saline (0.9% NaCl 0.2 mL/day s.c. 2x/day) and an aerobic treadmill training program; and HPA, homocysteine (0.45 µmol/g b.w./day s.c. 2x/day) and an aerobic treadmill training program. The HPA group had an increased level of malondialdehyde (5.568 ± 0.872 µmol/mg protein, p = 0.0128 vs. CPA (3.080 ± 0.887 µmol/mg protein)), catalase activity (3.195 ± 0.533 U/mg protein, p < 0.0001 vs. C (1.467 ± 0.501 U/mg protein), p = 0.0012 vs. H (1.955 ± 0.293 U/mg protein), and p = 0.0003 vs. CPA (1.789 ± 0.256 U/mg protein)), and total superoxide dismutase activity (9.857 ± 1.566 U/mg protein, p < 0.0001 vs. C (6.738 ± 0.339 U/mg protein), p < 0.0001 vs. H (6.015 ± 0.424 U/mg protein), and p < 0.0001 vs. CPA (5.172 ± 0.284 U/mg protein)) were detected in the rat colon. In the HPA group, higher activities of lactate dehydrogenase (2.675 ± 1.364 mU/mg protein) were detected in comparison to the CPA group (1.198 ± 0.217 mU/mg protein, p = 0.0234) and higher activities of malate dehydrogenase (9.962 (5.752-10.220) mU/mg protein) were detected in comparison to the CPA group (4.727 (4.562-5.299) mU/mg protein, p = 0.0385). Subchronic treadmill training in the rats with hyperhomocysteinemia triggers the colon tissue antioxidant response (by increasing the activities of superoxide dismutase and catalase) and elicits an increase in metabolic enzyme activities (lactate dehydrogenase and malate dehydrogenase). This study offers a comprehensive assessment of the effects of aerobic exercise on colonic tissues in a rat model of hyperhomocysteinemia, evaluating a range of biological indicators including antioxidant enzyme activity, metabolic enzyme activity, and morphometric parameters, which suggested that exercise may confer protective effects at both the physiological and morphological levels.

Biochemical, structural and dynamical characterizations of the lactate dehydrogenase from Selenomonas ruminantium provide information about an intermediate evolutionary step prior to complete allosteric regulation acquisition in the super family of lactate and malate dehydrogenases.

In this work, we investigated the lactate dehydrogenase (LDH) from Selenomonas ruminantium (S. rum), an enzyme that differs at key amino acid positions from canonical allosteric LDHs. The wild type (Wt) of this enzyme recognises pyuvate as all LDHs. However, introducing a single point mutation in the active site loop (I85R) allows S. Rum LDH to recognize the oxaloacetate substrate as a typical malate dehydrogenase (MalDH), whilst maintaining homotropic activation as an LDH. We report the tertiary structure of the Wt and I85RLDH mutant. The Wt S. rum enzyme structure binds NADH and malonate, whilst also resembling the typical compact R-active state of canonical LDHs. The structure of the mutant with I85R was solved in the Apo State (without ligand), and shows no large conformational reorganization such as that observed with canonical allosteric LDHs in Apo state. This is due to a local structural feature typical of S. rum LDH that prevents large-scale conformational reorganization. The S. rum LDH was also studied using Molecular Dynamics simulations, probing specific local deformations of the active site that allow the S. rum LDH to sample the T-inactive state. We propose that, with respect to the LDH/MalDH superfamily, the S. rum enzyme possesses a specificstructural and dynamical way to ensure homotropic activation.

Deciphering functional redundancy and energetics of malate oxidation in mycobacteria.

