Publications

• Characterization of a novel esterase Rv0045c from Mycobacterium tuberculosis.

  Guo J., Zheng X., Xu L., Liu Z., Xu K., Li S., Wen T., Liu S., Pang H.

  <h4>Background</h4>It was proposed that there are at least 250 enzymes in M. tuberculosis involved in lipid metabolism. Rv0045c was predicted to be a hydrolase by amino acid sequence similarity, although its precise biochemical characterization and function remained to be defined.<h4>Methodology/p ... >> More

  <h4>Background</h4>It was proposed that there are at least 250 enzymes in M. tuberculosis involved in lipid metabolism. Rv0045c was predicted to be a hydrolase by amino acid sequence similarity, although its precise biochemical characterization and function remained to be defined.<h4>Methodology/principal findings</h4>We expressed the Rv0045c protein to high levels in E. coli and purified the protein to high purity. We confirmed that the prepared protein was the Rv0045c protein by mass spectrometry analysis. Circular dichroism spectroscopy analysis showed that the protein possessed abundant β-sheet secondary structure, and confirmed that its conformation was stable in the range pH 6.0-10.0 and at temperatures ≤ 40 °C. Enzyme activity analysis indicated that the Rv0045c protein could efficiently hydrolyze short chain p-nitrophenyl esters (C₂-C₈), and its suitable substrate was p-nitrophenyl caproate (C₆) with optimal catalytic conditions of 39 °C and pH 8.0.<h4>Conclusions/significance</h4>Our results demonstrated that the Rv0045c protein is a novel esterase. These experiments will be helpful in understanding ester/lipid metabolism related to M. tuberculosis. << Less

  PLoS ONE 5:e13143-e13143(2010) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Two cutinase-like proteins secreted by Mycobacterium tuberculosis show very different lipolytic activities reflecting their physiological function.

  Schue M., Maurin D., Dhouib R., Bakala N'Goma J.C., Delorme V., Lambeau G., Carriere F., Canaan S.

  Cutinases are extracellular enzymes that are able to degrade cutin, a polyester protecting plant leaves and many kinds of lipids. Although cutinases are mainly found in phytopathogenic fungi or bacteria, 7 genes related to the cutinase family have been predicted in the genome of Mycobacterium tube ... >> More

  Cutinases are extracellular enzymes that are able to degrade cutin, a polyester protecting plant leaves and many kinds of lipids. Although cutinases are mainly found in phytopathogenic fungi or bacteria, 7 genes related to the cutinase family have been predicted in the genome of Mycobacterium tuberculosis. These genes may encode proteins that are involved in the complex lipid metabolism of the bacterium. Here, we report on the biochemical characterization of two secreted proteins of M. tuberculosis, Rv1984c and Rv3452, belonging to the cutinase family. Although their amino acid sequence shows 50% identity with that of the well-characterized cutinase from Fusarium solani pisi, and a high level of homology has been found to exist between these two enzymes, they show distinct substrate specificities. Rv1984c preferentially hydrolyzes medium-chain carboxylic esters and monoacylglycerols, whereas Rv3452 behaves like a phospholipase A(2), and it is able to induce macrophage lysis. The tetrahydrolipstatin inhibitor, a specific lipase inhibitor, abolishes the activity of both enzymes. Site-directed mutagenesis was performed to identify the catalytic triad of Rv1984c. Structural models for Rv1984c and Rv3452 were built, based on the crystal structure of F. solani cutinase, with a view to investigating the contribution of specific residues to the substrate specificity. Our findings open new prospects for investigating the physiological roles of cutinase-like proteins in the lipid metabolism and virulence of M. tuberculosis. << Less

  FASEB J. 24:1893-1903(2010) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Characterization of a novel exported esterase Rv3036c from Mycobacterium tuberculosis.

  Chen L., Dang G., Deng X., Cao J., Yu S., Wu D., Pang H., Liu S.

