Tuberculosis, the second leading infectious disease killer after HIV, remains a top public health priority. The causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), which can cause both acute and clinically latent infections, reprograms metabolism in response to the host niche. Phosphoenolpyruvate carboxykinase (Pck) is the enzyme at the center of the phosphoenolpyruvate-pyruvate-oxaloacetate node, which is involved in regulating the carbon flow distribution to catabolism, anabolism, or respiration in different states of Mtb infection. Under standard growth conditions, Mtb Pck is associated with gluconeogenesis and catalyzes the metal-dependent formation of phosphoenolpyruvate. In non-replicating Mtb, Pck can catalyze anaplerotic biosynthesis of oxaloacetate. Here, we present insights into the regulation of Mtb Pck activity by divalent cations. Through analysis of the X-ray structure of Pck-GDP and Pck-GDP-Mn2+ complexes, mutational analysis of the GDP binding site, and quantum mechanical (QM)-based analysis, we explored the structural determinants of efficient Mtb Pck catalysis. We demonstrate that Mtb Pck requires presence of Mn2+ and Mg2+ cations for efficient catalysis of gluconeogenic and anaplerotic reactions. The anaplerotic reaction, which preferably functions in reducing conditions that are characteristic for slowed or stopped Mtb replication, is also effectively activated by Fe2+ in the presence of Mn2+ or Mg2+ cations. In contrast, simultaneous presence of Fe2+ and Mn2+ or Mg2+ inhibits the gluconeogenic reaction. These results suggest that inorganic ions can contribute to regulation of central carbon metabolism by influencing the activity of Pck. Furthermore, the X-ray structure determination, biochemical characterization, and QM analysis of Pck mutants confirmed the important role of the Phe triad for proper binding of the GDP-Mn2+ complex in the nucleotide binding site and efficient catalysis of the anaplerotic reaction.

• MeSH
• aktivace enzymů MeSH
• fosfoenolpyruvátkarboxykinasa (závislá na ATP) chemie   genetika   metabolismus   MeSH
• glukoneogeneze MeSH
• katalýza MeSH
• kationty dvojmocné MeSH
• kineze MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• multimerizace proteinu MeSH
• mutace MeSH
• Mycobacterium tuberculosis enzymologie   genetika   MeSH
• nukleotidy metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• vazebná místa MeSH
• vodíková vazba MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Tuberculosis remains a major health concern worldwide. Eradication of its causative agent, the bacterial pathogen Mycobacterium tuberculosis, is particularly challenging due to a vast reservoir of latent carriers of the disease. Despite the misleading terminology of a so-called dormant state associated with latent infections, the bacteria have to maintain basic metabolic activities. Hypoxic conditions have been widely used as an in vitro system to study this dormancy. Such studies identified a rearrangement of central carbon metabolism to exploit fermentative processes caused by the lack of oxygen. Phosphoenolpyruvate carboxykinase (Pck; EC 4.1.1.32) is the enzyme at the center of these metabolic rearrangements. Although Pck is associated with gluconeogenesis under standard growth conditions, the enzyme can catalyze the reverse reaction, supporting anaplerosis of the tricarboxylic acid cycle, under conditions leading to slowed or stopped bacterial replication. To study the mechanisms that regulate the switch between two Pck functions, we systematically investigated factors influencing the gluconeogenic and anaplerotic reaction kinetics. We demonstrate that a reducing environment, as found under hypoxia-triggered non-replicating conditions, accelerates the reaction in the anaplerotic direction. Furthermore, we identified proteins that interact with Pck. The interaction between Pck and the reduced form of mycobacterial thioredoxin, gene expression of which is increased under hypoxic conditions, also increased the Pck anaplerotic activity. We thus propose that a reducing environment and the protein-protein interaction with thioredoxin in particular enable the Pck anaplerotic function under fermentative growth conditions.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• citrátový cyklus fyziologie   MeSH
• fosfoenolpyruvátkarboxykinasa (závislá na ATP) genetika   metabolismus   MeSH
• Mycobacterium tuberculosis enzymologie   genetika   MeSH
• oxidace-redukce MeSH
• regulace genové exprese enzymů fyziologie   MeSH
• regulace genové exprese u bakterií fyziologie   MeSH
• thioredoxiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) from mature maize seeds (Zea mays L.) was purified to homogeneity and a final specific activity of 13.3 μmol min⁻¹ mg⁻¹. Purified PEPC was treated with phosphatase from bovine intestinal mucosa or protein kinase A to study its apparent phosphorylation level. Kinetic parameters of the enzyme reaction catalyzed by phosphorylated and dephosphorylated forms under different conditions were compared, as well as an effect of modulators. The enzyme dephosphorylation resulted in the change of hyperbolic kinetics to the sigmoidal one (with respect to PEP), following with the decrease of maximal reaction rate and the increase of sensitivity to L-malate inhibition. The hyperbolic kinetics of native PEPC present in dry maize seeds was not changed after the protein kinase A treatment, while it was converted to the sigmoidal one after dephosphorylation. Level of PEPC phosphorylation was not affected during seed imbibition.