Oxidation of malate to oxaloacetate, catalyzed by either malate dehydrogenase (Mdh) or malate quinone oxidoreductase (Mqo), is a critical step of the tricarboxylic acid cycle. Both Mqo and Mdh are found in most bacterial genomes, but the level of functional redundancy between these enzymes remains unclear. A bioinformatic survey revealed that Mqo was not as widespread as Mdh in bacteria but that it was highly conserved in mycobacteria. We therefore used mycobacteria as a model genera to study the functional role(s) of Mqo and its redundancy with Mdh. We deleted mqo from the environmental saprophyte Mycobacterium smegmatis, which lacks Mdh, and found that Mqo was essential for growth on nonfermentable carbon sources. On fermentable carbon sources, the Δmqo mutant exhibited delayed growth and lowered oxygen consumption and secreted malate and fumarate as terminal end products. Furthermore, heterologous expression of Mdh from the pathogenic species Mycobacterium tuberculosis shortened the delayed growth on fermentable carbon sources and restored growth on nonfermentable carbon sources at a reduced growth rate. In M. tuberculosis, CRISPR interference of either mdh or mqo expression resulted in a slower growth rate compared to controls, which was further inhibited when both genes were knocked down simultaneously. These data reveal that exergonic Mqo activity powers mycobacterial growth under nonenergy limiting conditions and that endergonic Mdh activity complements Mqo activity, but at an energetic cost for mycobacterial growth. We propose Mdh is maintained in slow-growing mycobacterial pathogens for use under conditions such as hypoxia that require reductive tricarboxylic acid cycle activity.

Acetylation of conserved lysines fine-tunes mitochondrial malate dehydrogenase activity in land plants.

Plants need to rapidly and flexibly adjust their metabolism to changes of their immediate environment. Since this necessity results from the sessile lifestyle of land plants, key mechanisms for orchestrating central metabolic acclimation are likely to have evolved early. Here, we explore the role of lysine acetylation as a post-translational modification to directly modulate metabolic function. We generated a lysine acetylome of the moss Physcomitrium patens and identified 638 lysine acetylation sites, mostly found in mitochondrial and plastidial proteins. A comparison with available angiosperm data pinpointed lysine acetylation as a conserved regulatory strategy in land plants. Focusing on mitochondrial central metabolism, we functionally analyzed acetylation of mitochondrial malate dehydrogenase (mMDH), which acts as a hub of plant metabolic flexibility. In P. patens mMDH1, we detected a single acetylated lysine located next to one of the four acetylation sites detected in Arabidopsis thaliana mMDH1. We assessed the kinetic behavior of recombinant A. thaliana and P. patens mMDH1 with site-specifically incorporated acetyl-lysines. Acetylation of A. thaliana mMDH1 at K169, K170, and K334 decreases its oxaloacetate reduction activity, while acetylation of P. patens mMDH1 at K172 increases this activity. We found modulation of the malate oxidation activity only in A. thaliana mMDH1, where acetylation of K334 strongly activated it. Comparative homology modeling of MDH proteins revealed that evolutionarily conserved lysines serve as hotspots of acetylation. Our combined analyses indicate lysine acetylation as a common strategy to fine-tune the activity of central metabolic enzymes with likely impact on plant acclimation capacity.

Protein Conformational Space at the Edge of Allostery: Turning a Nonallosteric Malate Dehydrogenase into an "Allosterized" Enzyme Using Evolution-Guided Punctual Mutations.

We unveil the intimate relationship between protein dynamics and allostery by following the trajectories of model proteins in their conformational and sequence spaces. Starting from a nonallosteric hyperthermophilic malate dehydrogenase, we have tracked the role of protein dynamics in the evolution of the allosteric capacity. Based on a large phylogenetic analysis of the malate (MalDH) and lactate dehydrogenase (LDH) superfamily, we identified two amino acid positions that could have had a major role for the emergence of allostery in LDHs, which we targeted for investigation by site-directed mutagenesis. Wild-type MalDH and the single and double mutants were tested with respect to their substrate recognition profiles. The double mutant displayed a sigmoid-shaped profile typical of homotropic activation in LDH. By using molecular dynamics simulations, we showed that the mutations induce a drastic change in the protein sampling of its conformational landscape, making transiently T-like (inactive) conformers, typical of allosteric LDHs, accessible. Our data fit well with the seminal key concept linking protein dynamics and evolvability. We showed that the selection of a new phenotype can be achieved by a few key dynamics-enhancing mutations causing the enrichment of low-populated conformational substates.

Human cytosolic transaminases: side activities and patterns of discrimination towards physiologically available alternative substrates.

