Tuberculosis (TB) remains one of the major health concerns worldwide. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can flexibly change its metabolic processes during different life stages. Regulation of key metabolic enzyme activities by intracellular conditions, allosteric inhibition or feedback control can effectively contribute to Mtb survival under different conditions. Phosphofructokinase (Pfk) is one of the key enzymes regulating glycolysis. Mtb encodes two Pfk isoenzymes, Pfk A/Rv3010c and Pfk B/Rv2029c, which are differently expressed upon transition to the hypoxia-induced non-replicating state of the bacteria. While pfkB gene and protein expression are upregulated under hypoxic conditions, Pfk A levels decrease. Here, we present biochemical characterization of both Pfk isoenzymes, revealing that Pfk A and Pfk B display different kinetic properties. Although the glycolytic activity of Pfk A is higher than that of Pfk B, it is markedly inhibited by an excess of both substrates (fructose-6-phosphate and ATP), reaction products (fructose-1,6-bisphosphate and ADP) and common metabolic allosteric regulators. In contrast, synthesis of fructose-1,6-bisphosphatase catalyzed by Pfk B is not regulated by higher levels of substrates, and metabolites. Importantly, we found that only Pfk B can catalyze the reverse gluconeogenic reaction. Pfk B thus can support glycolysis under conditions inhibiting Pfk A function.
- MeSH
- adenosindifosfát metabolismus farmakologie MeSH
- adenosintrifosfát metabolismus farmakologie MeSH
- alosterická regulace MeSH
- bakteriální proteiny antagonisté a inhibitory metabolismus MeSH
- enzymová indukce MeSH
- fosfofruktokinasy antagonisté a inhibitory metabolismus MeSH
- fruktosadifosfáty biosyntéza farmakologie MeSH
- fruktosafosfáty metabolismus farmakologie MeSH
- glukoneogeneze MeSH
- glykolýza MeSH
- hexosafosfáty metabolismus MeSH
- izoenzymy antagonisté a inhibitory metabolismus MeSH
- katalýza MeSH
- kinetika MeSH
- kyslík farmakologie MeSH
- L-laktátdehydrogenasa metabolismus MeSH
- Mycobacterium tuberculosis účinky léků enzymologie MeSH
- pyruvátkinasa metabolismus MeSH
- rekombinantní proteiny metabolismus MeSH
- substrátová specifita MeSH
- zpětná vazba fyziologická MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Mycobacterium tuberculosis (Mtb) causes both acute tuberculosis and latent, symptom-free infection that affects roughly one-third of the world's population. It is a globally important pathogen that poses multiple dangers. Mtb reprograms its metabolism in response to the host niche, and this adaptation contributes to its pathogenicity. Knowledge of the metabolic regulation mechanisms in Mtb is still limited. Pyruvate kinase, involved in the late stage of glycolysis, helps link various metabolic routes together. Here, we demonstrate that Mtb pyruvate kinase (Mtb PYK) predominantly catalyzes the reaction leading to the production of pyruvate, but its activity is influenced by multiple metabolites from closely interlinked pathways that act as allosteric regulators (activators and inhibitors). We identified allosteric activators and inhibitors of Mtb PYK originating from glycolysis, citrate cycle, nucleotide/nucleoside inter-conversion related pathways that had not been described so far. Enzyme was found to be activated by fructose-1,6-bisphosphate, ribose-5-phosphate, adenine, adenosine, hypoxanthine, inosine, L-2-phosphoglycerate, l-aspartate, glycerol-2-phosphate, glycerol-3-phosphate. On the other hand thiamine pyrophosphate, glyceraldehyde-3-phosphate and L-malate were identified as inhibitors of Mtb PYK. The detailed kinetic analysis indicated a morpheein model of Mtb PYK allosteric control which is strictly dependent on Mg2+ and substantially increased by the co-presence of Mg2+ and K+.
