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Evidence for a link between cellular senescence and metabolic regulation is provided, through the observation that p53 represses the expression of malic enzymes, thereby regulating NADPH, lipid and glutamine metabolism; in turn, this repression further activates p53, promoting cellular senescence. Malic enzyme interactions with p53 The tumour suppressor p53 is known to regulate metabolic processes as well as cellular senescence. Peng Jiang et al . now link the two activities, showing that p53 represses the expression of the malic enzymes ME1 and ME2 and thereby regulates NADPH production, lipid and glutamine metabolism. ME1 and ME2 downregulation can, in turn, further activate p53 and thereby promote cellular senescence. ME1 and ME2 are often overexpressed in cancers and can suppress senescence and promote tumour growth. Cellular senescence both protects multicellular organisms from cancer and contributes to their ageing 1 . The pre-eminent tumour suppressor p53 has an important role in the induction and maintenance of senescence, but how it carries out this function remains poorly understood 1 , 2 , 3 . In addition, although increasing evidence supports the idea that metabolic changes underlie many cell-fate decisions and p53-mediated tumour suppression, few connections between metabolic enzymes and senescence have been established. Here we describe a new mechanism by which p53 links these functions. We show that p53 represses the expression of the tricarboxylic-acid-cycle-associated malic enzymes ME1 and ME2 in human and mouse cells. Both malic enzymes are important for NADPH production, lipogenesis and glutamine metabolism, but ME2 has a more profound effect. Through the inhibition of malic enzymes, p53 regulates cell metabolism and proliferation. Downregulation of ME1 and ME2 reciprocally activates p53 through distinct MDM2- and AMP-activated protein kinase-mediated mechanisms in a feed-forward manner, bolstering this pathway and enhancing p53 activation. Downregulation of ME1 and ME2 also modulates the outcome of p53 activation, leading to strong induction of senescence, but not apoptosis, whereas enforced expression of either malic enzyme suppresses senescence. Our findings define physiological functions of malic enzymes, demonstrate a positive-feedback mechanism that sustains p53 activation, and reveal a connection between metabolism and senescence mediated by p53.

Abstract The evolution of C4 photosynthesis required extensive modification of ancestral enzymes enabling the development of an efficient carbon concentrating mechanism. A key example is NADP-malic enzyme (NADP-ME), which, in maize and sorghum—members of the same C4 lineage—underwent gene duplication and neofunctionalization, resulting in 2 plastidic isoforms with distinct oligomeric states: a tetrameric C4-specific isoform and a dimeric housekeeping (nonC4) isoform. In this study, we resolve the structural basis of this oligomeric divergence using X-ray crystallography, cryo-electron microscopy, and molecular modeling combined with targeted biochemical analysis. Our findings demonstrate that the N-terminal region of nonC4-NADP-ME is involved in its oligomeric organization, whereas a suite of adaptive substitutions at the dimer interface drives the transition to the stable tetramer characteristic of the C4 isoform. Moreover, the C-terminal region stabilizes the oligomeric states of C4- and nonC4-NADP-ME through specific interactions with adaptive residues. We propose that tetramerization mitigates aggregation at the high expression levels demanded by the C4 cycle and likely creates a scaffold for the emergence of regulatory properties. Collectively, the data show that remodeling of terminal domains and inter-subunit interfaces rewires the quaternary architecture of the enzymes, illustrating how subtle structural changes can drive the evolution of complex innovations such as C4 photosynthesis.

Four enzymes, namely, the maize C4-specific phosphoenolpyruvate carboxylase (PEPC), the maize C4-specific pyruvate, orthophosphate dikinase (PPDK), the sorghum NADP-malate dehydrogenase (MDH), and the rice C3-specific NADP-malic enzyme (ME), were overproduced in the mesophyll cells of rice plants independently or in combination. Overproduction individually of PPDK, MDH or ME did not affect the rate of photosynthetic CO2 assimilation, while in the case of PEPC it was slightly reduced. The reduction in CO2 assimilation in PEPC overproduction lines remained unaffected by overproduction of PPDK, ME or a combination of both, however it was significantly restored by the combined overproduction of PPDK, ME, and MDH to reach levels comparable to or slightly higher than that of non-transgenic rice. The extent of the restoration of CO2 assimilation, however, was more marked at higher CO2 concentrations, an indication that overproduction of the four enzymes in combination did not act to concentrate CO2 inside the chloroplast. Transgenic rice plants overproducing the four enzymes showed slight stunting. Comparison of transformants overproducing different combinations of enzymes indicated that overproduction of PEPC together with ME was responsible for stunting, and that overproduction of MDH had some mitigating effects. Possible mechanisms underlying these phenotypic effects, as well as possibilities and limitations of introducing the C4-like photosynthetic pathway into C3 plants, are discussed.

