Tetrahydrolipstatin Inhibition, Functional Analyses, and Three-dimensional Structure of a Lipase Essential for Mycobacterial Viability

The highly complex and unique mycobacterial cell wall is critical to the survival of Mycobacteria in host cells. However, the biosynthetic pathways responsible for its synthesis are, in general, incompletely characterized. Rv3802c from Mycobacterium tuberculosis is a partially characterized phospholipase/thioesterase encoded within a genetic cluster dedicated to the synthesis of core structures of the mycobacterial cell wall, including mycolic acids and arabinogalactan. Enzymatic assays performed with purified recombinant proteins Rv3802c and its close homologs from Mycobacterium smegmatis (MSMEG_6394) and Corynebacterium glutamicum (NCgl2775) show that they all have significant lipase activities that are inhibited by tetrahydrolipstatin, an anti-obesity drug that coincidently inhibits mycobacterial cell wall biosynthesis. The crystal structure of MSMEG_6394, solved to 2.9 Å resolution, revealed an α/β hydrolase fold and a catalytic triad typically present in esterases and lipases. Furthermore, we demonstrate direct evidence of gene essentiality in M. smegmatis and show the structural consequences of loss of MSMEG_6394 function on the cellular integrity of the organism. These findings, combined with the predicted essentiality of Rv3802c in M. tuberculosis, indicate that the Rv3802c family performs a fundamental and indispensable lipase-associated function in mycobacteria. The highly complex and unique mycobacterial cell wall is critical to the survival of Mycobacteria in host cells. However, the biosynthetic pathways responsible for its synthesis are, in general, incompletely characterized. Rv3802c from Mycobacterium tuberculosis is a partially characterized phospholipase/thioesterase encoded within a genetic cluster dedicated to the synthesis of core structures of the mycobacterial cell wall, including mycolic acids and arabinogalactan. Enzymatic assays performed with purified recombinant proteins Rv3802c and its close homologs from Mycobacterium smegmatis (MSMEG_6394) and Corynebacterium glutamicum (NCgl2775) show that they all have significant lipase activities that are inhibited by tetrahydrolipstatin, an anti-obesity drug that coincidently inhibits mycobacterial cell wall biosynthesis. The crystal structure of MSMEG_6394, solved to 2.9 Å resolution, revealed an α/β hydrolase fold and a catalytic triad typically present in esterases and lipases. Furthermore, we demonstrate direct evidence of gene essentiality in M. smegmatis and show the structural consequences of loss of MSMEG_6394 function on the cellular integrity of the organism. These findings, combined with the predicted essentiality of Rv3802c in M. tuberculosis, indicate that the Rv3802c family performs a fundamental and indispensable lipase-associated function in mycobacteria. IntroductionThe genus Mycobacterium contains a number of medically significant species, most notably the devastating human pathogen Mycobacterium tuberculosis that causes around 2 million deaths each year, the most by any single infectious agent. Despite the availability of a vaccine, the number of infected individuals worldwide continues to increase, as does the prevalence of drug-resistant forms of M. tuberculosis (1World Health Organization WHO Report 2009: Global Tuberculosis Control, Epidemiology, Strategy, Financing. World Health Organization, Geneva, Switzerland2009: 1-314Google Scholar). A key virulence factor is the unique mycobacterial cell wall that consists of a core structure as follows: peptidoglycan covalently linked to arabinogalactan esterified with mycolic acids to form the mycolyl-arabinogalactan-peptidoglycan or “mAGP” complex and a series of free glycolipids, including trehalose monomycolates, trehalose dimycolates, phosphatidylinositol mannosides, and lipoarabinomannans (2Brennan P.J. Tuberculosis. 