Abstract:Mycobacterium tuberculosis (Mtb) is the leading cause of death due to a bacterial infection. The success of the Mtb pathogen has largely been attributed to the nonreplicating, persistence phase of the life cycle, for which the glyoxylate shunt is required. In Escherichia coli flux through the shunt is controlled by regulation of isocitrate dehydrogenase (ICDH). In Mtb, the mechanism of regulation is unknown, and currently there is no mechanistic or structural information on ICDH. We optimized expression and pu… Show more
“…Nevertheless, the overall charge in the active site is positive, which facilitates binding of the negatively charged IPM. In the case of malate dehydrogenase , 6‐phosphogluconate dehydrogenase and various isocitrate dehydrogenase enzymes it has been proposed that the nearby lysine residue, deprotonated by an aspartic acid residue, may act as a general base to remove the 2‐hydroxyl proton of the substrate. Indeed, in the obtained structure of IPMDH, the basic nitrogen atom of Lys185′ is located at a distance of approximately 3 Å from the 2‐hydroxyl group, suggesting its possible role as a base.…”
The three-dimensional structure of the enzyme 3-isopropylmalate dehydrogenase from the bacterium Thermus thermophilus in complex with Mn 2+ , its substrate isopropylmalate and its co-factor product NADH at 2.0 A resolution features a fully closed conformation of the enzyme. Upon closure of the two domains, the substrate and the co-factor are brought into precise relative orientation and close proximity, with a distance between the C2 atom of the substrate and the C4N atom of the pyridine ring of the co-factor of approximately 3.0A. The structure further shows binding of a K + ion close to the active site, and provides an explanation for its known activating effect. Hence, this structure is an excellent mimic for the enzymatically competent complex. Using high-level QM/MM calculations, it may be demonstrated that, in the observed arrangement of the reactants, transfer of a hydride from the C2 atom of 3-isopropylmalate to the C4N atom of the pyridine ring of NAD + is easily possible, with an activation energy of approximately 15 kcalÁmol À1 . The activation energy increases by approximately 4-6 kcalÁmol À1 when the K + ion is omitted from the calculations. In the most plausible scenario, prior to hydride transfer the eamino group of Lys185 acts as a general base in the reaction, aiding the deprotonation reaction of 3-isopropylmalate prior to hydride transfer by employing a low-barrier proton shuttle mechanism involving a water molecule.
DatabaseStructural data have been submitted to the Protein Data Bank under accession number 4F7I.Abbreviations IPMDH, 3-isopropylmalate dehydrogenase; IPM, 3-isopropylmalate; QM/MM, hybrid quantum mechanics molecular mechanics.
“…Nevertheless, the overall charge in the active site is positive, which facilitates binding of the negatively charged IPM. In the case of malate dehydrogenase , 6‐phosphogluconate dehydrogenase and various isocitrate dehydrogenase enzymes it has been proposed that the nearby lysine residue, deprotonated by an aspartic acid residue, may act as a general base to remove the 2‐hydroxyl proton of the substrate. Indeed, in the obtained structure of IPMDH, the basic nitrogen atom of Lys185′ is located at a distance of approximately 3 Å from the 2‐hydroxyl group, suggesting its possible role as a base.…”
The three-dimensional structure of the enzyme 3-isopropylmalate dehydrogenase from the bacterium Thermus thermophilus in complex with Mn 2+ , its substrate isopropylmalate and its co-factor product NADH at 2.0 A resolution features a fully closed conformation of the enzyme. Upon closure of the two domains, the substrate and the co-factor are brought into precise relative orientation and close proximity, with a distance between the C2 atom of the substrate and the C4N atom of the pyridine ring of the co-factor of approximately 3.0A. The structure further shows binding of a K + ion close to the active site, and provides an explanation for its known activating effect. Hence, this structure is an excellent mimic for the enzymatically competent complex. Using high-level QM/MM calculations, it may be demonstrated that, in the observed arrangement of the reactants, transfer of a hydride from the C2 atom of 3-isopropylmalate to the C4N atom of the pyridine ring of NAD + is easily possible, with an activation energy of approximately 15 kcalÁmol À1 . The activation energy increases by approximately 4-6 kcalÁmol À1 when the K + ion is omitted from the calculations. In the most plausible scenario, prior to hydride transfer the eamino group of Lys185 acts as a general base in the reaction, aiding the deprotonation reaction of 3-isopropylmalate prior to hydride transfer by employing a low-barrier proton shuttle mechanism involving a water molecule.
DatabaseStructural data have been submitted to the Protein Data Bank under accession number 4F7I.Abbreviations IPMDH, 3-isopropylmalate dehydrogenase; IPM, 3-isopropylmalate; QM/MM, hybrid quantum mechanics molecular mechanics.