Adenosine kinase (ADK) from Mycobacterium tuberculosis (Mtb) was selected as a target for design of antimycobacterial nucleosides. Screening of 7-(het)aryl-7-deazaadenine ribonucleosides with Mtb and human (h) ADKs and testing with wild-type and drug-resistant Mtb strains identified specific inhibitors of Mtb ADK with micromolar antimycobacterial activity and low cytotoxicity. X-ray structures of complexes of Mtb and hADKs with 7-ethynyl-7-deazaadenosine showed differences in inhibitor interactions in the adenosine binding sites. 1D (1)H STD NMR experiments revealed that these inhibitors are readily accommodated into the ATP and adenosine binding sites of Mtb ADK, whereas they bind preferentially into the adenosine site of hADK. Occupation of the Mtb ADK ATP site with inhibitors and formation of catalytically less competent semiopen conformation of MtbADK after inhibitor binding in the adenosine site explain the lack of phosphorylation of 7-substituted-7-deazaadenosines. Semiempirical quantum mechanical analysis confirmed different affinity of nucleosides for the Mtb ADK adenosine and ATP sites.
- MeSH
- adenin analogy a deriváty chemie MeSH
- adenosinkinasa antagonisté a inhibitory chemie metabolismus MeSH
- adenosintrifosfát metabolismus MeSH
- antituberkulotika chemie farmakologie MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- Mycobacterium tuberculosis účinky léků enzymologie MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- preklinické hodnocení léčiv MeSH
- ribonukleosidy chemie farmakologie MeSH
- vazebná místa MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
HIV integrase (IN) catalyzes the insertion of proviral DNA into the host cell chromosome. While IN has strict sequence requirements for the viral cDNA ends, the integration site preference has been shown to be very diverse. Here, we mapped the HIV IN strand transfer reaction requirements using various short oligonucleotides (ON) that mimic the target DNA. Most double stranded DNA dodecamers served as excellent IN targets with variable integration efficiency depending mostly on the ON sequences. The preferred integration was lost with any changes in the geometry of the DNA double helical structures. Various hairpin-loop-forming ONs also served as efficient integration targets. Similar integration preferences were also observed for ONs, in which the nucleotide hairpin loop was replaced with a flexible aliphatic linker. The integration biases with all target DNA structures tested were significantly influenced by changes in the resulting secondary ON structures.
- MeSH
- dimerizace MeSH
- DNA virů genetika MeSH
- HIV-1 genetika MeSH
- HIV-integrasa genetika izolace a purifikace MeSH
- integrace viru genetika MeSH
- katalýza MeSH
- konformace nukleové kyseliny MeSH
- molekulární sekvence - údaje MeSH
- oligonukleotidy genetika MeSH
- sekvence nukleotidů MeSH
- substrátová specifita genetika MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- práce podpořená grantem MeSH
Retroviral integrase participates in two catalytic reactions, which require interactions with the two ends of the viral DNA in the 3'processing reaction, and with a targeted host DNA in the strand transfer reaction. The 3'-hydroxyl group of 2'-deoxyadenosine resulting from the specific removing of GT dinucleotide from the viral DNA in the processing reaction provides the attachment site for the host DNA in a transesterification reaction. We synthesized oligonucleotides (ONs) of various lengths that mimic the processed HIV-1 U5 terminus of the proviral long terminal repeat (LTR) and are ended by 2'-deoxyadenosine containing a 3'-O-phosphonomethyl group. The duplex stability of phosphonomethyl ONs was increased by covalent linkage of the modified strand with its complementary strand by a triethylene glycol loop (TEG). Modified ONs containing up to 10 bases inhibited in vitro the strand transfer reaction catalyzed by HIV-1 integrase at nanomolar concentrations.
- MeSH
- financování organizované MeSH
- HIV - dlouhá koncová repetice genetika MeSH
- HIV-integrasa účinky léků MeSH
- inhibitory HIV-integrasy farmakologie chemická syntéza MeSH
- konformace nukleové kyseliny MeSH
- kyseliny fosforu MeSH
- molekulární mimikry MeSH
- oligonukleotidy farmakologie chemická syntéza MeSH