2003; 83: 91-97Crossref PubMed Scopus (614) Google Scholar), that facilitate vital interactions with host cells to initiate and maintain an infection. The essentiality of the core for mycobacterial growth and survival leads to the biosynthetic enzymes involved and being considered as ideal targets for drug development (3Brennan P.J. Crick D.C. Curr. Top. Med. Chem. 2007; 7: 475-488Crossref PubMed Scopus (109) Google Scholar).A subset of genes required for the late steps of mycolic acid and arabinogalactan biosynthesis are located in proximity to the genomes of mycobacteria and corynebacteria. These genes include a well characterized cell wall biosynthesis cluster encoding enzymes required for the activation (AccD4 and FadD32) and condensation (Pks13) (4Portevin D. De Sousa-D'Auria C. Houssin C. Grimaldi C. Chami M. Daffé M. Guilhot C. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 314-319Crossref PubMed Scopus (276) Google Scholar) of mycolic acid intermediates prior to the final reduction step (5Lea-Smith D.J. Pyke J.S. Tull D. McConville M.J. Coppel R.L. Crellin P.K. J. Biol. Chem. 2007; 282: 11000-11008Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), and transfer of mature mycolic acids (6Jackson M. Raynaud C. Lanéelle M.A. Guilhot C. Laurent-Winter C. Ensergueix D. Gicquel B. Daffé M. Mol. Microbiol. 1999; 31: 1573-1587Crossref PubMed Scopus (223) Google Scholar). Also within the cluster are genes required for arabinogalactan biosynthesis (atfB, glf, glfT, and Rv3806c) (7Seidel M. Alderwick L.J. Birch H.L. Sahm H. Eggeling L. Besra G.S. J. Biol. Chem. 2007; 282: 14729-14740Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 8Mikusová K. Yagi T. Stern R. McNeil M.R. Besra G.S. Crick D.C. Brennan P.J. J. Biol. Chem. 2000; 275: 33890-33897Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 9Kremer L. Dover L.G. Morehouse C. Hitchin P. Everett M. Morris H.R. Dell A. Brennan P.J. McNeil M.R. Flaherty C. Duncan K. Besra G.S. J. Biol. Chem. 2001; 276: 26430-26440Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 10Sassetti C.M. Boyd D.H. Rubin E.J. Mol. Microbiol. 2003; 48: 77-84Crossref PubMed Scopus (1975) Google Scholar) which, like the mycolic acid biosynthesis genes, are essential for growth of M. tuberculosis (10Sassetti C.M. Boyd D.H. Rubin E.J. Mol. Microbiol. 2003; 48: 77-84Crossref PubMed Scopus (1975) Google Scholar).Despite extensive functional characterization of this cluster over the last decade, the in vivo function of the product of one gene, Rv3802c, remains unknown, although mycolyltransferase (11Takayama K. Wang C. Besra G.S. Clin. Microbiol. Rev. 2005; 18: 81-101Crossref PubMed Scopus (493) Google Scholar) or Pks13-associated thioesterase (12Parker S.K. Barkley R.M. Rino J.G. Vasil M.L. PLoS One. 2009; 4e4281Crossref PubMed Scopus (52) Google Scholar) functions have been suggested. Although its genomic location strongly suggests a role in cell wall biosynthesis, definitive proof of such a role is lacking. The putative product of Rv3802c has a predicted signal sequence that contains a possible transmembrane domain, and it has been expressed to assess immunological responses (13West N.P. Wozniak T.M. Valenzuela J. Feng C.G. Sher A. Ribeiro J.M. Britton W.J. Vaccine. 