Regulation of glutamate metabolism by protein kinases in mycobacteria

Protein kinase G of Mycobacterium tuberculosis has been implicated in virulence and in regulation of glutamate metabolism. Here we show that this kinase undergoes a pattern of autophosphorylation that is distinct from that of other M. tuberculosis protein kinases characterized to date and we identify GarA as a substrate for phosphorylation by PknG. Autophosphorylation of PknG has little effect on kinase activity but promotes binding to GarA, an interaction that is also detected in living mycobacteria. PknG phosphorylates GarA at threonine 21, adjacent to the residue phosphorylated by PknB (T22), and these two phosphorylation events are mutually exclusive. Like the homologue OdhI from Corynebacterium glutamicum, the unphosphorylated form of GarA is shown to inhibit α‐ketoglutarate decarboxylase in the TCA cycle. Additionally GarA is found to bind and modulate the activity of a large NAD⁺‐specific glutamate dehydrogenase with an unusually low affinity for glutamate. Previous reports of a defect in glutamate metabolism caused by pknG deletion may thus be explained by the effect of unphosphorylated GarA on these two enzyme activities, which may also contribute to the attenuation of virulence.     

Influence of Allosteric Regulators on Individual Steps in the Reaction Catalyzed by Mycobacterium tuberculosis 2-Hydroxy-3-oxoadipate Synthase

Allosteric regulation often controls key branch points in metabolic processes. Mycobacterium tuberculosis 2-hydroxy-3-oxoadipate synthase (HOAS), a thiamin diphosphate (ThDP)-dependent enzyme, produces 2-hydroxy-3-oxoadipate using 2-ketoglutarate and glyoxylate. The proposed chemical mechanism in analogy with other ThDP-dependent carboligases involves multiple ThDP-bound covalent intermediates. Acetyl coenzyme A is an activator, and GarA, a forkhead association domain-containing protein known to regulate glutamate metabolism, is an allosteric inhibitor of HOAS. Steady state kinetics using assays to study the first half and the full catalytic cycle suggested that the regulators act at different steps in the overall mechanism. To explore the modes of regulation and to test the effects on individual catalytic steps, we performed circular dichroism (CD) studies using a non-decarboxylatable 2-ketoglutarate analog and determined the distribution of ThDP-bound covalent intermediates during the steady state of the HOAS reaction using one-dimensional ¹H gradient carbon heteronuclear single quantum coherence NMR. The results suggest that acetyl coenzyme A acts as a mixed V and K type activator and predominantly affects the predecarboxylation steps. GarA does not inhibit the formation of the predecarboxylation analog and does not affect the accumulation of the postdecarboxylation covalent intermediate derived from 2-ketoglutarate; however, it decreases the abundance of the product ThDP adduct in the HOAS pathway. Thus, the two regulators act on different halves of the catalytic cycle in an unusual regulatory regime.     

The FHA-containing protein GarA acts as a phosphorylation-dependent molecular switch in mycobacterial signaling

Fork-head associated (FHA) domains are widely found in bacteria, but their cellular functions remain unclear. Here, we focus on Mycobacterium tuberculosis GarA, an FHA-containing protein conserved in actinomycetes that is phosphorylated by different Ser/Thr protein kinases. Using various physicochemical approaches, we show that phosphorylation significantly stabilizes GarA, and that its FHA domain interacts strongly with the phosphorylated N-terminal extension. Altogether, our results indicate that phosphorylation triggers an intra-molecular protein closure, blocking the phosphothreonine-binding site and switching off the regulatory properties of GarA. The model can explain the reported functions of this mycobacterial protein as regulator of glycogen degradation and glutamate metabolism.

Structured summary:

MINT-6804218: GarA (uniprotkb:P64897) and GarA (uniprotkb:P64897) bind (MI:0407) by isothermal titration calorimetry (MI:0065)     

Structures of citrate synthase and malate dehydrogenase of Mycobacterium tuberculosis

The tricarboxylic acid (TCA) cycle is a central metabolic pathway of all aerobic organisms and is responsible for the synthesis of many important precursors and molecules. TCA cycle plays a key role in the metabolism of Mycobacterium tuberculosis and is involved in the adaptation process of the bacteria to the host immune response. We present here the first crystal structures of M. tuberculosis malate dehydrogenase and citrate synthase, two consecutive enzymes of the TCA, at 2.6 Å and 1.5 Å resolution, respectively. General analogies and local differences with the previously reported homologous protein structures are described. Proteins 2015; 83:389–394. © 2014 Wiley Periodicals, Inc.     