• MeSH
• fosfoenolpyruvátkarboxylasa antagonisté a inhibitory   izolace a purifikace   metabolismus   MeSH
• fosforylace MeSH
• kinetika MeSH
• kukuřice setá enzymologie   MeSH
• maláty farmakologie   MeSH
• proteinkinasy závislé na cyklickém AMP farmakologie   MeSH
• semena rostlinná enzymologie   MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mycobacterium tuberculosis (MTb), the causative agent of tuberculosis, can persist in macrophages for decades, maintaining its basic metabolic activities. Phosphoenolpyruvate carboxykinase (Pck; EC 4.1.1.32) is a key player in central carbon metabolism regulation. In replicating MTb, Pck is associated with gluconeogenesis, but in non-replicating MTb, it also catalyzes the reverse anaplerotic reaction. Here, we explored the role of selected cysteine residues in function of MTb Pck under different redox conditions. Using mass spectrometry analysis we confirmed formation of S-S bridge between cysteines C391 and C397 localized in the C-terminal subdomain. Molecular dynamics simulations of C391-C397 bridged model indicated local conformation changes needed for formation of the disulfide. Further, we used circular dichroism and Raman spectroscopy to analyze the influence of C391 and C397 mutations on Pck secondary and tertiary structures, and on enzyme activity and specificity. We demonstrate the regulatory role of C391 and C397 that form the S-S bridge and in the reduced form stabilize Pck tertiary structure and conformation for gluconeogenic and anaplerotic reactions.

• MeSH
• aminokyselinové motivy MeSH
• biokatalýza * MeSH
• cystein metabolismus   MeSH
• disulfidy metabolismus   MeSH
• fosfoenolpyruvátkarboxykinasa (závislá na ATP) chemie   metabolismus   MeSH
• kinetika MeSH
• molekulární modely MeSH
• mutace genetika   MeSH
• mutageneze cílená MeSH
• mutantní proteiny chemie   MeSH
• Mycobacterium tuberculosis enzymologie   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• stabilita enzymů MeSH
• substrátová specifita MeSH
• tandemová hmotnostní spektrometrie MeSH
• terciární struktura proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• fosfoenolpyruvát metabolismus   MeSH
• glukosa farmakologie   MeSH
• kůra ledviny fyziologie   chemie   účinky léků   MeSH
• lidé MeSH
• Check Tag
• lidé MeSH