2008; 26: 3853-3859Crossref PubMed Scopus (27) Google Scholar) and enzymatic activities. The enzyme is one of seven cutinase-like proteins in M. tuberculosis and is retained in the cell wall, following translocation across the cell membrane (14West N.P. Chow F.M. Randall E.J. Wu J. Chen J. Ribeiro J.M. Britton W.J. FASEB J. 2009; 23: 1694-1704Crossref PubMed Scopus (57) Google Scholar). Previous studies have shown it to have phospholipase A and thioesterase activities (12Parker S.K. Barkley R.M. Rino J.G. Vasil M.L. PLoS One. 2009; 4e4281Crossref PubMed Scopus (52) Google Scholar), consistent with a role in mycolic acid biosynthesis, and significant lipase activity completely dependent on its Ser-Asp-His catalytic triad (14West N.P. Chow F.M. Randall E.J. Wu J. Chen J. Ribeiro J.M. Britton W.J. FASEB J. 2009; 23: 1694-1704Crossref PubMed Scopus (57) Google Scholar). A very recent study has suggested a role for Rv3802c in regulation of outer lipid composition in response to stress because the induction of the Corynebacterium glutamicum ortholog triggered an increase in mycolic acid biosynthesis as part of an outer membrane remodeling response to heat stress (15Meniche X. Labarre C. de Sousa-d'Auria C. Huc E. Laval F. Tropis M. Bayan N. Portevin D. Guilhot C. Daffe M. Houssin C. J. Bacteriol. 2009; 191: 7323-7332Crossref PubMed Scopus (26) Google Scholar).Recently, Rv3802c was identified as a major target of tetrahydrolipstatin (THL) 6The abbreviations used are: THLtetrahydrolipstatinKnkanamycinSmstreptomycinPDBProtein Data Bankr.m.s.d.root mean square deviationBistris propane1,3-bis[tris(hydroxymethyl)methylamino]propaneTEMtransmission electron microscopy. (12Parker S.K. Barkley R.M. Rino J.G. Vasil M.L. PLoS One. 2009; 4e4281Crossref PubMed Scopus (52) Google Scholar). THL is a well characterized and irreversible inhibitor of serine esterases (16Hadváry P. Lengsfeld H. Wolfer H. Biochem. J. 1988; 256: 357-361Crossref PubMed Scopus (300) Google Scholar), originally identified for its specificity for pancreatic lipases, and thus was developed as an anti-obesity drug. THL has been reported to bind covalently to the catalytic serine residue of pancreatic lipase (17Hadváry P. Sidler W. Meister W. Vetter W. Wolfer H. J. Biol. Chem. 1991; 266: 2021-2027Abstract Full Text PDF PubMed Google Scholar) and was found to have similar affinity for human fatty-acid synthase (18Pemble 4th, C.W. Johnson L.C. Kridel S.J. Lowther W.T. Nat. Struct. Mol. Biol. 2007; 14: 704-709Crossref PubMed Scopus (137) Google Scholar). In addition to its actions in humans, THL inhibits and disrupts cell wall formation in several mycobacterial species, with the exception of the nonpathogenic model species Mycobacterium smegmatis (19Kremer L. de Chastellier C. Dobson G. Gibson K.J. Bifani P. Balor S. Gorvel J.P. Locht C. Minnikin D.E. Besra G.S. Mol. Microbiol. 2005; 57: 1113-1126Crossref PubMed Scopus (46) Google Scholar). Rv3802c was strongly inhibited by THL, whereas the nonorthologous but cutinase motif-bearing M. smegmatis lipase MSMEG_1403 was not inhibited at up to a 500:1 inhibitor to enzyme molar ratio (12Parker S.K. Barkley R.M. Rino J.G. Vasil M.L. PLoS One. 2009; 4e4281Crossref PubMed Scopus (52) Google Scholar).To better understand the enzymology of Rv3802c, we expressed and purified the M. tuberculosis enzyme, its homolog in the nonpathogenic model species M. smegmatis, and the ortholog from a related species, C. glutamicum, and we assessed their inhibition by THL. We report here that the closest M. smegmatis homolog to Rv3802c, MSMEG_6394, is inhibited by THL. The crystal structure of MSMEG_6394, along with direct evidence of the essentiality of MSMEG_6394 in M. smegmatis, indicates a fundamental role of this lipase in Mycobacteria.DISCUSSIONThe unique and highly impermeable mycobacterial cell wall is a key virulence factor that forms the interface between host and pathogen. It enables the bacterium to resist destruction by the host and