Transaminases play key roles in central metabolism, transferring the amino group from a donor substrate to an acceptor. These enzymes can often act, with low efficiency, on compounds different from the preferred substrates. To understand what might have shaped the substrate specificity of this class of enzymes, we examined the reactivity of six human cytosolic transaminases towards amino acids whose main degradative pathways do not include any transamination. We also tested whether sugars and sugar phosphates could serve as alternative amino group acceptors for these cytosolic enzymes. Each of the six aminotransferases reacted appreciably with at least three of the alternative amino acid substrates in vitro, albeit at usually feeble rates. Reactions with L-Thr, L-Arg, L-Lys and L-Asn were consistently very slow-a bias explained in part by the structural differences between these amino acids and the preferred substrates of the transaminases. On the other hand, L-His and L-Trp reacted more efficiently, particularly with GTK (glutamine transaminase K; also known as KYAT1). This points towards a role of GTK in the salvage of L-Trp (in cooperation with ω-amidase and possibly with the cytosolic malate dehydrogenase, MDH1, which efficiently reduced the product of L-Trp transamination). Finally, the transaminases were extremely ineffective at utilizing sugars and sugar derivatives, with the exception of the glycolytic intermediate dihydroxyacetone phosphate, which was slowly but appreciably transaminated by some of the enzymes to yield serinol phosphate. Evidence for the formation of this compound in a human cell line was also obtained. We discuss the biological and evolutionary implications of our results.

Genome-Wide Identification and Expression Analysis of Malate Dehydrogenase Gene Family in Sweet Potato and Its Two Diploid Relatives.

Malate dehydrogenase (MDH; EC 1.1.1.37) plays a vital role in plant growth and development as well as abiotic stress responses, and it is widely present in plants. However, the MDH family genes have not been explored in sweet potato. In this study, nine, ten, and ten MDH genes in sweet potato (Ipomoea batatas) and its two diploid wild relatives, Ipomoea trifida and Ipomoea triloba, respectively, were identified. These MDH genes were unevenly distributed on seven different chromosomes among the three species. The gene duplications and nucleotide substitution analysis (Ka/Ks) revealed that the MDH genes went through segmental duplications during their evolution under purifying selection. A phylogenetic and conserved structure divided these MDH genes into five subgroups. An expression analysis indicated that the MDH genes were omni-presently expressed in distinct tissues and responded to various abiotic stresses. A transcription factor prediction analysis proved that Dof, MADS-box, and MYB were the main transcription factors of sweet potato MDH genes. These findings provide molecular features of the MDH family in sweet potato and its two diploid wild relatives, which further supports functional characterizations.

Light-Dependent Expression and Promoter Methylation of the Genes Encoding Succinate Dehydrogenase, Fumarase, and NAD-Malate Dehydrogenase in Maize (Zea mays L.) Leaves.

The expression and methylation of promoters of the genes encoding succinate dehydrogenase, fumarase, and NAD-malate dehydrogenase in maize (Zea mays L.) leaves depending on the light regime were studied. The genes encoding the catalytic subunits of succinate dehydrogenase showed suppression of expression upon irradiation by red light, which was abolished by far-red light. This was accompanied by an increase in promoter methylation of the gene Sdh1-2 encoding the flavoprotein subunit A, while methylation was low for Sdh2-3 encoding the iron-sulfur subunit B under all conditions. The expression of Sdh3-1 and Sdh4 encoding the anchoring subunits C and D was not affected by red light. The expression of Fum1 encoding the mitochondrial form of fumarase was regulated by red and far-red light via methylation of its promoter. Only one gene encoding the mitochondrial NAD-malate dehydrogenase gene (mMdh1) was regulated by red and far-red light, while the second gene (mMdh2) did not respond to irradiation, and neither gene was controlled by promoter methylation. It is concluded that the dicarboxylic branch of the tricarboxylic acid cycle is regulated by light via the phytochrome mechanism, and promoter methylation is involved with the flavoprotein subunit of succinate dehydrogenase and the mitochondrial fumarase.

Reconstitution of oxaloacetate metabolism in the tricarboxylic acid cycle in Synechocystis sp. PCC 6803: discovery of important factors that directly affect the conversion of oxaloacetate.