“…41,42) In addition, septin-2 make up the cytoskeleton of basal epithelial cells or acts as a scaffold to anchor the actomyosin ring to the plasma membrane during furrowing. 43,44) Rab guanosine 5′-triphosphatases (GTPases), one of the Ras superfamily members of monomeric GTPases, are small G proteins and involved in membrane trafficking.…”
Central post-stroke pain (CPSP) is one of the complications of cerebral ischemia and neuropathic pain syndrome. At present, there are few studies of pain in regions such as the spinal cord or sciatic nerve in cerebral ischemic animal models. To identify proteomic changes in the spinal cord and sciatic nerve in global cerebral ischemic model mice, in the present study we performed an investigation using proteomic methods. In a comparison between the intensity of protein spots obtained from a sham and that from a bilateral carotid artery occulusion (BCAO) in spinal cord and sciatic nerve, the levels of 10 (spinal cord) and 7 (sciatic nerve) protein spots were altered. The protein levels in the spinal cord were significantly increased in N G ,N Gdimethylarginine dimethylaminohydrolase 1 (DDAH1), 6-phosphogluconolactonase isoform 1, and precursor apoprotein A-I and decreased in dihydropyrimidinase-related protein 2 (CRMP-2), enolase 1B, rab guanosine 5′-diphosphate (GDP) dissociation inhibitor beta, septin-2 isoform a, isocitrate dehydrogenase subunit alpha, cytosolic malate dehydrogenase, and adenosine triphosphate synthase. The protein levels in the sciatic nerve were significantly increased in a mimecan precursor, myosin light chain 1/3, and myosin regulatory light chain 2 (MLC2), and decreased in dihydropyrimidinase-related protein 3 (CRMP-4), protein disulfideisomerase A3, 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1, and B-type creatine kinase. In addition, CRMP-2 and CRMP-4 protein levels were decreased, and DDAH1 and MLC2 protein levels were increased on day 1 after BCAO using Western blotting. These results suggested that changes in these proteins may be involved in the regulation of CPSP.Key words global ischemia; pain; spinal cord; sciatic nerve; proteome Cerebral stroke remains one of the leading causes of death and disability worldwide.1) Recent insights into the basic mechanism involved in ischemic stroke indicate that endothelial dysfunctions along with oxidative stress and neuroinflammation are key elements in the occurrence and development of ischemic brain damage that results in cell damage and death.2,3) Many clinical symptoms can manifest after strokes, including motor deficits, cognitive dysfunction, language problems, emotional disturbances, social maladjustment, somatosensory dysfunction, and central pain. 4,5) Cerebral stroke occasionally induces central post-stroke pain (CPSP) that manifests as burning pain, throbbing pain, aching pain, slashing pain, allodynia, and hyperalgesia, either continuously or intermittently on the affected side.6,7) CPSP is known to be one of the neuropathic pain.8) Neuropathic pain is defined as "pain initiated or caused by a primary lesion or dysfunction of the nervous system." It can be a debilitating and difficult condition to treat and is often resistant to simple analgesics, requiring additional analgesic treatment.
9)Some hypotheses of the pathophysiological mechanisms of somatosensory dysfunction have been proposed to explain CPSP symptoms, such as the ...
“…These data, coupled with the absence of an AceK homologue in Mtb, suggests that the regulation of Mtb ICDH1 is unlikely to undergo in similar mechanism to E. coli ICDH. Furthermore, in ICDH of E. coli AceK phosphorylates the protein at Ser113, which is the binding site for isocitrate, and the Mtb ICDH1 lacks a comparable acceptor residue 37–39 .Figure 5Rv2170 acetylates Mtb ICDH1 (Rv3339c) at K30 and K129. ( A ) Protein sequence of ICDH1.…”
Recent data indicate that the metabolism of Mycobacterium tuberculosis (Mtb) inside its host cell is heavily dependent on cholesterol and fatty acids. Mtb exhibits a unique capacity to co-metabolize different carbon sources and the products from these substrates are compartmentalized metabolically. Isocitrate lies at one of the key nodes of carbon metabolism and can feed into either the glyoxylate shunt (via isocitrate lyase) or the TCA cycle (via isocitrate dehydrogenase (ICDH) activity) and we sought to better understand the regulation at this junction. An isocitrate lyase-deficient mutant of Mtb (Δicl1) exhibited a delayed growth phenotype in stearic acid (C18 fatty acid) media and we isolated rescue mutants that had lost this growth delay. We found that mutations in the gene rv2170 promoted Mtb replication under these conditions and rescued the growth delay in a Δicl1 background. The Mtb Rv2170 protein shows lysine acetyltransferase activity, which is capable of post-translationally modifying lysine residues of the ICDH protein leading to a reduction in its enzymatic activity. Our data show that contrary to most bacteria that regulate ICDH activity through phosphorylation, Mtb is capable of regulating ICDH activity by acetylation. This mechanism of regulation is similar to that utilized for mammalian mitochondrial ICDH.
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