also contains unusual molecules that promote uptake by host macrophages and modify host responses to create a favorable environment for bacterial survival and replication. As a result, the biosynthetic processes involved in the synthesis of the mycobacterial cell wall have been the subject of intensive research, and many of the key cell wall enzymes are now known. For example, nearly all genes within an ∼20-kb genetic locus dedicated to cell wall biosynthesis have now been characterized. In this study, we have focused on one incompletely characterized gene of this cluster, Rv3802c from M. tuberculosis.Homology searches using M. tuberculosis Rv3802c revealed that this gene is very well conserved in Actinomycetes genomes, and we chose to focus on previously uncharacterized homologs from M. smegmatis and C. glutamicum. Previous studies using p-nitrophenyl butyrate substrates had revealed a significant lipase activity of Rv3802c (14West N.P. Chow F.M. Randall E.J. Wu J. Chen J. Ribeiro J.M. Britton W.J. FASEB J. 2009; 23: 1694-1704Crossref PubMed Scopus (57) Google Scholar), and THL-inhibited phospholipase A/thioesterase activities have also been reported (12Parker S.K. Barkley R.M. Rino J.G. Vasil M.L. PLoS One. 2009; 4e4281Crossref PubMed Scopus (52) Google Scholar). Here, we have shown that all three enzymes have significant lipase activities with similar affinity for the substrate and turnover rate. In addition, all three enzymes are inhibited by micromolar concentrations of THL. This suggests that the active sites of all three enzymes are relatively similar. Although Parker et al. (12Parker S.K. Barkley R.M. Rino J.G. Vasil M.L. PLoS One. 2009; 4e4281Crossref PubMed Scopus (52) Google Scholar) reported a lack of THL inhibition against an M. smegmatis enzyme, the enzyme tested (MSMEG_1403) is not the homolog of Rv3802c but rather a culture supernatant enzyme with phospholipase A activity. They suggested that a lack of MSMEG_1403 inhibition by THL is significant given that M. smegmatis growth is also not inhibited by the drug. However, our finding that MSMEG_6394 is inhibited by THL as well as being an essential enzyme in M. smegmatis would suggest that growth should be inhibited. We propose that the lack of growth inhibition in this organism is due to the inability of THL to reach its target(s) rather than a lack of activity against any particular enzyme. Because THL is active against M. tuberculosis, which shares a very similar cell wall architecture with M. smegmatis (42Brennan P.J. Nikaido H. Annu. Rev. Biochem. 1995; 64: 29-63Crossref PubMed Scopus (1545) Google Scholar), lack of cell entry seems an unlikely possibility. We suggest that efflux of the drug via one of the many transporters in M. smegmatis is the most likely explanation for the lack of activity against this organism.The annotation of Rv3802c as a putative cutinase and the observed enzyme activities of Rv3802c and its orthologs are entirely consistent with the three-dimensional structure that showed MSMEG_6394 to be a member of the cutinase family of α/β one of the closest structural was to K. J. Mol. Microbiol. 2009; PubMed Scopus Google Scholar), a that the mycobacterial cell wall, free mycolic acids from the arabinogalactan has been to facilitate mycobacterial by the outer of the cell wall from the complex K. J. Mol. Microbiol. 