The tricarboxylic acid (TCA) cycle is one of the most important metabolic pathways in nature. Oxygenic photoautotrophic bacteria, cyanobacteria, have an unusual TCA cycle. The TCA cycle in cyanobacteria contains two unique enzymes that are not part of the TCA cycle in other organisms. In recent years, sustainable metabolite production from carbon dioxide using cyanobacteria has been looked at as a means to reduce the environmental burden of this gas. Among cyanobacteria, the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) is an optimal host for sustainable metabolite production. Recently, metabolite production using the TCA cycle in Synechocystis 6803 has been carried out. Previous studies revealed that the branch point of the oxidative and reductive TCA cycles, oxaloacetate metabolism, plays a key role in metabolite production. However, the biochemical mechanisms regulating oxaloacetate metabolism in Synechocystis 6803 are poorly understood. Concentrations of oxaloacetate in Synechocystis 6803 are extremely low, such that in vivo analysis of oxaloacetate metabolism does not seem realistic. Therefore, using purified enzymes, we reconstituted oxaloacetate metabolism in Synechocystis 6803 in vitro to reveal the regulatory mechanisms involved. Reconstitution of oxaloacetate metabolism revealed that pH, Mg2+ and phosphoenolpyruvate are important factors affecting the conversion of oxaloacetate in the TCA cycle. Biochemical analyses of the enzymes involved in oxaloacetate metabolism in this and previous studies revealed the biochemical mechanisms underlying the effects of these factors on oxaloacetate conversion. In addition, we clarified the function of two l-malate dehydrogenase isozymes in oxaloacetate metabolism. These findings serve as a basis for various applications of the cyanobacterial TCA cycle.

Resurrection of Ancestral Malate Dehydrogenases Reveals the Evolutionary History of Halobacterial Proteins: Deciphering Gene Trajectories and Changes in Biochemical Properties.

Extreme halophilic Archaea thrive in high salt, where, through proteomic adaptation, they cope with the strong osmolarity and extreme ionic conditions of their environment. In spite of wide fundamental interest, however, studies providing insights into this adaptation are scarce, because of practical difficulties inherent to the purification and characterization of halophilic enzymes. In this work, we describe the evolutionary history of malate dehydrogenases (MalDH) within Halobacteria (a class of the Euryarchaeota phylum). We resurrected nine ancestors along the inferred halobacterial MalDH phylogeny, including the Last Common Ancestral MalDH of Halobacteria (LCAHa) and compared their biochemical properties with those of five modern halobacterial MalDHs. We monitored the stability of these various MalDHs, their oligomeric states and enzymatic properties, as a function of concentration for different salts in the solvent. We found that a variety of evolutionary processes, such as amino acid replacement, gene duplication, loss of MalDH gene and replacement owing to horizontal transfer resulted in significant differences in solubility, stability and catalytic properties between these enzymes in the three Halobacteriales, Haloferacales, and Natrialbales orders since the LCAHa MalDH. We also showed how a stability trade-off might favor the emergence of new properties during adaptation to diverse environmental conditions. Altogether, our results suggest a new view of halophilic protein adaptation in Archaea.

A piggybacking mechanism enables peroxisomal localization of the glyoxylate cycle enzyme Mdh2 in yeast.

Eukaryotic cells have evolved organelles that allow the compartmentalization and regulation of metabolic processes. Knowledge of molecular mechanisms that allow temporal and spatial organization of enzymes within organelles is therefore crucial for understanding eukaryotic metabolism. Here, we show that the yeast malate dehydrogenase 2 (Mdh2) is dually localized to the cytosol and to peroxisomes and is targeted to peroxisomes via association with Mdh3 and a Pex5-dependent piggybacking mechanism. This dual localization of Mdh2 contributes to our understanding of the glyoxylate cycle and provides a new perspective on compartmentalization of cellular metabolism, which is critical for the perception of metabolic disorders and aging.

Comparing mutagenesis and simulations as tools for identifying functionally important sequence changes for protein thermal adaptation.

