Identification of Rv3852 as an Agrimophol-Binding Protein in Mycobacterium tuberculosis

Mycobacterial tuberculosis (Mtb) is able to preserve its intrabacterial pH (pH_IB) near neutrality in the acidic phagosomes of immunologically activated macrophages and to cause lethal pathology in immunocompetent mice. In contrast, when its ability to maintain pH_IB homeostasis is genetically compromised, Mtb dies in acidic phagosomes and is attenuated in the mouse. Compounds that phenocopy the genetic disruption of Mtb’s pH_IB homeostasis could serve as starting points for drug development in their own right or through identification of their targets. A previously reported screen of a natural product library identified a phloroglucinol, agrimophol, that lowered Mtb’s pH_IB and killed Mtb at an acidic extrabacterial pH. Inability to identify agrimophol-resistant mutants of Mtb suggested that the compound may have more than one target. Given that polyphenolic compounds may undergo covalent reactions, we attempted an affinity-based method for target identification. The structure-activity relationship of synthetically tractable polyhydroxy diphenylmethane analogs with equivalent bioactivity informed the design of a bioactive agrimophol alkyne. After click-chemistry reaction with azido-biotin and capture on streptavidin, the biotinylated agrimophol analog pulled down the Mtb protein Rv3852, a predicted membrane protein that binds DNA in vitro. A ligand-protein interaction between agrimophol and recombinant Rv3852 was confirmed by isothermal calorimetry (ITC) and led to disruption of Rv3852’s DNA binding function. However, genetic deletion of rv3852 in Mtb did not phenocopy the effect of agrimophol on Mtb, perhaps because of redundancy of its function.     

Characterization of a novel esterase Rv1497 of Mycobacterium tuberculosisH37Rv demonstrating β-lactamase activity

The Rv1497 (LipL) of the Mycobacterium tuberculosis H37Rv was predicted to be similar to hypothetical esterases and penicillin binding proteins ofM. tuberculosis as well as to be involved in lipid metabolism. Sequence alignment revealed that Rv1497 protein contains characteristic consensus β-lactamase motif ‘SXXK’ in addition to a conserve pentapeptide –GXSXG-, characteristic of lipolytic enzymes, at the C-terminus of protein in contrast to its usual N-terminus location. For detailed characterization of protein, the rv1497 gene was cloned, expressed with N-terminal His-tag and purified to homogeneity on Ni-NTA column. Rv1497 demonstrated both esterase and β-lactamase activities. A serine located within consensus β-lactamase motif ‘SXXK’ was identified as catalytic residue in both esterase and β-lactamase enzymatic activities whereas serine residue located within conserved pentapeptide did not show any effect on both enzyme activities. The catalytic residues of Rv1497 for β-lactamase activity were determined to be Ser88, Tyr-175 and His355 residues by site-directed mutagenesis. The enzyme demonstrated preference for short chain esters (pNP-butyrate). The expression of lipL gene was significantly up-regulated during acidic stress as compared to normal conditions in in vitro culture of M. tuberculosis H37Ra. This is perhaps the first report demonstrating an esterase of mycobacterium showing β-lactamase activity.     

The Mycobacterium tuberculosis Rv2745c Plays an Important Role in Responding to Redox Stress

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death from an infectious disease worldwide. Over the course of its life cycle in vivo, Mtb is exposed to a plethora of environmental stress conditions. Temporal regulation of genes involved in sensing and responding to such conditions is therefore crucial for Mtb to establish an infection. The Rv2745c (clgR) gene encodes a Clp protease gene regulator that is induced in response to a variety of stress conditions and potentially plays a role in Mtb pathogenesis. Our isogenic mutant, Mtb:ΔRv2745c, is significantly more sensitive to in vitro redox stress generated by diamide, relative to wild-type Mtb as well as to a complemented strain. Together with the fact that the expression of Rv2745c is strongly induced in response to redox stress, these results strongly implicate a role for ClgR in the management of intraphagosomal redox stress. Additionally, we observed that redox stress led to the dysregulation of the expression of the σ^H/σ^E regulon in the isogenic mutant, Mtb:ΔRv2745c. Furthermore, induction of clgR in Mtb and Mtb:ΔRv2745c (comp) did not lead to Clp protease induction, indicating that clgR has additional functions that need to be elucidated. Our data, when taken together with that obtained by other groups, indicates that ClgR plays diverse roles in multiple regulatory networks in response to different stress conditions. In addition to redox stress, the expression of Rv2745c correlates with the expression of genes involved in sulfate assimilation as well as in response to hypoxia and reaeration. Clearly, the Mtb Rv2745c-encoded ClgR performs different functions during stress response and is important for the pathogenicity of Mtb in-vivo, regardless of its induction of the Clp proteolytic pathway.     