2009; PubMed Scopus Google Scholar), from its The structural the of a similar function for Rv3802c, and to a role for this enzyme mycobacterial Because the of cell wall and of the a Rv3802c this the unusual cell wall of of the Actinomycetes would the of a dedicated enzyme to this of This role is consistent with the of Rv3802c in the cell wall, although cutinase-like proteins are the culture of M. tuberculosis (14West N.P. Chow F.M. Randall E.J. Wu J. Chen J. Ribeiro J.M. Britton W.J. FASEB J. 2009; 23: 1694-1704Crossref PubMed Scopus (57) Google Scholar). This function would also the essentiality of Rv3802c and the of our in M. smegmatis at the as is to be essential for in M. tuberculosis because of the of the Rv3802c gene to in (10Sassetti C.M. Boyd D.H. Rubin E.J. Mol. Microbiol. 2003; 48: 77-84Crossref PubMed Scopus (1975) Google Scholar), although direct evidence of essentiality is lacking. inability to the gene in M. smegmatis (MSMEG_6394) suggested essentiality in this species as and we this by and a was found to be on the of MSMEG_6394, and the with the of loss of cell wall and of electron in the of cells. of is that the loss of the MSMEG_6394 gene in cell via the formation of the electron by to the over the suggested role of Rv3802c in mycolic acid biosynthesis (12Parker S.K. Barkley R.M. Rino J.G. Vasil M.L. PLoS One. 2009; 4e4281Crossref PubMed Scopus (52) Google Scholar), we to lipid with a lipid but the The composition of is not but they to be of a that the for et al. (19Kremer L. de Chastellier C. Dobson G. Gibson K.J. Bifani P. Balor S. Gorvel J.P. Locht C. Minnikin D.E. Besra G.S. Mol. Microbiol. 2005; 57: 1113-1126Crossref PubMed Scopus (46) Google Scholar) reported and the of in Mycobacterium although to be lipid and not as well as the electron We also to cell wall following loss of the to and an species, but cell in a of cell wall most with cellular and not related to the loss of recent study has suggested a role for Rv3802c and its orthologs in outer membrane lipid composition stress (15Meniche X. Labarre C. de Sousa-d'Auria C. Huc E. Laval F. Tropis M. Bayan N. Portevin D. Guilhot C. Daffe M. Houssin C. J. Bacteriol. 2009; 191: 7323-7332Crossref PubMed Scopus (26) Google Scholar). The found that the C. glutamicum ortholog be to a with in cell wall and we have as our of C. glutamicum was not This study evidence of induction of heat stress to an increase in mycolic acid biosynthesis and a in (15Meniche X. Labarre C. de Sousa-d'Auria C. Huc E. Laval F. Tropis M. Bayan N. Portevin D. Guilhot C. Daffe M. Houssin C. J. Bacteriol. 2009; 191: 7323-7332Crossref PubMed Scopus (26) Google Scholar). However, this finding does not for the essential of MSMEG_6394 and Rv3802c in M. smegmatis and M. tuberculosis, growth This strongly suggests that a fundamental role for Rv3802c and its orthologs remains We propose a role for the enzyme that is essential for the of cell wall active mycobacterial The essentiality of Rv3802c and the its THL, also M. tuberculosis (19Kremer L. de Chastellier C. Dobson G. Gibson K.J. Bifani P. Balor S. Gorvel J.P. Locht C. Minnikin D.E. Besra G.S. Mol. Microbiol. 2005; 57: 1113-1126Crossref PubMed Scopus (46) Google Scholar) Rv3802c an target to to increase the against drug-resistant M. tuberculosis (1World Health Organization WHO Report 2009: Global Tuberculosis Control, Epidemiology, Strategy, Financing. World Health Organization, Geneva, Switzerland2009: 1-314Google Scholar). IntroductionThe genus Mycobacterium contains a number of medically significant species, most notably the devastating human pathogen Mycobacterium tuberculosis that causes around 2 million deaths each year, the most by any single infectious agent. Despite the availability of a vaccine, the number of infected individuals worldwide continues to increase, as does the prevalence of drug-resistant forms of M. tuberculosis (1World Health Organization WHO Report 2009: Global Tuberculosis Control, Epidemiology, Strategy, Financing. World Health Organization, Geneva, Switzerland2009: 1-314Google Scholar). A key virulence factor is the unique mycobacterial cell wall that consists of a core structure as follows: peptidoglycan covalently linked to arabinogalactan esterified with mycolic acids to form the mycolyl-arabinogalactan-peptidoglycan or “mAGP” complex and a series of free glycolipids, including trehalose monomycolates, trehalose dimycolates, phosphatidylinositol mannosides, and lipoarabinomannans (2Brennan P.J. Tuberculosis. 