Poster Sessions BP01: Energy Metabolism. NMR investigation of metabolic alterations in the brain of thiamine‐deficient rats

Thiamine deficiency (TD) results in region‐selective impairment of brain metabolism. Since thiamine is a cofactor for enzymes involved in glucose metabolism, ¹H and ¹³C‐NMR was used to investigate metabolic fluxes through the major pathways of glucose metabolism in vulnerable (medial thalamus, MT; inferior colliculus, IC) and nonvulnerable brain structures of rats made thiamine deficient following treatment with the central thiamine antagonist pyrithiamine vs. pair‐fed controls. Symptomatic stages of TD resulted in decreased glutamate and GABA in MT an IC confirming previous biochemical studies. ¹³C‐isotopomer analysis revealed decreased de novo synthesis of [4–¹³C]glutamate (30%p < 0.02) and [2–¹³C]GABA (60%p < 0.01) in MT and IC consistent with decreased activities of pyruvate‐ and α‐ketoglutarate dehydrogenases. These changes were accompanied by decreased consumption of glucose and increased synthesis of lactate from [1–¹³C]glucose confirming decreased mitochondrial metabolism. Accumulation of glyceraldehyde‐3‐phosphate suggested inhibition of glucose flux through the thiamine‐deficient enzyme transketolase. Onset of symptoms of TD and significant cell death was accompanied by decreased neuronal marker molecules NAA and NAAG in MT. Focal lactate accumulation resulting from decreased activities of mitochondrial thiamine‐dependent enzymes appears to play a key role in the pathogenesis of selective neuronal cell death in TD. [funded by CIHR Canada].     

Viability,morphology, and proteome of Mycobacterium smegmatis MSMEG_0319 knockout strain

Mycobacterium tuberculosis Rv0228, a membrane protein, is predicted as a drug target through computational methods. MSMEG_0319 (MS0319) in Mycobacterium smegmatis mc²155 is the ortholog of Rv0228. To study the effect of MS0319 protein on M. smegmatis, an MS0319 gene knockout strain (ΔMS0319) was generated via a homologous recombination technique in this study. The results showed that the lack of MS0319 protein in mc²155 cells led to the loss of viability at nonpermissive temperature. Scanning electron microscopy and transmission electron microscopy observations showed drastic changes in cellular shape especially cell wall disruption in ΔMS0319 cells. Proteomic analysis of ΔMS0319 cells through LC‐MS/MS revealed that 462 proteins had changes in their expressions by lacking MS0319 protein. The M. tuberculosis orthologs of these 462 proteins were found through BLASTp search and functional clustering and metabolic pathway enrichment were performed on the orthologs. The results revealed that most of them were enzymes involved in metabolism of carbohydrates and amino acids, indicating that Rv0228 played an important role in cellular metabolism. All these results suggested Rv0228 as a potential target for development of antituberculosis drugs.     

An immunoinformatics approach to define T cell epitopes from polyketide and non‐ribosomal peptide synthesis proteins of Mycobacterium tuberculosis as potential vaccine candidates

The role of polyketide and non‐ribosomal proteins from the class of small molecule metabolism of Mycobacterium tuberculosis is well documented in envelope organization, virulence, and pathogenesis. Consequently, the identification of T cell epitopes from these proteins could serve to define potential antigens for the development of vaccines. Fourty‐one proteins from polyketide and non‐ribosomal peptide synthesis of small molecule metabolism proteins of M tuberculosis H37Rv were analyzed computationally for the presence of HLA class I binding nanomeric peptides. All possible overlapping nanomeric peptide sequences from 41 small molecule metabolic proteins were generated through in silico and analyzed for their ability to bind to 33 alleles belonging to A, B, and C loci of HLA class I molecule. Polyketide and non‐ribosomal protein analyses revealed that 20% of generated peptides were predicted to bind HLA with halftime of dissociation T_1/2 ≥ 100 minutes, and 77% of them were mono‐allelic in their binding. The structural bases for recognition of nanomers by different HLA molecules were studied by structural modeling of HLA class I‐peptide complexes. Pathogen peptides that could mimic as self‐peptides or partially self‐peptides in the host were excluded using a comparative study with the human proteome; thus, subunit or DNA vaccines will have more chance of success.     

