E1 of ?-ketoglutarate dehydrogenase defends Mycobacterium tuberculosis against glutamate anaplerosis and nitroxidative stress.
ABSTRACT: Enzymes of central carbon metabolism (CCM) in Mycobacterium tuberculosis (Mtb) make an important contribution to the pathogen's virulence. Evidence is emerging that some of these enzymes are not simply playing the metabolic roles for which they are annotated, but can protect the pathogen via additional functions. Here, we found that deficiency of 2-hydroxy-3-oxoadipate synthase (HOAS), the E1 component of the ?-ketoglutarate (?-KG) dehydrogenase complex (KDHC), did not lead to general metabolic perturbation or growth impairment of Mtb, but only to the specific inability to cope with glutamate anaplerosis and nitroxidative stress. In the former role, HOAS acts to prevent accumulation of aldehydes, including growth-inhibitory succinate semialdehyde (SSA). In the latter role, HOAS can participate in an alternative four-component peroxidase system, HOAS/dihydrolipoyl acetyl transferase (DlaT)/alkylhydroperoxide reductase colorless subunit gene (ahpC)-neighboring subunit (AhpD)/AhpC, using ?-KG as a previously undescribed source of electrons for reductase action. Thus, instead of a canonical role in CCM, the E1 component of Mtb's KDHC serves key roles in situational defense that contribute to its requirement for virulence in the host. We also show that pyruvate decarboxylase (AceE), the E1 component of pyruvate dehydrogenase (PDHC), can participate in AceE/DlaT/AhpD/AhpC, using pyruvate as a source of electrons for reductase action. Identification of these systems leads us to suggest that Mtb can recruit components of its CCM for reactive nitrogen defense using central carbon metabolites.
Project description:Mycobacterium tuberculosis (Mtb) adapts to persist in a nutritionally limited macrophage compartment. Lipoamide dehydrogenase (Lpd), the third enzyme (E3) in Mtb's pyruvate dehydrogenase complex (PDH), also serves as E1 of peroxynitrite reductase/peroxidase (PNR/P), which helps Mtb resist host-reactive nitrogen intermediates. In contrast to Mtb lacking dihydrolipoamide acyltransferase (DlaT), the E2 of PDH and PNR/P, Lpd-deficient Mtb is severely attenuated in wild-type and immunodeficient mice. This suggests that Lpd has a function that DlaT does not share. When DlaT is absent, Mtb upregulates an Lpd-dependent branched-chain keto acid dehydrogenase (BCKADH) encoded by pdhA, pdhB, pdhC, and lpdC. Without Lpd, Mtb cannot metabolize branched-chain amino acids and potentially toxic branched-chain intermediates accumulate. Mtb deficient in both DlaT and PdhC phenocopies Lpd-deficient Mtb. Thus, Mtb critically requires BCKADH along with PDH and PNR/P for pathogenesis. These findings position Lpd as a potential target for anti-infectives against Mtb.
Project description:Burkholderia thailandensis is a model organism for human pathogens Burkholderia mallei and Burkholderia pseudomallei. The study of B. thailandensis peroxiredoxin is helpful for understanding the survival, pathogenic infection, and antibiotic resistance of its homologous species. Alkyl hydroperoxide reductase subunit C (AhpC) is an important peroxiredoxin involved in oxidative damage defense. Here, we report that BthAhpC exhibits broad specificity for peroxide substrates, including inorganic and organic peroxides and peroxynitrite. AhpC catalyzes the reduction of oxidants using the N-terminal conserved Cys57 as a peroxidatic Cys and the C-terminal conserved Cys171 and Cys173 as resolving Cys. These three conserved Cys residues play critical roles in the catalytic mechanism. AhpD directly interacts with AhpC as an electron donor, and the conserved Cys residues in active site of AhpD are important for AhpC reduction. AhpC is directly repressed by OxyR as shown by identifying the OxyR binding site in the ahpC promoter with a DNA binding assay. This work sheds light on the function of AhpC in the peroxides and peroxynitrite damage response in B. thailandensis and homologous species.