2003; 83: 91-97Crossref PubMed Scopus (614) Google Scholar), that facilitate vital interactions with host cells to initiate and maintain an infection. The essentiality of the core for mycobacterial growth and survival leads to the biosynthetic enzymes involved and being considered as ideal targets for drug development (3Brennan P.J. Crick D.C. Curr. Top. Med. Chem. 2007; 7: 475-488Crossref PubMed Scopus (109) Google Scholar).A subset of genes required for the late steps of mycolic acid and arabinogalactan biosynthesis are located in proximity to the genomes of mycobacteria and corynebacteria. These genes include a well characterized cell wall biosynthesis cluster encoding enzymes required for the activation (AccD4 and FadD32) and condensation (Pks13) (4Portevin D. De Sousa-D'Auria C. Houssin C. Grimaldi C. Chami M. Daffé M. Guilhot C. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 314-319Crossref PubMed Scopus (276) Google Scholar) of mycolic acid intermediates prior to the final reduction step (5Lea-Smith D.J. Pyke J.S. Tull D. McConville M.J. Coppel R.L. Crellin P.K. J. Biol. Chem. 2007; 282: 11000-11008Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), and transfer of mature mycolic acids (6Jackson M. Raynaud C. Lanéelle M.A. Guilhot C. Laurent-Winter C. Ensergueix D. Gicquel B. Daffé M. Mol. Microbiol. 1999; 31: 1573-1587Crossref PubMed Scopus (223) Google Scholar). Also within the cluster are genes required for arabinogalactan biosynthesis (atfB, glf, glfT, and Rv3806c) (7Seidel M. Alderwick L.J. Birch H.L. Sahm H. Eggeling L. Besra G.S. J. Biol. Chem. 2007; 282: 14729-14740Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 8Mikusová K. Yagi T. Stern R. McNeil M.R. Besra G.S. Crick D.C. Brennan P.J. J. Biol. Chem. 2000; 275: 33890-33897Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 9Kremer L. Dover L.G. Morehouse C. Hitchin P. Everett M. Morris H.R. Dell A. Brennan P.J. McNeil M.R. Flaherty C. Duncan K. Besra G.S. J. Biol. Chem. 2001; 276: 26430-26440Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 10Sassetti C.M. Boyd D.H. Rubin E.J. Mol. Microbiol. 2003; 48: 77-84Crossref PubMed Scopus (1975) Google Scholar) which, like the mycolic acid biosynthesis genes, are essential for growth of M. tuberculosis (10Sassetti C.M. Boyd D.H. Rubin E.J. Mol. Microbiol. 2003; 48: 77-84Crossref PubMed Scopus (1975) Google Scholar).Despite extensive functional characterization of this cluster over the last decade, the in vivo function of the product of one gene, Rv3802c, remains unknown, although mycolyltransferase (11Takayama K. Wang C. Besra G.S. Clin. Microbiol. Rev. 2005; 18: 81-101Crossref PubMed Scopus (493) Google Scholar) or Pks13-associated thioesterase (12Parker S.K. Barkley R.M. Rino J.G. Vasil M.L. PLoS One. 2009; 4e4281Crossref PubMed Scopus (52) Google Scholar) functions have been suggested. Although its genomic location strongly suggests a role in cell wall biosynthesis, definitive proof of such a role is lacking. The putative product of Rv3802c has a predicted signal sequence that contains a possible transmembrane domain, and it has been expressed to assess immunological responses (13West N.P. Wozniak T.M. Valenzuela J. Feng C.G. Sher A. Ribeiro J.M. Britton W.J. Vaccine. 