Molecular insights into the binding of coenzyme F420 to the conserved protein Rv1155 from Mycobacterium tuberculosis

Coenzyme F₄₂₀ is a deazaflavin hydride carrier with a lower reduction potential than most flavins. In Mycobacterium tuberculosis (Mtb), F₄₂₀ plays an important role in activating PA-824, an antituberculosis drug currently used in clinical trials. Although F₄₂₀ is important to Mtb redox metabolism, little is known about the enzymes that bind F₄₂₀ and the reactions that they catalyze. We have identified a novel F₄₂₀-binding protein, Rv1155, which is annotated in the Mtb genome sequence as a putative flavin mononucleotide (FMN)-binding protein. Using biophysical techniques, we have demonstrated that instead of binding FMN or other flavins, Rv1155 binds coenzyme F₄₂₀. The crystal structure of the complex of Rv1155 and F₄₂₀ reveals one F₄₂₀ molecule bound to each monomer of the Rv1155 dimer. Structural, biophysical, and bioinformatic analyses of the Rv1155–F₄₂₀ complex provide clues about its role in the bacterium.     

Characterization of culture filtrate proteins Rv1197 and Rv1198 of ESAT-6 family from Mycobacterium tuberculosis H37Rv

BackgroundWe have characterized two immunogenic proteins, Rv1197 and Rv1198, of the Esx-5 system of the ESAT-6 family of Mycobacterium tuberculosis H37Rv.MethodsThe complex formation between Rv1197 and Rv1198 was characterized by biophysical techniques. The reactivity of serum from TB patients towards these proteins was characterized by ELISA. Lymphocyte proliferation and cytokine induction were followed in restimulated splenocytes from immunized mice by using MTT assay and CBA flowcytometry, respectively.ResultsRv1197 and Rv1198 strongly interact to form a heterodimeric complex under reducing conditions, which is characterized by a dissociation constant of 97 × 10^− 9 M and melting temperature, Tm, of 50.5 °C. Strong humoral responses to Rv1197, Rv1198, CFP-10 and MoaC1 (Rv3111) antigens were found in Indian patients with active pulmonary tuberculosis (n = 44), in comparison to non-infected healthy individuals (n = 20). The seroreactivity to Rv1198 was characterized by a sensitivity of 75% and specificity of 90%. In BALB/c mice, immunization with Rv1198-FIA induced a pro-inflammatory response with elevated levels of TNF and IL-6, along with low induction of IFN-γ, IL-2 and IL-10, but no induction of IL-4.ConclusionRv1197 and Rv1198 form a stable complex, which is regulated by the redox state of Rv1198. Rv1198 is immunogenic with highly specific seroreactivity towards TB patients' serum. Rv1198 elicits a pro-inflammatory recall response in immunized mice.General SignificanceThis study characterizes the interaction of Rv1197 and Rv1198, and establishes the immunogenic nature of Rv1198.     

Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP-Binding: Requirement for Establishing Chronic Persistent Infection

Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosis universal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-Å-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i) M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii) Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii) Rv2623 binds ATP; and iv) the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.     

Mycobacterium tuberculosis Rv2179c Protein Establishes a New Exoribonuclease Family with Broad Phylogenetic Distribution

Ribonucleases (RNases) maintain the cellular RNA pool by RNA processing and degradation. In many bacteria, including the human pathogen Mycobacterium tuberculosis (Mtb), the enzymes mediating several central RNA processing functions are still unknown. Here, we identify the hypothetical Mtb protein Rv2179c as a highly divergent exoribonuclease. Although the primary sequence of Rv2179c has no detectable similarity to any known RNase, the Rv2179c crystal structure reveals an RNase fold. Active site residues are equivalent to those in the DEDD family of RNases, and Rv2179c has close structural homology to Escherichia coli RNase T. Consistent with the DEDD fold, Rv2179c has exoribonuclease activity, cleaving the 3′ single-strand overhangs of duplex RNA. Functional orthologs of Rv2179c are prevalent in actinobacteria and found in bacteria as phylogenetically distant as proteobacteria. Thus, Rv2179c is the founding member of a new, large RNase family with hundreds of members across the bacterial kingdom.     