  Mycobacterium tuberculosis possesses an unusually high number of genes involved in the metabolism of lipids. Driven by a newly described esterase motif SXXK in the amino acid sequence and a predicted signal peptide, the gene rv3036c from M. tuberculosis was cloned and characterized biochemically. ... >> More

  Mycobacterium tuberculosis possesses an unusually high number of genes involved in the metabolism of lipids. Driven by a newly described esterase motif SXXK in the amino acid sequence and a predicted signal peptide, the gene rv3036c from M. tuberculosis was cloned and characterized biochemically. Rv3036c efficiently hydrolyzes soluble p-nitrophenyl esters but not emulsified lipid. The highest activity of this enzyme was observed when p-nitrophenyl acetate (C2) was used as the substrate. Based on the activities, Rv3036c was classified as a nonlipolytic hydrolase. The results of immunoreactivity studies on the subcellular mycobacterial fractions suggested that the enzyme was present in the cell wall and cell membrane in mycobacteria. In summary, Rv3036c was characterized as a novel cell wall-anchored esterase from M. tuberculosis. << Less

  Protein Expr. Purif. 104:50-56(2014) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• Characterization of LipN (Rv2970c) of Mycobacterium tuberculosis H37Rv and its probable role in xenobiotic degradation.

  Jadeja D., Dogra N., Arya S., Singh G., Singh G., Kaur J.

  LipN (Rv2970c) belongs to the Lip family of M. tuberculosis H37Rv and is homologous to the human Hormone Sensitive Lipase. The enzyme demonstrated preference for short carbon chain substrates with optimal activity at 45°C/pH 8.0 and stability between pH 6.0-9.0. The specific activity of the enzyme ... >> More

  LipN (Rv2970c) belongs to the Lip family of M. tuberculosis H37Rv and is homologous to the human Hormone Sensitive Lipase. The enzyme demonstrated preference for short carbon chain substrates with optimal activity at 45°C/pH 8.0 and stability between pH 6.0-9.0. The specific activity of the enzyme was 217 U/mg protein with pNP-butyrate as substrate. It hydrolyzed tributyrin to di- and monobutyrin. The active-site residues of the enzyme were confirmed to be Ser216, Asp316, and His346. Tetrahydrolipstatin, RHC-80267 and N-bromosuccinimide inhibited LipN enzyme activity completely. Interestingly, Trp145, a non active-site residue, demonstrated functional role to retain enzyme activity. The enzyme was localized in cytosolic fraction of M. tuberculosis H37Rv. The enzyme was able to synthesize ester of butyric acid, methyl butyrate, in presence of methanol. LipN was able to hydrolyze 4-hydroxyphenylacetate to hydroquinone. The gene was not expressed in in-vitro growth conditions while the expression of rv2970c gene was observed post 6h of macrophage infection by M. tuberculosis H37Ra. Under individual in-vitro stress conditions, the gene was expressed during acidic stress condition only. These findings suggested that LipN is a cytosolic, acid inducible carboxylesterase with no positional specificity in demonstrating activity with short carbon chain substrates. It requires Trp145, a non active site residue, for it's enzyme activity. << Less

  J. Cell. Biochem. 117:390-401(2016) [PubMed] [EuropePMC]

  This publication is cited by 9 other entries.

• Characterization of an acid inducible lipase Rv3203 from Mycobacterium tuberculosis H37Rv.

  Singh G., Arya S., Narang D., Jadeja D., Singh G., Gupta U.D., Singh K., Kaur J.

  The Rv3203 (LipV) of Mycobacterium tuberculosis (Mtb) H37Rv, is annotated as a member of Lip family based on the presence of characteristic consensus esterase motif ‘GXSXG’. In vitro culture studies of Mtb H37Ra indicated that expression of Rv3203 gene was up-regulated during acidic stress as comp ... >> More

  The Rv3203 (LipV) of Mycobacterium tuberculosis (Mtb) H37Rv, is annotated as a member of Lip family based on the presence of characteristic consensus esterase motif ‘GXSXG’. In vitro culture studies of Mtb H37Ra indicated that expression of Rv3203 gene was up-regulated during acidic stress as compared to normal whereas no expression was observed under nutrient and oxidative stress conditions. Therefore, detailed characterization of Rv3203 was done by gene cloning and its further expression and purification as his-tagged protein in microbial expression system. The enzyme was purified to homogeneity by affinity chromatography. It demonstrated broad substrate specificity and preferentially hydrolyzed p-nitrophenyl myristate. The purified enzyme demonstrated an optimum activity at pH 8.0 and temperature 50 °C. The specific activity, K m and V max of enzyme was determined to be 21.29 U mg(-1) protein, 714.28 μM and 62.5 μmol ml(-1) min(-1), respectively. The pH stability assay and circular dichroism spectroscopic analysis revealed that Rv3203 protein is more stable in acidic condition. Tetrahydrolipstatin, a specific lipase inhibitor and RHC80267, a diacylglycerol lipase inhibitor abolished the activity of this enzyme. The catalytic triad residues were determined to be Ser50, Asp180 and His203 residues by site-directed mutagenesis. << Less

  Mol. Biol. Rep. 41:285-296(2014) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• Identification and characterization of lipase activity and immunogenicity of LipL from Mycobacterium tuberculosis.