Succinic acid (SA), a dicarboxylic acid of industrial importance, can be efficiently produced by metabolically engineered Mannheimia succiniciproducens . Malate dehydrogenase (MDH) is one of the key enzymes for SA production, but has not been well characterized. Here we report biochemical and structural analyses of various MDHs and development of hyper-SA producing M. succiniciproducens by introducing the best MDH. Corynebacterium glutamicum MDH ( Cg MDH) shows the highest specific activity and least substrate inhibition, whereas M. succiniciproducens MDH ( Ms MDH) shows low specific activity at physiological pH and strong uncompetitive inhibition toward oxaloacetate ( ki of 67.4 and 588.9 μM for Ms MDH and Cg MDH, respectively). Structural comparison of the two MDHs reveals a key residue influencing the specific activity and susceptibility to substrate inhibition. A high-inoculum fed-batch fermentation of the final strain expressing cgmdh produces 134.25 g L −1 of SA with the maximum productivity of 21.3 g L −1  h −1 , demonstrating the importance of enzyme optimization in strain development. Malate dehydrogenase (MDH) is one of the key enzymes for succinic acid (SA) bioproduction. Here, the authors report biochemical and structural analyses of various MDHs to reveal amino acids influencing the specific activity and susceptibility to substrate inhibition, and achieve industrial-level SA production.

Abstract Allosteric regulation is a powerful mechanism for controlling the efficiency of enzymes. Deciphering the evolutionary mechanisms by which allosteric properties have been acquired in enzymes is of fundamental importance. We used the malate (MalDH) and lactate deydrogenases (LDHs) superfamily as model to elucidate this phenomenon. By introducing a few of mutations associated to the emergence of allosteric LDHs into the non-allosteric MalDH from Methanopyrus kandleri, we have gradually shifted its enzymatic profile toward that typical of allosteric LDHs. We first investigated the process triggering homotropic activation. The structures of the resulting mutants show the typical compact organization of the R-active state of LDHs, but a distorted (T-like) catalytic site demonstrating that they corresponds to hybrid states. Molecular dynamics simulations and free energy calculations confirmed the capability of these mutants to sample the T-inactive state. By adding a final single mutation to fine-tune the flexibility of the catalytic site, we obtained an enzyme with both sigmoid (homotropic) and hyperbolic (heterotropic) substrate activation profiles. Its structure shows a typical extended T-state as in LDHs, whereas its catalytic state has as a restored configuration favorable for catalysis. Free energy calculations indicate that the T and R catalytic site configurations are in an equilibrium that depends on solvent conditions. We observed long-range communication between monomers as required for allosteric activation. Our work links the evolution of allosteric regulation in the LDH/MDH superfamily to the ensemble model of allostery at molecular level, and highlights the important role of the underlying protein dynamics.

Enzymes of the central metabolism tend to assemble into transient supramolecular complexes. However, the functional significance of the interactions, particularly between enzymes catalyzing non-consecutive reactions, remains unclear. Here, by co-localizing two non-consecutive enzymes of the TCA cycle from Bacillus subtilis , malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICD), in phase separated droplets we show that MDH-ICD interaction leads to enzyme agglomeration with a concomitant enhancement of ICD catalytic rate and an apparent sequestration of its reaction product, 2-oxoglutarate. Theory demonstrates that MDH-mediated clustering of ICD molecules explains the observed phenomena. In vivo analyses reveal that MDH overexpression leads to accumulation of 2-oxoglutarate and reduction of fluxes flowing through both the catabolic and anabolic branches of the carbon-nitrogen intersection occupied by 2-oxoglutarate, resulting in impeded ammonium assimilation and reduced biomass production. Our findings suggest that the MDH-ICD interaction is an important coordinator of carbon-nitrogen metabolism. Enzymes of central metabolism tend to assemble into transient supramolecular complexes. Here, the authors stoichiometrically perturbed the supramolecular complex of TCA cycle enzymes in B. subtilis and propose that MDH-ICD clustering causes 2-oxoglutartae sequestration by reducing its diffusion rate, a mechanism that has evolved to regulate flux through the carbon-nitrogen metabolic branch-point.

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.