Mycobacterium tuberculosis Rv3406 Is a Type II Alkyl Sulfatase Capable of Sulfate Scavenging

The genome of Mycobacterium tuberculosis (Mtb) encodes nine putative sulfatases, none of which have a known function or substrate. Here, we characterize Mtb’s single putative type II sulfatase, Rv3406, as a non-heme iron (II) and α-ketoglutarate-dependent dioxygenase that catalyzes the oxidation and subsequent cleavage of alkyl sulfate esters. Rv3406 was identified based on its homology to the alkyl sulfatase AtsK from Pseudomonas putida. Using an in vitro biochemical assay, we confirmed that Rv3406 is a sulfatase with a preference for alkyl sulfate substrates similar to those processed by AtsK. We determined the crystal structure of the apo Rv3406 sulfatase at 2.5 Å. The active site residues of Rv3406 and AtsK are essentially superimposable, suggesting that the two sulfatases share the same catalytic mechanism. Finally, we generated an Rv3406 mutant (Δrv3406) in Mtb to study the sulfatase’s role in sulfate scavenging. The Δrv3406 strain did not replicate in minimal media with 2-ethyl hexyl sulfate as the sole sulfur source, in contrast to wild type Mtb or the complemented strain. We conclude that Rv3406 is an iron and α-ketoglutarate-dependent sulfate ester dioxygenase that has unique substrate specificity that is likely distinct from other Mtb sulfatases.     

Rv0180c contributes to Mycobacterium tuberculosis cell shape and to infectivity in mice and macrophages

Mycobacterium tuberculosis, the main causative agent of human tuberculosis, is transmitted from person to person via small droplets containing very few bacteria. Optimizing the chance to seed in the lungs is therefore a major adaptation to favor survival and dissemination in the human population. Here we used TnSeq to identify genes important for the early events leading to bacterial seeding in the lungs. Beside several genes encoding known virulence factors, we found three new candidates not previously described: rv0180c, rv1779c and rv1592c. We focused on the gene, rv0180c, of unknown function. First, we found that deletion of rv0180c in M. tuberculosis substantially reduced the initiation of infection in the lungs of mice. Next, we established that Rv0180c enhances entry into macrophages through the use of complement-receptor 3 (CR3), a major phagocytic receptor for M. tuberculosis. Silencing CR3 or blocking the CR3 lectin site abolished the difference in entry between the wild-type parental strain and the Δrv0180c::km mutant. However, we detected no difference in the production of both CR3-known carbohydrate ligands (glucan, arabinomannan, mannan), CR3-modulating lipids (phthiocerol dimycocerosate), or proteins in the capsule of the Δrv0180c::km mutant in comparison to the wild-type or complemented strains. By contrast, we established that Rv0180c contributes to the functionality of the bacterial cell envelope regarding resistance to toxic molecule attack and cell shape. This alteration of bacterial shape could impair the engagement of membrane receptors that M. tuberculosis uses to invade host cells, and open a new perspective on the modulation of bacterial infectivity.     

Potential of Mycobacterium tuberculosis resuscitation-promoting factors as antigens in novel tuberculosis sub-unit vaccines