  Cao J., Dang G., Li H., Li T., Yue Z., Li N., Liu Y., Liu S., Chen L.

  Lipids and lipid-metabolizing esterases/lipases are highly important for the mycobacterial life cycle and, possibly, for mycobacterial virulence. In this study, we expressed 10 members of the Lip family of Mycobacterium tuberculosis. Among the 10 proteins, LipL displayed a significantly high enzym ... >> More

  Lipids and lipid-metabolizing esterases/lipases are highly important for the mycobacterial life cycle and, possibly, for mycobacterial virulence. In this study, we expressed 10 members of the Lip family of Mycobacterium tuberculosis. Among the 10 proteins, LipL displayed a significantly high enzymatic activity for the hydrolysis of long-chain lipids. The optimal temperature for the lipase activity of LipL was demonstrated to be 37°C, and the optimal pH was 8.0. The lipase active center was not the conserved motif G-x-S-x-G, but rather the S-x-x-K and GGG motifs, and the key catalytic amino acid residues were identified as G50, S88, and K91, as demonstrated through site-directed mutagenesis experiments. A three-dimensional modeling structure of LipL was constructed, which showed that the GGG motif was located in the surface of a pocket structure. Furthermore, the subcellular localization of LipL was demonstrated to be on the mycobacterial surface by Western blot analysis. Our results revealed that the LipL protein could induce a strong humoral immune response in humans and activate a CD8+ T cell-mediated response in mice. Overall, our study identified and characterized a novel lipase denoted LipL from M. tuberculosis, and demonstrated that LipL functions as an immunogen that activates both humoral and cell-mediated responses. << Less

  PLoS ONE 10:E0138151-E0138151(2015) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.

• A novel extracellular esterase from Bacillus subtilis and its conversion to a monoacylglycerol hydrolase.

  Eggert T., Pencreac'h G., Douchet I., Verger R., Jaeger K.-E.

  A novel gene lipB, which encodes an extracellular lipolytic enzyme, was identified in the Bacillus subtilis genomic DNA sequence. We have cloned and overexpressed lipB in B. subtilis and Escherichia coli and have also purified the enzyme from a B. subtilis culture supernatant to electrophoretic ho ... >> More

  A novel gene lipB, which encodes an extracellular lipolytic enzyme, was identified in the Bacillus subtilis genomic DNA sequence. We have cloned and overexpressed lipB in B. subtilis and Escherichia coli and have also purified the enzyme from a B. subtilis culture supernatant to electrophoretic homogeneity. Four different lipase assays were used to determine its catalytic activity: pH-stat, spectrophotometry, fluorimetry and the monomolecular film technique. LipB preferentially hydrolysed triacylglycerol-esters and p-nitrophenyl-esters of fatty acids with short chain lengths of </= 10 carbon atoms. Triolein, which is a typical substrate for true lipases, was not hydrolysed at all. These results led us to classify LipB as an esterase rather than a lipase. The catalytic triad of LipB consists of residues Ser78, Asp134, and His157 as demonstrated by amino-acid sequence alignments and site-directed mutagenesis. The nucleophile Ser78 is located in a lipase-specific consensus sequence, which is Ala-X-Ser-X-Gly for most Bacillus lipases. All other bacterial lipases contain a glycine residue instead of the alanine at position-2 with respect to the catalytic serine. We have investigated the role of this alanine residue by constructing LipB variant A76G, thereby restoring the lipase-specific consensus motif. When compared with LipB this variant showed a markedly reduced thermostability but an increased stability at pH 5-7. Determination of the specific activities of wild-type LipB and variant A76G using a monomolecular film of the substrate monoolein revealed an interesting result: the A76G substitution had converted the esterase LipB into a monoacylglycerol hydrolase. << Less

  Eur. J. Biochem. 267:6459-6469(2000) [PubMed] [EuropePMC]

  This publication is cited by 10 other entries.