Project description:Genes encoding a homolog of Escherichia coli OxyR (oxyR) and an alkyl hydroperoxide reductase system (ahpC and ahpD) have been isolated from Streptomyces coelicolor A3(2). The ahpC and ahpD genes constitute an operon transcribed divergently from the oxyR gene. Expression of both ahpCD and oxyR genes was maximal at early exponential phase and decreased rapidly as cells entered mid-exponential phase. Overproduction of OxyR in Streptomyces lividans conferred resistance against cumene hydroperoxide and H2O2. The oxyR mutant produced fewer ahpCD and oxyR transcripts than the wild type, suggesting that OxyR acts as a positive regulator for their expression. Both oxyR and ahpCD transcripts increased more than fivefold within 10 min of H2O2 treatment and decreased to the normal level in 50 min, with kinetics similar to those of the CatR-mediated induction of the catalase A gene (catA) by H2O2. The oxyR mutant failed to induce oxyR and ahpCD genes in response to H2O2, indicating that OxyR is the modulator for the H2O2-dependent induction of these genes. Purified OxyR protein bound specifically to the intergenic region between ahpC and oxyR, suggesting its direct role in regulating these genes. These results demonstrate that in S. coelicolor OxyR mediates H2O2 induction of its own gene and genes for alkyl hydroperoxide reductase system, but not the catalase gene (catA), unlike in Escherichia coli and Salmonella enterica serovar Typhimurium.
Project description:Host-generated oxidative stress is considered one of the main mechanisms constraining Mycobacterium tuberculosis (Mtb) growth. The redox-sensing mechanisms in Mtb are not completely understood. Here we show that WhiB4 responds to oxygen (O?) and nitric oxide (NO) via its 4Fe-4S cluster and controls the oxidative stress response in Mtb. The WhiB4 mutant (Mtb?whiB4) displayed an altered redox balance and a reduced membrane potential. Microarray analysis demonstrated that Mtb?whiB4 overexpresses the antioxidant systems including alkyl hydroperoxidase (ahpC-ahpD) and rubredoxins (rubA-rubB). DNA binding assays showed that WhiB4 [4Fe-4S] cluster is dispensable for DNA binding. However, oxidation of the apo-WhiB4 Cys thiols induced disulphide-linked oligomerization, DNA binding and transcriptional repression, whereas reduction reversed the effect. Furthermore, WhiB4 binds DNA with a preference for GC-rich sequences. Expression analysis showed that oxidative stress repressed whiB4 and induced antioxidants in Mtb, while their hyper-induction was observed in Mtb?whiB4. Mtb?whiB4 showed increased resistance to oxidative stress in vitro and enhanced survival inside the macrophages. Lastly, Mtb?whiB4 displayed hypervirulence in the lungs of guinea pigs, but showed a defect in dissemination to their spleen. These findings suggest that WhiB4 systematically calibrates the activation of oxidative stress response in Mtb to maintain redox balance, and to modulate virulence.