Novel vaccines are needed to control tuberculosis (TB), the bacterial infectious disease that together with malaria and HIV is worldwide responsible for high levels of morbidity and mortality. TB can result from the reactivation of an initially controlled latent infection by Mycobacterium tuberculosis (Mtb). Mtb proteins for which a possible role in this reactivation process has been hypothesized are the five homologs of the resuscitation-promoting factor of Micrococcus luteus, namely Mtb Rv0867c (rpfA), Rv1009 (rpfB), Rv1884c (rpfC), Rv2389c (rpfD) and Rv2450c (rpfE). Analysis of the immune recognition of these 5 proteins following Mtb infection or Mycobacterium bovis BCG vaccination of mice showed that Rv1009 (rpfB) and Rv2389c (rpfD) are the most antigenic in the tested models. We therefore selected rpfB and rpfD for testing their vaccine potential as plasmid DNA vaccines. Elevated cellular immune responses and modest but significant protection against intra-tracheal Mtb challenge were induced by immunization with the rpfB encoding DNA vaccine. The results indicate that rpfB is the most promising candidate of the five rpf-like proteins of Mtb in terms of its immunogenicity and protective efficacy and warrants further analysis for inclusion as an antigen in novel TB vaccines.     

Molecular characterization of novel immunodominant molybdenum cofactor biosynthesis protein C1 (Rv3111) from Mycobacterium tuberculosis H37Rv

BackgroundIn the molybdenum cofactor biosynthesis pathway, MoaA and MoaC catalyze the first step of transformation of GTP to cPMP. In M. tuberculosis H37Rv, three different genes (Rv3111, Rv0864 and Rv3324c) encode for MoaC homologs. Out of these three only MoaC1 (Rv3111) is secretory in nature.MethodsWe have characterized MoaC1 protein through biophysical, in-silico, and immunological techniques.ResultsWe have characterized the conformation and thermodynamic stability of MoaC1, and have established its secretory nature by demonstrating the presence of anti-MoaC1 antibodies in human tuberculosis patients' sera. Further, MoaC1 elicited a dominant Th1 immune response in mice characterized by increased induction of IL-2 and IFN-γ.ConclusionIntegrating these results, we conclude that MoaC1 is a structured secretory protein capable of binding with GTP and eliciting induced immune response.General significanceThis study would be useful for the development of vaccines against tuberculosis and to improve methods used for diagnosis of tuberculosis.     

An Extracellular Disulfide Bond Forming Protein (DsbF) from Mycobacterium tuberculosis: Structural, Biochemical, and Gene Expression Analysis

Disulfide bond forming (Dsb) proteins ensure correct folding and disulfide bond formation of secreted proteins. Previously, we showed that Mycobacterium tuberculosis DsbE (Mtb DsbE, Rv2878c) aids in vitro oxidative folding of proteins. Here, we present structural, biochemical, and gene expression analyses of another putative Mtb secreted disulfide bond isomerase protein homologous to Mtb DsbE, Mtb DsbF (Rv1677). The X-ray crystal structure of Mtb DsbF reveals a conserved thioredoxin fold although the active-site cysteines may be modeled in both oxidized and reduced forms, in contrast to the solely reduced form in Mtb DsbE. Furthermore, the shorter loop region in Mtb DsbF results in a more solvent-exposed active site. Biochemical analyses show that, similar to Mtb DsbE, Mtb DsbF can oxidatively refold reduced, unfolded hirudin and has a comparable pK_a for the active-site solvent-exposed cysteine. However, contrary to Mtb DsbE, the Mtb DsbF redox potential is more oxidizing and its reduced state is more stable. From computational genomics analysis of the M. tuberculosis genome, we identified a potential Mtb DsbF interaction partner, Rv1676, a predicted peroxiredoxin. Complex formation is supported by protein coexpression studies and inferred by gene expression profiles, whereby Mtb DsbF and Rv1676 are upregulated under similar environments. Additionally, comparison of Mtb DsbF and Mtb DsbE gene expression data indicates anticorrelated gene expression patterns, suggesting that these two proteins and their functionally linked partners constitute analogous pathways that may function under different conditions.     

Rv2477c is an antibiotic-sensitive manganese-dependent ABC-F ATPase in Mycobacterium tuberculosis