Comparative studies of orthologous proteins of species evolved at different temperatures have revealed consistent patterns of temperature-related variation in thermal stabilities of structure and function. However, the precise mechanisms by which interspecific variations in sequence foster these adaptive changes remain largely unknown. Here, we compare orthologs of cytosolic malate dehydrogenase (cMDH) from marine molluscs adapted to temperatures ranging from -1.9 °C (Antarctica) to ∼55 °C (South China coast) and show how amino acid usage in different regions of the enzyme (surface, intermediate depth, and protein core) varies with adaptation temperature. This eukaryotic enzyme follows some but not all of the rules established in comparisons of archaeal and bacterial proteins. To link the effects of specific amino acid substitutions with adaptive variations in enzyme thermal stability, we combined site-directed mutagenesis (SDM) and in vitro protein experimentation with in silico mutagenesis using molecular dynamics simulation (MDS) techniques. SDM and MDS methods generally but not invariably yielded common effects on protein stability. MDS analysis is shown to provide insights into how specific amino acid substitutions affect the conformational flexibilities of mobile regions (MRs) of the enzyme that are essential for binding and catalysis. Whereas these substitutions invariably lie outside of the MRs, they effectively transmit their flexibility-modulating effects to the MRs through linked interactions among surface residues. This discovery illustrates that regions of the protein surface lying outside of the site of catalysis can help establish an enzyme's thermal responses and foster evolutionary adaptation of function.

Identification and Characterization of Malate Dehydrogenases in Tomato (Solanum lycopersicum L.).

Malate dehydrogenase, which facilitates the reversible conversion of malate to oxaloacetate, is essential for energy balance, plant growth, and cold and salt tolerance. However, the genome-wide study of the MDH family has not yet been carried out in tomato (Solanum lycopersicum L.). In this study, 12 MDH genes were identified from the S. lycopersicum genome and renamed according to their chromosomal location. The tomato MDH genes were split into five groups based on phylogenetic analysis and the genes that clustered together showed similar lengths, and structures, and conserved motifs in the encoded proteins. From the 12 tomato MDH genes on the chromosomes, three pairs of segmental duplication events involving four genes were found. Each pair of genes had a Ka/Ks ratio < 1, indicating that the MDH gene family of tomato was purified during evolution. Gene expression analysis exhibited that tomato MDHs were differentially expressed in different tissues, at various stages of fruit development, and differentially regulated in response to abiotic stresses. Molecular docking of four highly expressed MDHs revealed their substrate and co-factor specificity in the reversible conversion process of malate to oxaloacetate. Further, co-localization of tomato MDH genes with quantitative trait loci (QTL) of salt stress-related phenotypes revealed their broader functions in salt stress tolerance. This study lays the foundation for functional analysis of MDH genes and genetic improvement in tomato.

Effect of acute hypoxia on the brain energy metabolism of the scorpionfish Scorpaena porcus Linnaeus, 1758: the pattern of oxidoreductase activity and adenylate system.

The activity of oxidoreductases, malate dehydrogenase and lactate dehydrogenase (MDH, 1.1.1.37; LDH, 1.1.1.27), as well as parameters of adenylate system-[ATP], [ADP], [AMP], total adenylate pool (AP), and adenylate energy charge (AEC) in medulla oblongata (MB) and forebrain, midbrain, and diencephalon (FDMB)-were studied in the scorpionfish under acute hypoxia (0.9-1.2 mg O2·L-1, 90 min). A higher MDH activity level was observed in MB and FDMB, as compared to LDH (p < 0.05). At the same time, MB showed a higher adenylate content and increased AP (p < 0.05). AEC did not exceed ~ 0.7 (vs. the maximum of this index ~ 0.9-1.0) in the brain of the scorpionfish indicating adaptation of the tissue energy status to hypoxia. A rapid decrease in MDH activity (p < 0.05) was observed in MB under acute hypoxia. These changes were accompanied by insignificant LDH activation. A pronounced LDH activation (p < 0.05), a decrease in MDH activity, and the highest AP raise (p < 0.05) were observed in FDMB, suggesting activation of glycolysis and simultaneous decrease in the rate of ATP consumption. MB and FDMB demonstrated the ability to a relative retention of AEC during hypoxia. The unidirectional metabolic adaptation was based on the intensification of glycolysis, a decrease of ATP consumption, and a subsequent increase in adenylate concentration that allowed the scorpionfish brain structures to maintain the energy status under acute hypoxia.

Urinary malate dehydrogenase 2 is a new biomarker for early detection of non-small-cell lung cancer.