Project description:Legionella pneumophila expresses two catalase-peroxidase enzymes that exhibit strong peroxidatic but weak catalatic activities, suggesting that other enzymes participate in decomposition of hydrogen peroxide (H2O2). Comparative genomics revealed that L. pneumophila and its close relative Coxiella burnetii each contain two peroxide-scavenging alkyl hydroperoxide reductase (AhpC) systems: AhpC1, which is similar to the Helicobacter pylori AhpC system, and AhpC2 AhpD (AhpC2D), which is similar to the AhpC AhpD system of Mycobacterium tuberculosis. To establish a catalatic function for these two systems, we expressed L. pneumophila ahpC1 or ahpC2 in a catalase/peroxidase mutant of Escherichia coli and demonstrated restoration of H2O2 resistance by a disk diffusion assay. ahpC1::Km and ahpC2D::Km chromosomal deletion mutants were two- to eightfold more sensitive to H2O2, tert-butyl hydroperoxide, cumene hydroperoxide, and paraquat than the wild-type L. pneumophila, a phenotype that could be restored by trans-complementation. Reciprocal strategies to construct double mutants were unsuccessful. Mutant strains were not enfeebled for growth in vitro or in a U937 cell infection model. Green fluorescence protein reporter assays revealed expression to be dependent on the stage of growth, with ahpC1 appearing after the exponential phase and ahpC2 appearing during early exponential phase. Quantitative real-time PCR showed that ahpC1 mRNA levels were approximately 7- to 10-fold higher than ahpC2D mRNA levels. However, expression of ahpC2D was significantly increased in the ahpC1 mutant, whereas ahpC1 expression was unchanged in the ahpC2D mutant. These results indicate that AhpC1 or AhpC2D (or both) provide an essential hydrogen peroxide-scavenging function to L. pneumophila and that the compensatory activity of the ahpC2D system is most likely induced in response to oxidative stress.
Project description:The E1p enzyme is an essential part of the pyruvate dehydrogenase complex (PDHC) and catalyzes the oxidative decarboxylation of pyruvate with concomitant acetylation of the E2p enzyme within the complex. We analyzed the Corynebacterium glutamicum aceE gene, encoding the E1p enzyme, and constructed and characterized an E1p-deficient mutant. Sequence analysis of the C. glutamicum aceE gene and adjacent regions revealed that aceE is not flanked by genes encoding other enzymes of the PDHC. Transcriptional analysis revealed that aceE from C. glutamicum is monocistronic and that its transcription is initiated 121 nucleotides upstream of the translational start site. Inactivation of the chromosomal aceE gene led to the inability to grow on glucose and to the absence of PDHC and E1p activities, indicating that only a single E1p enzyme is present in C. glutamicum and that the PDHC is essential for the growth of this organism on carbohydrate substrates. Surprisingly, the E1p enzyme of C. glutamicum showed up to 51% identity to homodimeric E1p proteins from gram-negative bacteria but no similarity to E1 alpha- or beta-subunits of heterotetrameric E1p enzymes which are generally assumed to be typical for gram-positives. To investigate the distribution of E1p enzymes in bacteria, we compiled and analyzed the phylogeny of 46 homodimeric E1p proteins and of 58 alpha-subunits of heterotetrameric E1p proteins deposited in public databases. The results revealed that the distribution of homodimeric and heterotetrameric E1p subunits in bacteria is not in accordance with the rRNA-based phylogeny of bacteria and is more heterogeneous than previously assumed.
Project description:Sirtuins (SIRTs) are critical enzymes that govern genome regulation, metabolism, and aging. Despite conserved deacetylase domains, mitochondrial SIRT4 and SIRT5 have little to no deacetylase activity, and a robust catalytic activity for SIRT4 has been elusive. Here, we establish SIRT4 as a cellular lipoamidase that regulates the pyruvate dehydrogenase complex (PDH). Importantly, SIRT4 catalytic efficiency for lipoyl- and biotinyl-lysine modifications is superior to its deacetylation activity. PDH, which converts pyruvate to acetyl-CoA, has been known to be primarily regulated by phosphorylation of its E1 component. We determine that SIRT4 enzymatically hydrolyzes the lipoamide cofactors from the E2 component dihydrolipoyllysine acetyltransferase (DLAT), diminishing PDH activity. We demonstrate SIRT4-mediated regulation of DLAT lipoyl levels and PDH activity in cells and in vivo, in mouse liver. Furthermore, metabolic flux switching via glutamine stimulation induces SIRT4 lipoamidase activity to inhibit PDH, highlighting SIRT4 as a guardian of cellular metabolism.