The Rv2477c protein of Mycobacterium tuberculosis (Mtb) belongs to the ATP-binding cassette (ABC) subfamily F that contains proteins with tandem nucleotide-binding domains but lacking transmembrane domains. ABC-F subfamily proteins have been implicated in diverse cellular processes such as translation, antibiotic resistance, cell growth and nutrient sensing. In order to investigate the biochemical characteristics of Rv2477c, we expressed it in Escherichia coli, purified it and characterized its enzymatic functions. We show that Rv2477c displays strong ATPase activity (V_max = 45.5 nmol/mg/min; K_m = 90.5 μM) that is sensitive to orthovanadate. The ATPase activity was maximal in the presence of Mn²⁺ at pH 5.2. The Rv2477c protein was also able to hydrolyze GTP, TTP and CTP but at lower rates. Glutamate to glutamine substitutions at amino acid residues 185 and 468 in the two Walker B motifs of Rv2477c severely inhibited its ATPase activity. The antibiotics tetracycline and erythromycin, which target protein translation, were able to inhibit the ATPase activity of Rv2477c. We postulate that Rv2477c could be involved in mycobacterial protein translation and in resistance to tetracyclines and macrolides. This is the first report of the biochemical characterization of an ABC-F subfamily protein in Mtb.     

Molecular characterization of secretory proteins Rv3619c and Rv3620c from Mycobacterium tuberculosis H37Rv

Rv3619c and Rv3620c are the secretory, antigenic proteins of the ESAT-6/CFP-10 family of Mycobacterium tuberculosis H37Rv. In this article, we show that Rv3619c interacts with Rv3620c to form a 1 : 1 heterodimeric complex with a dissociation constant (K(d)) of 4.8 × 10(-7) M. The thermal unfolding of the heterodimer was completely reversible, with a T(m) of 48 °C. The comparative thermodynamics and thermal unfolding analysis of the Rv3619c-Rv3620c dimer, the ESAT-6-CFP-10 dimer and another ESAT family heterodimer, Rv0287-Rv0288, revealed that the binding strength and stability of Rv3619c-Rv3620c are relatively lower than those of the other two pairs. Molecular modeling and docking studies predict the structure of Rv3619c-Rv3620c to be similar to that of ESAT-6-CFP-10. Spectroscopic studies revealed that, in an acidic environment, Rv3619c and Rv3620c lose their secondary structure and interact weakly to form a complex with a lower helical content, indicating that Rv3619c-Rv3620c is destabilized at low pH. These results, combined with those of previous studies, suggest that unfolding of the proteins is required for dissociation of the complex and membrane binding. In the presence of membrane mimetics, the α-helical contents of Rv3619c and Rv3620 increased by 42% and 35%, respectively. In mice, the immune response against Rv3619c protein is characterized by increased levels of interferon-γ, interleukin-12 and IgG(2a) , indicating a dominant Th1 response, which is mandatory for protection against mycobacterial infection. This study therefore emphasizes the potential of Rv3619c as a subunit vaccine candidate.     

Mycobacterial Metabolic Syndrome: LprG and Rv1410 Regulate Triacylglyceride Levels,Growth Rate and Virulence in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) mutants lacking rv1411c, which encodes the lipoprotein LprG, and rv1410c, which encodes a putative efflux pump, are dramatically attenuated for growth in mice. Here we show that loss of LprG-Rv1410 in Mtb leads to intracellular triacylglyceride (TAG) accumulation, and overexpression of the locus increases the levels of TAG in the culture medium, demonstrating a role of this locus in TAG transport. LprG binds TAG within a large hydrophobic cleft and is sufficient to transfer TAG from donor to acceptor membranes. Further, LprG-Rv1410 is critical for broadly regulating bacterial growth and metabolism in vitro during carbon restriction and in vivo during infection of mice. The growth defect in mice is due to disrupted bacterial metabolism and occurs independently of key immune regulators. The in vivo essentiality of this locus suggests that this export system and other regulators of metabolism should be considered as targets for novel therapeutics.     

Pro- and Anti-Inflammatory Cytokines against Rv2031 Are Elevated during Latent Tuberculosis: A Study in Cohorts of Tuberculosis Patients,Household Contacts and Community Controls in an Endemic Setting