The obligate intracellular pathogen, Anaplasma phagocytophilum, is the causative agent of life-threatening diseases in humans and animals. A. phagocytophilum is an emerging tick-borne pathogen in the United States, Europe, Africa and Asia, with increasing numbers of infected people and animals every year. It is increasingly recognized that intracellular pathogens modify host cell metabolic pathways to increase infection and transmission in both vertebrate and invertebrate hosts. Recent reports have shown that amino acids are central to the host-pathogen metabolic interaction. In this study, a genome-wide search for components of amino acid metabolic pathways was performed in Ixodes scapularis, the main tick vector of A. phagocytophilum in the United States, for which the genome was recently published. The enzymes involved in the synthesis and degradation pathways of the twenty amino acids were identified. Then, the available transcriptomics and proteomics data was used to characterize the mRNA and protein levels of I. scapularis amino acid metabolic pathway components in response to A. phagocytophilum infection of tick tissues and ISE6 tick cells. Our analysis was focused on the interplay between carbohydrate and amino acid metabolism during A. phagocytophilum infection in ISE6 cells. The results showed that tick cells increase the synthesis of phosphoenolpyruvate (PEP) from tyrosine to control A. phagocytophilum infection. Metabolic pathway analysis suggested that this is achieved by (i) increasing the transcript and protein levels of mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), (ii) shunting tyrosine into the tricarboxylic acid (TCA) cycle to increase fumarate and oxaloacetate which will be converted into PEP by PEPCK-M, and (iii) blocking all the pathways that use PEP downstream gluconeogenesis (i.e., de novo serine synthesis pathway (SSP), glyceroneogenesis and gluconeogenesis). While sequestering host PEP may be critical for this bacterium because it cannot actively carry out glycolysis to produce PEP, excess of this metabolite may be toxic for A. phagocytophilum. The present work provides a more comprehensive view of the major amino acid metabolic pathways involved in the response to pathogen infection in ticks, and provides the basis for further studies to develop novel strategies for the control of granulocytic anaplasmosis.

• MeSH
• aminokyseliny metabolismus   MeSH
• Anaplasma phagocytophilum účinky léků   genetika   metabolismus   patogenita   MeSH
• anaplasmóza MeSH
• apoptóza MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• buněčné linie MeSH
• citrátový cyklus MeSH
• fosfoenolpyruvát metabolismus   farmakologie   MeSH
• fosfoenolpyruvátkarboxykinasa (závislá na ATP) metabolismus   MeSH
• genom bakteriální MeSH
• glukoneogeneze MeSH
• glykolýza MeSH
• interakce hostitele a patogenu fyziologie   MeSH
• klíště mikrobiologie   MeSH
• kyselina oxaloctová metabolismus   MeSH
• messenger RNA genetika   MeSH
• metabolické sítě a dráhy genetika   MeSH
• metabolismus sacharidů MeSH
• mitochondrie metabolismus   MeSH
• proteomika metody   MeSH
• serin metabolismus   MeSH
• transkriptom MeSH
• tyrosin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

• Publikační typ
• abstrakty MeSH

Drought stress is one of the most frequent forms of abiotic stresses, which occurs under condition of limited water availability. In this work, the possible participation of phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPC), NADP-malic enzyme (EC 1.1.1.40; NADP-ME), and pyruvate, phosphate dikinase (EC 2.7.9.1; PPDK) in response to drought of tobacco plants (Nicotiana tabacum L., cv. W38) was investigated. Enzyme specific activities in tobacco leaves of drought stressed plants were significantly increased after 11 days of stress, PEPC 2.3-fold, NADP-ME 3.9-fold, and PPDK 2.7-fold compared to control plants. The regulation of PEPC and NADP-ME activities were studied on transcriptional level by the quantitative RT PCR and on translational level - immunochemically. The amount of NADP-ME protein and transcription of mRNA for chloroplastic NADP-ME isoform were increased indicating their enhanced synthesis de novo. On the other hand, mRNA for cytosolic isoform of NADP-ME was decreased. The changes in PEPC protein and PEPC mRNA were not substantial. Therefore regulation of PEPC activity by phosphorylation was evaluated and found to be involved in the stress response. During recovery, activities of the tested enzymes returned close to their basal levels.

• MeSH
• aklimatizace MeSH
• chloroplasty metabolismus   MeSH
• fosfoenolpyruvátkarboxylasa genetika   metabolismus   MeSH
• fyziologický stres fyziologie   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• listy rostlin enzymologie   MeSH
• malátdehydrogenasa genetika   metabolismus   MeSH
• období sucha * MeSH
• pyruvátfosfátdikinasa genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• tabák enzymologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• chromatografie MeSH
• fosfáty metabolismus   MeSH
• karboxylyasy izolace a purifikace   metabolismus   MeSH
• kinetika MeSH
• koenzym A metabolismus   MeSH
• mangan farmakologie   MeSH
• pyruváty MeSH
• Streptomyces enzymologie   metabolismus   MeSH