High‐throughput respiration screening of single mitochondrial substrates using permeabilized CHO cells highlights control of mitochondria metabolism

Respiration analysis using isolated mitochondria and electrochemical oxygen sensing has contributed significantly to the knowledge about mitochondrial metabolism, which is involved in energy generation but also in ageing and numerous diseases. Here, we present a high‐throughput respiration screening for functional in situ mitochondrial studies in permeabilized Chinese hamster ovary cells. The determination of oxygen uptake rates allowed a quantitative comparison between different conditions and a distinction of substrates into three groups providing an insight into tricarboxylic acid (TCA) cycle regulation. The mitochondrial metabolization of citrate, isocitrate, glutamine, and glutamate was highly stimulated by ADP supply. In contrast, the metabolization of α‐ketoglutarate, succinate, fumarate, and malate was little controlled by the energy and redox state. Metabolization of pyruvate was very strictly regulated by several independent mechanisms: phosphorylation, feedback inhibition, but also by the availability of CoA. A moderate stimulation of pyruvate metabolization was accomplished by feeding both pyruvate and aspartate simultaneously. The presented high‐throughput respiration screening provides comprehensive information about the effect of single or mixed substrates on mitochondrial metabolic activities, including transport and TCA cycle regulation, and metabolic bottlenecks. This supports the design of efficient mammalian producer strains or feeding strategies, but also the investigation of pathological and toxicological effects related to mitochondrial metabolism.     

Survival of mycobacteria depends on proteasome‐mediated amino acid recycling under nutrient limitation

Intracellular protein degradation is an essential process in all life domains. While in all eukaryotes regulated protein degradation involves ubiquitin tagging and the 26S‐proteasome, bacterial prokaryotic ubiquitin‐like protein (Pup) tagging and proteasomes are conserved only in species belonging to the phyla Actinobacteria and Nitrospira. In Mycobacterium tuberculosis, the Pup‐proteasome system (PPS) is important for virulence, yet its physiological role in non‐pathogenic species has remained an enigma. We now report, using Mycobacterium smegmatis as a model organism, that the PPS is essential for survival under starvation. Upon nitrogen limitation, PPS activity is induced, leading to accelerated tagging and degradation of many cytoplasmic proteins. We suggest a model in which the PPS functions to recycle amino acids under nitrogen starvation, thereby enabling the cell to maintain basal metabolic activities. We also find that the PPS auto‐regulates its own activity via pupylation and degradation of its components in a manner that promotes the oscillatory expression of PPS components. As such, the destructive activity of the PPS is carefully balanced to maintain cellular functions during starvation.     

Lysine acetylation of the Mycobacterium tuberculosis HU protein modulates its DNA binding and genome organization

Nucleoid‐associated protein HU, a conserved protein across eubacteria is necessary for maintaining the nucleoid organization and global regulation of gene expression. Mycobacterium tuberculosis HU (MtHU) is distinct from the other orthologues having 114 amino acid long carboxyl terminal extensions with a high degree of sequence similarity to eukaryotic histones. In this study, we demonstrate that the DNA binding property of MtHU is regulated by posttranslational modifications akin to eukaryotic histones. MtHU purified from M. tuberculosis cells is found to be acetylated on multiple lysine residues unlike the E. coli expressed recombinant protein. Using coimmunoprecipitation assay, we identified Eis as one of the acetyl transferases that interacts with MtHU and modifies it. Although Eis is known to acetylate aminoglycosides, the kinetics of acetylation showed that its protein acetylation activity on MtHU is robust. In vitro Eis modified MtHU at various lysine residues, primarily those located at the carboxyl terminal domain. Acetylation of MtHU caused reduced DNA interaction and alteration in DNA compaction ability of the NAP. Over‐expression of the Eis leads to hyperacetylation of HU and decompaction of genome. These results provide first insights into the modulation of the nucleoid structure by lysine acetylation in bacteria.     