Tuberculosis (TB) is among the leading causes of morbidity and mortality. The causative agent, Mycobacterium tuberculosis (Mtb), has evolved virulent factors for entry, survival, multiplication and immune evasion. Rv2031 (also called alpha crystallin, hspX, 16-kDa antigen), one of the most immunogenic latency antigens, is believed to play a key role in long-term viability of Mtb. Here, we report the dynamics of pro-inflammatory (IFN-γ, TNF-α) and anti-inflammatory (IL-10) cytokines against Rv2031 using whole blood assay in human cohorts in a TB endemic setting. Cytokine responses to ESAT-6-CFP-10 were also measured for comparison. Blood samples were collected from smear positive pulmonary TB patients and their contacts at baseline, 6 and 12 months, and from community controls at entry. At baseline, 54.4% of controls and 73.2% of contacts were QFT-GIT test positive. Baseline IFN-γ, TNF-α and IL-10 responses to Rv2031 were significantly higher in controls compared to contacts and untreated patients (p<0.001). Furthermore, untreated patients had significantly higher TNF-α and IL-10 responses to Rv2031 compared to contacts (p<0.001). In contacts and treated patients, IFN-γ, TNF-α and IL-10 responses to Rv2031 significantly increased over 12 months (p<0.0001) and became comparable with the corresponding levels in controls. There was a positive and significant correlation between Rv2031 and ESAT-6-CFP-10 specific cytokine responses in each study group. The fact that the levels of IFN-γ, TNF-α and IL-10 against Rv2031 were highest during latent TB infection may indicate their potential as markers of protection against TB. Taken together, the findings of this study suggest the potential of IFN-γ, TNF-α and IL-10 against Rv2031 as biomarkers of the host response to Mtb during convalescence from, and the absence of, active tuberculosis.     

Mutational analysis of the (p)ppGpp synthetase activity of the Rel enzyme of Mycobacterium tuberculosis

Rel_Mtb, a GTP pyrophosphokinase encoded by the Mycobacterium tuberculosis (Mtb) genome, catalyzes synthesis of (p)ppGpp from ATP and GDP(GTP) and its hydrolysis to GDP(GTP) and pyrophosphate to mediate stringent response, which helps bacteria to survive during nutrient limitation. Like other members of Rel_Spo homologs, Rel_Mtb has four distinct domains: HD, Rel_Spo (RSD), TGS and ACT. The N-terminal HD and RSD are responsible for (p)ppGpp hydrolysis and synthesis, respectively. In this study, we have dissected the rel _Mtb gene function and determined the minimal region essential for (p)ppGpp synthetic activity. The Rel_Mtb and its truncated derivatives were expressed from an arabinose inducible promoter (P _BAD ), and in vivo functional analyses were done in a (p)ppGpp null Escherichia coli strain. Our results indicate that only 243 amino acids (188–430 residues) containing fragment are sufficient for Rel_Mtb (p)ppGpp synthetic activity. The results were further confirmed by in vitro assays using purified proteins. We further characterized the RSD of Rel_Mtb by substituting several conserved amino acids with structurally related residues and identified six such residues, which appeared to be critical for maintaining its catalytic activity. Furthermore, we have also extended our analysis to an RSD encoding gene rv1366 of Mtb, and experimental results indicated that the encoded protein Rv1366 is unable to synthesize (p)ppGpp.     

Residues Asn214, Gln211, Glu219 and Gln221 contained in the subfamily 3 catalytic signature of the isocitrate lyase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> are involved in its catalytic and thermal properties

Isocitrate lyase, encoded by the aceA gene, plays an important role in the ability of Pseudomonas aeruginosa to grow on fatty acids, acetate, acyclic terpenes, and amino acids. Phylogenetic analysis indicated that the ICL superfamily is divided in two families: the ICL family, which includes five subfamilies, and the 2-methylisocitrate lyase (MICL) family. ICL from P. aeruginosa (ICL-Pa) was identified in a different ICL node (subfamily 3) than other Pseudomonas ICL enzymes (grouped in subfamily 1). Analysis also showed that psychrophilic bacteria are mainly grouped in ICL subfamily 3, whose ICL proteins contain the highly conserved catalytic pattern Q_IEN_QV_SDE_KQ_CGHQD. We performed site-directed mutagenesis, enzymatic activity, and structure modeling of conserved residues in mutated ICLs by using ICL-Pa as a model. Our results indicated that the N214 residue is essential for catalytic function, while mutating the Q211, E219, and Q221 residues impairs its catalytic and thermostability properties. Our findings suggest that conserved residues in the subfamily 3 signature of ICL-Pa play important roles in catalysis and thermostability and are likely associated with the catalytic loop structural conformation.