Project description:BACKGROUND:Alkyl hydroperoxidase activity provides an important antioxidant defense for bacterial cells. The catalytic mechanism requires two peroxidases, AhpC and AhpD, where AhpD plays the role of an essential adaptor protein. RESULTS:The crystal structure of a putative AhpD from Pseudomonas aeruginosa has been determined at 1.9 Å. The protein has an all-helical fold with a chain topology similar to a known AhpD from Mycobacterium tuberculosis despite a low overall sequence identity of 9%. A conserved two ?-helical motif responsible for function is present in both. However, in the P. aeruginosa protein, helices H3, H4 of this motif are located at the N-terminal part of the chain, while in M. tuberculosis AhpD, the corresponding helices H8, H9 are situated at the C-terminus. Residues 24-62 of the putative catalytic region of P. aeruginosa have a higher sequence identity of 33% where the functional activity is supplied by a proton relay system of five residues, Glu36, Cys48, Tyr50, Cys51, and His55, and one structural water molecule. A comparison of five other related hypothetical proteins from various species, assigned to the alkyl hydroperoxidase D-like protein family, shows they contain the same conserved structural motif and catalytic sequence Cys-X-X-Cys. We have shown that AhpD from P. aeruginosa exhibits a weak ability to reduce H(2)O(2) as tested using a ferrous oxidation-xylenol orange (FOX) assay, and this activity is blocked by thiol alkylating reagents. CONCLUSION:Thus, this hypothetical protein was assigned to the AhpD-like protein family with peroxidase-related activity. The functional relationship of specific oligomeric structures of AhpD-like structural family is discussed.
Project description:Oxoglutarate dehydrogenase (ODH) and pyruvate dehydrogenase (PDH) complexes catalyze key reactions in central metabolism, and in Corynebacterium glutamicum there is indication of an unusual supercomplex consisting of AceE (E1), AceF (E2), and Lpd (E3) together with OdhA. OdhA is a fusion protein of additional E1 and E2 domains, and odhA orthologs are present in all Corynebacterineae, including, for instance, Mycobacterium tuberculosis. Here we show that deletion of any of the individual domains of OdhA in C. glutamicum resulted in loss of ODH activity, whereas PDH was still functional. On the other hand, deletion of AceF disabled both PDH activity and ODH activity as well, although isolated AceF protein had solely transacetylase activity and no transsuccinylase activity. Surprisingly, the isolated OdhA protein was inactive with 2-oxoglutarate as the substrate, but it gained transsuccinylase activity upon addition of dihydrolipoamide. Further enzymatic analysis of mutant proteins and mutant cells revealed that OdhA specifically catalyzes the E1 and E2 reaction to convert 2-oxoglutarate to succinyl-coenzyme A (CoA) but fully relies on the lipoyl residues provided by AceF involved in the reactions to convert pyruvate to acetyl-CoA. It therefore appears that in the putative supercomplex in C. glutamicum, in addition to dihydrolipoyl dehydrogenase E3, lipoyl domains are also shared, thus confirming the unique evolutionary position of bacteria such as C. glutamicum and M. tuberculosis.
Project description:Antibiotics are typically more effective against replicating rather than nonreplicating bacteria. However, a major need in global health is to eradicate persistent or nonreplicating subpopulations of bacteria such as Mycobacterium tuberculosis (Mtb). Hence, identifying chemical inhibitors that selectively kill bacteria that are not replicating is of practical importance. To address this, we screened for inhibitors of dihydrolipoamide acyltransferase (DlaT), an enzyme required by Mtb to cause tuberculosis in guinea pigs and used by the bacterium to resist nitric oxide-derived reactive nitrogen intermediates, a stress encountered in the host. Chemical screening for inhibitors of Mtb DlaT identified select rhodanines as compounds that almost exclusively kill nonreplicating mycobacteria in synergy with products of host immunity, such as nitric oxide and hypoxia, and are effective on bacteria within macrophages, a cellular reservoir for latent Mtb. Compounds that kill nonreplicating pathogens in cooperation with host immunity could complement the conventional chemotherapy of infectious disease.