Reliable and noninvasive biomarkers for the early diagnosis of non-small-cell lung cancer (NSCLC) are an unmet need. This study aimed to screen and validate potential urinary biomarkers for the early diagnosis of NSCLC. Using protein mass spectrometry, urinary MDH2 was found to be abundant both in patients with lung cancer and lung cancer model mice compared with controls. Urine samples obtained as retrospective and prospective cohorts including 1091 NSCLC patients and 736 healthy controls were measured using ELISA. Patients with stage I NSCLC had higher urinary MDH2 compared with healthy controls. The area under the receiver-operating characteristic curve (AUC) for the urinary MDH2 was 0.7679 and 0.7234 in retrospective and prospective cohorts to distinguish stage I cases from controls. Urinary MDH2 levels correlated with gender and smoking history. MDH2 expression levels were elevated in lung cancer tissues. MDH2 knockdown using shRNA inhibited the proliferation of lung cancer cells. Our study demonstrated that urinary MDH2 concentration was higher in early-stage NSCLC patients compared with that in controls and that MDH2 could serve as a potential biomarker for early detection of NSCLC.

Distinct sequence and structural feature of trypanosoma malate dehydrogenase.

Glycosomal malate dehydrogenase from Trypanosoma cruzi (tcgMDH) catalyzes the oxidation/reduction of malate/oxaloacetate, a crucial step of the glycolytic process occurring in the glycosome of the human parasite. Inhibition of tcgMDH is considered a druggable trait for the development of trypanocidal drugs. Sequence comparison of MDHs from different organisms revealed a distinct insertion of a prolin rich 9-mer (62-KLPPVPRDP-70) in tcgMDH as compared to other eukaryotic MDHs. Crystal structure of tcgMDH is solved here at 2.6 Å resolution with Rwork/Rfree values of 0.206/0.216. The tcgMDH forms homo-dimer with the solvation free energy (ΔGo) gain of -9.77 kcal/mol. The dimeric form is also confirmed in solution by biochemical assays, chemical-crosslinking and dynamic light scattering. The inserted 9-mer adopts a structure of a solvent accessible loop in the vicinity of NAD+ binding site. The distinct sequence and structural feature of tcgMDH, revealed in the present report, provides an anchor point for the development of inhibitors specific for tcgMDH, possible trypanocidal agents of the future.

Profile of protein lysine propionylation in Aeromonas hydrophila and its role in enzymatic regulation.

Protein lysine propionylation (Kpr) modification is a novel post-translational modification (PTM) of prokaryotic cells that was recently discovered; however, it is not clear how this modification regulates bacterial life. In this study, the protein Kpr modification profile in Aeromonas hydrophila was identified by high specificity antibody-based affinity enrichment combined with high resolution LC MS/MS. A total of 98 lysine-propionylated peptides with 59 Kpr proteins were identified, most of which were associated with energy metabolism, transcription and translation processes. To further understand the role of Kpr modified proteins, the K168 site on malate dehydrogenase (MDH) and K608 site on acetyl-coenzyme A synthetase (AcsA) were subjected to site-directed mutation to arginine (R) and glutamine (Q) to simulate deacylation and propionylation, respectively. Subsequent measurement of the enzymatic activity showed that the K168 site of Kpr modification on MDH may negatively regulate the MDH enzymatic activity while also affecting the survival of mdh derivatives when using glucose as the carbon source, whereas Kpr modification of K608 of AcsA does not. Overall, the results of this study indicate that protein Kpr modification plays an important role in bacterial biological functions, especially those involved in the activity of metabolic enzymes.

Four-carbon dicarboxylic acid production through the reductive branch of the open cyanobacterial tricarboxylic acid cycle in Synechocystis sp. PCC 6803.

Succinate, fumarate, and malate are valuable four-carbon (C4) dicarboxylic acids used for producing plastics and food additives. C4 dicarboxylic acid is biologically produced by heterotrophic organisms. However, current biological production requires organic carbon sources that compete with food uses. Herein, we report C4 dicarboxylic acid production from CO2 using metabolically engineered Synechocystis sp. PCC 6803. Overexpression of citH, encoding malate dehydrogenase (MDH), resulted in the enhanced production of succinate, fumarate, and malate. citH overexpression increased the reductive branch of the open cyanobacterial tricarboxylic acid (TCA) cycle flux. Furthermore, product stripping by medium exchanges increased the C4 dicarboxylic acid levels; product inhibition and acidification of the media were the limiting factors for succinate production. Our results demonstrate that MDH is a key regulator that activates the reductive branch of the open cyanobacterial TCA cycle. The study findings suggest that cyanobacteria can act as a biocatalyst for converting CO2 to carboxylic acids.