• The PE16 (Rv1430) of Mycobacterium tuberculosis is an esterase belonging to serine hydrolase superfamily of proteins.

  Sultana R., Vemula M.H., Banerjee S., Guruprasad L.

  The PE and PPE multigene families, first discovered during the sequencing of M. tuberculosis H37Rv genome are responsible for antigenic variation and have been shown to induce increased humoral and cell mediated immune response in the host. Using the bioinformatics tools, we had earlier reported t ... >> More

  The PE and PPE multigene families, first discovered during the sequencing of M. tuberculosis H37Rv genome are responsible for antigenic variation and have been shown to induce increased humoral and cell mediated immune response in the host. Using the bioinformatics tools, we had earlier reported that the 225 amino acid residue PE-PPE domain (Pfam: PF08237) common to some PE and PPE proteins has a "serine α/β hydrolase" fold and conserved Ser, Asp and His catalytic triad characteristic of lipase, esterase and cutinase activities. In order to prove experimentally that PE-PPE domain is indeed a serine hydrolase, we have cloned the full-length Rv1430 and its PE-PPE domain into pET-28a vector, expressed the proteins in E. coli and purified to homogeneity. The activity assays of both purified proteins were carried out using p-nitrophenyl esters of aliphatic carboxylic acids with varying chain length (C2-C16) to study the substrate specificity. To characterize the active site of the PE-PPE domain, we mutated the Ser199 to Ala. The activity of the protein in the presence of serine protease inhibitor-PMSF and the mutant protein were measured. Our results reveal that Rv1430 and its PE-PPE domain possess esterase activity and hydrolyse short to medium chain fatty acid esters with the highest specific activity for pNPC6 at 37°C, 38°C and pH 7.0, 8.0. The details of this work and the observed results are reported in this manuscript. << Less

  PLoS ONE 8:E55320-E55320(2013) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Rv1288, a two domain, cell wall anchored, nutrient stress inducible carboxyl-esterase of Mycobacterium tuberculosis, modulates cell wall lipid.

  Maan P., Kumar A., Kaur J., Kaur J.

  Rv1288, a conserved hypothetical protein of <i>M. tuberculosis (M.tb)</i>, was recently characterized as two-domain esterase enzyme by <i>in silico</i> study. In the present study, Rv1288 and its domains (Est and Lyt) were cloned individually from <i>M.tb</i> into <i>E. coli</i> for expression and ... >> More

  Rv1288, a conserved hypothetical protein of <i>M. tuberculosis (M.tb)</i>, was recently characterized as two-domain esterase enzyme by <i>in silico</i> study. In the present study, Rv1288 and its domains (Est and Lyt) were cloned individually from <i>M.tb</i> into <i>E. coli</i> for expression and purification. The purified rRv1288 and rEst proteins exhibited lipolytic activity with medium chain length esters as optimum substrates, while Lyt domain did not show enzymatic activity. However, presence of Lyt domain resulted in enhanced rate of protein aggregation at higher temperature. Both rRv1288 and rEst followed the similar patterns of substrate specificity, temperature and pH activity. Site directed mutagenesis confirmed the Ser-294, Asp-391 and His-425 as catalytic site residues. Rv1288 was found to be present in cell wall fraction of <i>M.tb</i> H37Ra. Peptidoglycan binding activity of Rv1288 and its domains demonstrated that the Lyt domain is essential for anchoring protein to the cell wall. Expression of <i>rv1288</i> was up regulated in <i>M.tb</i> under nutrient starved condition. Over expression of <i>rv1288</i> in surrogate host <i>M. smegmatis</i> led to change in colony morphology, enhanced pellicle and aggregate formation that might be linked with the changed lipid composition of bacterial cell wall. Cell wall of <i>M. smegmatis</i> expressing <i>rv1288</i> had higher amount of lipids, with a significant increase in trehalose dimycolate content. Rv1288 also leads to increase in drug resistance of <i>M. smegmatis</i>. Rv1288 also enhanced the intracellular survival of <i>M. smegmatis</i> in Raw264.7 cell line. Overall, this study suggested that Rv1288, a cell wall localized carboxyl hydrolase with mycolyl-transferase activity, modulated the cell wall lipids to favor the survival of bacteria under stress condition. << Less

  Front. Cell. Infect. Microbiol. 8:421-421(2018) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.