Depletion of malic enzyme 3 in pancreatic cancer cells that have a deletion of the gene for malic enzyme 2 selectively kills the cells, suggesting that the enzyme might represent a therapeutic target for this subset of cancers. Gene deletion in pancreatic cancer In pancreatic cancer, a commonly deleted genomic region includes the gene that encodes metabolic malic enzyme 2, ME2. The authors show that pancreatic tumour cells depend on the activity of the other malic enzyme isoform, ME3, for survival. This finding reveals a metabolic collateral vulnerability that may be further explored to develop therapeutic strategies. The genome of pancreatic ductal adenocarcinoma (PDAC) frequently contains deletions of tumour suppressor gene loci, most notably SMAD4 , which is homozygously deleted in nearly one-third of cases 1 . As loss of neighbouring housekeeping genes can confer collateral lethality, we sought to determine whether loss of the metabolic gene malic enzyme 2 ( ME2 ) in the SMAD4 locus would create cancer-specific metabolic vulnerability upon targeting of its paralogous isoform ME3 . The mitochondrial malic enzymes (ME2 and ME3) are oxidative decarboxylases that catalyse the conversion of malate to pyruvate and are essential for NADPH regeneration and reactive oxygen species homeostasis 2 , 3 . Here we show that ME3 depletion selectively kills ME2 -null PDAC cells in a manner consistent with an essential function for ME3 in ME2 -null cancer cells. Mechanistically, integrated metabolomic and molecular investigation of cells deficient in mitochondrial malic enzymes revealed diminished NADPH production and consequent high levels of reactive oxygen species. These changes activate AMP activated protein kinase (AMPK), which in turn directly suppresses sterol regulatory element-binding protein 1 (SREBP1)-directed transcription of its direct targets including the BCAT2 branched-chain amino acid transaminase 2) gene. BCAT2 catalyses the transfer of the amino group from branched-chain amino acids to α-ketoglutarate (α-KG) 4 thereby regenerating glutamate, which functions in part to support de novo nucleotide synthesis. Thus, mitochondrial malic enzyme deficiency, which results in impaired NADPH production, provides a prime ‘collateral lethality’ therapeutic strategy for the treatment of a substantial fraction of patients diagnosed with this intractable disease.

Increased glucose consumption is a hallmark of cancer cells. The increased consumption and subsequent metabolism of glucose during proliferation creates the need for a constant supply of NAD, a co-factor in glycolysis. Regeneration of the NAD required to support enhanced glycolysis has been attributed to the terminal glycolytic enzyme, lactate dehydrogenase (LDH). However, loss of glucose carbons to biosynthetic pathways early in glycolysis reduces the carbon supply to LDH. Thus, alternative routes for NAD regeneration must exist to support the increased glycolytic rate while allowing for the diversion of glucose to generate biomass and support proliferation. Here we demonstrate, using a variety of cancer cell lines as well as activated primary T cells, that cytosolic malate dehydrogenase 1 (MDH1) is an alternative to LDH as a supplier of NAD. Moreover, our results indicate that MDH1 generates malate with carbons derived from glutamine, thus enabling utilization of glucose carbons for glycolysis and for biomass. Amplification of MDH1 occurs at an impressive frequency in human tumors and correlates with poor prognosis. Together, our findings suggest that proliferating cells rely on both MDH1 and LDH to replenish cytosolic NAD, and that therapies designed at targeting glycolysis must consider both dehydrogenases.

Summary In this study, we characterized the role of an apple cytosolic malate dehydrogenase gene (MdcyMDH) in the tolerance to salt and cold stresses and investigated its regulation mechanism in stress tolerance. The MdcyMDH transcript was induced by mild cold and salt treatments, and MdcyMDH‐overexpressing apple plants possessed improved cold and salt tolerance compared to wild‐type (WT) plants. A digital gene expression tag profiling analysis revealed that MdcyMDH overexpression largely altered some biological processes, including hormone signal transduction, photosynthesis, citrate cycle and oxidation–reduction. Further experiments verified that MdcyMDH overexpression modified the mitochondrial and chloroplast metabolisms and elevated the level of reducing power, primarily caused by increased ascorbate and glutathione, as well as the increased ratios of ascorbate/dehydroascorbate and glutathione/glutathione disulphide, under normal and especially stress conditions. Concurrently, the transgenic plants produced a high H2O2 content, but a low O2·− production rate was observed compared to the WT plants. On the other hand, the transgenic plants accumulated more free and total salicylic acid (SA) than the WT plants under normal and stress conditions. Taken together, MdcyMDH conferred the transgenic apple plants a higher stress tolerance by producing more reductive redox states and increasing the SA level; MdcyMDH could serve as a target gene to genetically engineer salt‐ and cold‐tolerant trees.