Deletion of glutamate dehydrogenase 1 (Glud1) in the central nervous system affects glutamate handling without altering synaptic transmission

Glutamate dehydrogenase (GDH), encoded by GLUD1, participates in the breakdown and synthesis of glutamate, the main excitatory neurotransmitter. In the CNS, besides its primary signaling function, glutamate is also at the crossroad of metabolic and neurotransmitter pathways. Importance of brain GDH was questioned here by generation of CNS‐specific GDH‐null mice (CnsGlud1^?/?); which were viable, fertile and without apparent behavioral problems. GDH immunoreactivity as well as enzymatic activity were absent in Cns‐Glud1^?/? brains. Immunohistochemical analyses on brain sections revealed that the pyramidal cells of control animals were positive for GDH, whereas the labeling was absent in hippocampal sections of Cns‐Glud1^?/? mice. Electrophysiological recordings showed that deletion of GDH within the CNS did not alter synaptic transmission in standard conditions. Cns‐Glud1^?/? mice exhibited deficient oxidative catabolism of glutamate in astrocytes, showing that GDH is required for Krebs cycle pathway. As revealed by NMR studies, brain glutamate levels remained unchanged, whereas glutamine levels were increased. This pattern was favored by up‐regulation of astrocyte‐type glutamate and glutamine transporters and of glutamine synthetase. Present data show that the lack of GDH in the CNS modifies the metabolic handling of glutamate without altering synaptic transmission.     

Allosteric inhibitors of Mycobacterium tuberculosis tryptophan synthase

Global dispersion of multidrug resistant bacteria is very common and evolution of antibiotic‐resistance is occurring at an alarming rate, presenting a formidable challenge for humanity. The development of new therapeuthics with novel molecular targets is urgently needed. Current drugs primarily affect protein, nucleic acid, and cell wall synthesis. Metabolic pathways, including those involved in amino acid biosynthesis, have recently sparked interest in the drug discovery community as potential reservoirs of such novel targets. Tryptophan biosynthesis, utilized by bacteria but absent in humans, represents one of the currently studied processes with a therapeutic focus. It has been shown that tryptophan synthase (TrpAB) is required for survival of Mycobacterium tuberculosis in macrophages and for evading host defense, and therefore is a promising drug target. Here we present crystal structures of TrpAB with two allosteric inhibitors of M. tuberculosis tryptophan synthase that belong to sulfolane and indole‐5‐sulfonamide chemical scaffolds. We compare our results with previously reported structural and biochemical studies of another, azetidine‐containing M. tuberculosis tryptophan synthase inhibitor. This work shows how structurally distinct ligands can occupy the same allosteric site and make specific interactions. It also highlights the potential benefit of targeting more variable allosteric sites of important metabolic enzymes.     

A systems biology framework for modeling metabolic enzyme inhibition of Mycobacterium tuberculosis

Background  

Because metabolism is fundamental in sustaining microbial life, drugs that target pathogen-specific metabolic enzymes and pathways can be very effective. In particular, the metabolic challenges faced by intracellular pathogens, such as Mycobacterium tuberculosis, residing in the infected host provide novel opportunities for therapeutic intervention.     

Polyphosphate kinase 2: a modulator of nucleoside diphosphate kinase activity in mycobacteria

Mycobacteria encode putative class II polyphosphate kinases (PPKs). We report that recombinant PPK2 of Mycobacterium tuberculosis catalyses the synthesis of GTP from GDP using polyphosphate rather than ATP as phosphate donor. Unlike that of PPK1, this is the favoured reaction of PPK2. The sites of autophosphorylation, H115 and H247, as well as G74 were critical for GTP‐synthesizing activity. Compromised survival of a ppk2 knockout (PPK2‐KO) of Mycobacterium smegmatis under heat or acid stress or hypoxia, and the ability of ppk2 of M. tuberculosis to complement this, confirmed that PPK2 plays a role in mycobacterial survival under stress. Intracellular ATP : GTP ratio was higher in PPK2‐KO compared with the wild‐type M. smegmatis, bringing to light a role of PPK2 in regulating the intracellular nucleotide pool. We present evidence that PPK2 does so by interacting with nucleoside diphosphate kinase (Ndk). Pull‐down assays and analysis by surface plasmon resonance demonstrated that the interaction requires G74 of PPK2_MTB and ¹⁰⁹LET¹¹¹ of Ndk_MTB. In summary, we unravel a novel mechanism of regulation of nucleotide pools in mycobacteria. Downregulation of ppk2 impairs survival of M. tuberculosis in macrophages, suggesting that PPK2 plays an important role in the physiology of the bacteria residing within macrophages.