2008; 26: 3853-3859Crossref PubMed Scopus (27) Google Scholar) and enzymatic activities. The enzyme is one of seven cutinase-like proteins in M. tuberculosis and is retained in the cell wall, following translocation across the cell membrane (14West N.P. Chow F.M. Randall E.J. Wu J. Chen J. Ribeiro J.M. Britton W.J. FASEB J. 2009; 23: 1694-1704Crossref PubMed Scopus (57) Google Scholar). Previous studies have shown it to have phospholipase A and thioesterase activities (12Parker S.K. Barkley R.M. Rino J.G. Vasil M.L. PLoS One. 2009; 4e4281Crossref PubMed Scopus (52) Google Scholar), consistent with a role in mycolic acid biosynthesis, and significant lipase activity completely dependent on its Ser-Asp-His catalytic triad (14West N.P. Chow F.M. Randall E.J. Wu J. Chen J. Ribeiro J.M. Britton W.J. FASEB J. 2009; 23: 1694-1704Crossref PubMed Scopus (57) Google Scholar). A very recent study has suggested a role for Rv3802c in regulation of outer lipid composition in response to stress because the induction of the Corynebacterium glutamicum ortholog triggered an increase in mycolic acid biosynthesis as part of an outer membrane remodeling response to heat stress (15Meniche X. Labarre C. de Sousa-d'Auria C. Huc E. Laval F. Tropis M. Bayan N. Portevin D. Guilhot C. Daffe M. Houssin C. J. Bacteriol. 2009; 191: 7323-7332Crossref PubMed Scopus (26) Google Scholar).Recently, Rv3802c was identified as a major target of tetrahydrolipstatin (THL) 6The abbreviations used are: THLtetrahydrolipstatinKnkanamycinSmstreptomycinPDBProtein Data Bankr.m.s.d.root mean square deviationBistris propane1,3-bis[tris(hydroxymethyl)methylamino]propaneTEMtransmission electron microscopy. (12Parker S.K. Barkley R.M. Rino J.G. Vasil M.L. PLoS One. 2009; 4e4281Crossref PubMed Scopus (52) Google Scholar). THL is a well characterized and irreversible inhibitor of serine esterases (16Hadváry P. Lengsfeld H. Wolfer H. Biochem. J. 1988; 256: 357-361Crossref PubMed Scopus (300) Google Scholar), originally identified for its specificity for pancreatic lipases, and thus was developed as an anti-obesity drug. THL has been reported to bind covalently to the catalytic serine residue of pancreatic lipase (17Hadváry P. Sidler W. Meister W. Vetter W. Wolfer H. J. Biol. Chem. 1991; 266: 2021-2027Abstract Full Text PDF PubMed Google Scholar) and was found to have similar affinity for human fatty-acid synthase (18Pemble 4th, C.W. Johnson L.C. Kridel S.J. Lowther W.T. Nat. Struct. Mol. Biol. 2007; 14: 704-709Crossref PubMed Scopus (137) Google Scholar). In addition to its actions in humans, THL inhibits and disrupts cell wall formation in several mycobacterial species, with the exception of the nonpathogenic model species Mycobacterium smegmatis (19Kremer L. de Chastellier C. Dobson G. Gibson K.J. Bifani P. Balor S. Gorvel J.P. Locht C. Minnikin D.E. Besra G.S. Mol. Microbiol. 2005; 57: 1113-1126Crossref PubMed Scopus (46) Google Scholar). Rv3802c was strongly inhibited by THL, whereas the nonorthologous but cutinase motif-bearing M. smegmatis lipase MSMEG_1403 was not inhibited at up to a 500:1 inhibitor to enzyme molar ratio (12Parker S.K. Barkley R.M. Rino J.G. Vasil M.L. PLoS One. 2009; 4e4281Crossref PubMed Scopus (52) Google Scholar).To better understand the enzymology of Rv3802c, we expressed and purified the M. tuberculosis enzyme, its homolog in the nonpathogenic model species M. smegmatis, and the ortholog from a related species, C. glutamicum, and we assessed their inhibition by THL. We report here that the closest M. smegmatis homolog to Rv3802c, MSMEG_6394, is inhibited by THL. The crystal structure of MSMEG_6394, along with direct evidence of the essentiality of MSMEG_6394 in M. smegmatis, indicates a fundamental role of this lipase in

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.