Purification and properties of a triacylglycerol lipase from Mycobacterium phlei

Paznokas, John L.; Kaplan, Arnold

doi:10.1016/0005-2760(77)90212-0

Cited by 8 publications

(8 citation statements)

References 14 publications

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“…Molecular weights of the mycobacterial Tween-hydrolyzing esterases ranged from 36,000 to 58,000 daltons. These values are comparable to those of common bacterial carboxylic acyl hydrolases: 34,500 for undecyl acetate esterase from Pseudomonas cepacia (II), 40,000 for acyl-CoA thioesterase from M. smegmatis (15), 40,000 for the active subunit of triacylglycerollipase from M. phlei (7), and 55,000 for acyl-CoA thioesterase from Pseudomonas aeruginosa (6).…”

Section: Discussionsupporting

confidence: 67%

Physicochemical Characterization of Tween 80‐Hydrolyzing Esterases Produced by Rapidly Growing Mycobacteria

Tomioka

Yoneyama

Asano

et al. 1983

Microbiology and Immunology

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Tween 80-hydrolyzing esterases produced by various species of rapidly growing mycobacteria were partially purified from sonicated cell lysates by diethylaminoethyl (DEAE) cellulose and subsequent Sephadex G-150 column chromatographies. The amount of the esterase produced per gram of bacterial cells varied markedly with each species. Mycobacterium smegmatis, M. chelonei, and M. phlei were high producers and M. chitae and M. diernhoferi were low producers of Tween-hydrolyzing esterase. The resistance of each mycobacterial strain to oleic acid correlated well with their esterase-producing ability. All the esterases studied were adsorbed on DEAE cellulose in 50 mM Tris-HCl buffer (pH 7.5), indicating that they are acidic proteins. Esterases of M. smegmatis, M. chitae, M. fortuitum, and M. phlei were eluted from DEAE at high concentrations (0.11-0.18 M) of ammonium sulfate, while those of M. parafortuitum and M. diernhoferi were eluted at lower concentrations (0.05-0.08 M). With Sephadex G-150 gel filtration, all esterases were shown to have similar molecular weights (36,000 to 58,000). On the basis of heat-stability and trypsin- or chymotrypsin-sensitivity, these esterases were divided into three groups: one was heat-stable and protease-sensitive (M. smegmatis and M. fortuitum), another was heat-labile and protease-resistant (M. chelonei), and the other was the intermediate of the above two groups (M. diernhoferi).

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Section: Discussionsupporting

confidence: 67%

Physicochemical Characterization of Tween 80‐Hydrolyzing Esterases Produced by Rapidly Growing Mycobacteria

Tomioka

Yoneyama

Asano

et al. 1983

Microbiology and Immunology

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“…Release of energy stored in TG requires the activity of long-chain TG hydrolase(s). Although the purification of an intracellular, cell wallassociated long-chain TG lipase from Mycobacterium phlei was reported several decades ago (32), there has been no report in the literature on the identification of a long-chain TG hydrolase from M. tuberculosis. We report here on the identification and characterization of the lipY (Rv3097c) product, which is such an enzyme demonstrating a high specific activity for trioleoylglycerol hydrolysis and the identification of lipL, lipG, lipC, lipK, and Rv1169c products, which possess low levels of long-chain TG hydrolase activity.…”

Section: Discussionmentioning

confidence: 99%

A Novel Lipase Belonging to the Hormone-sensitive Lipase Family Induced under Starvation to Utilize Stored Triacylglycerol in Mycobacterium tuberculosis

Deb¹,

Daniel²,

Sirakova

et al. 2006

Journal of Biological Chemistry

251

277

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Twenty-four putative lipase/esterase genes of Mycobacterium tuberculosis H37Rv were expressed in Escherichia coli and assayed for long-chain triacylglycerol (TG) hydrolase activity. We show here that the product of Rv3097c (LIPY) hydrolyzed long-chain TG with high specific activity. LIPY was purified after solubilization from inclusion bodies; the enzyme displayed a K m of 7.57 mM and V max of 653.3 nmol/mg/min for triolein with optimal activity between pH 8.0 and pH 9.0. LIPY was inhibited by active serine-directed reagents and was inactivated at temperatures above 37°C. Detergents above their critical micellar concentrations and divalent cations inhibited the activity of LIPY. The N-terminal half of LIPY showed sequence homology with the proline glutamic acid-polymorphic GC-rich repetitive sequences protein family of M. tuberculosis. The C-terminal half of LIPY possesses amino acid domains homologous with the hormone-sensitive lipase family and the conserved active-site motif GDSAG. LIPY shows low sequence identity with the annotated lipases of M. tuberculosis and with other bacterial lipases. We demonstrate that hypoxic cultures of M. tuberculosis, which had accumulated TG, hydrolyzed the stored TG when subjected to nutrient starvation. Under such conditions, lipY was induced more than all lipases, suggesting a central role for it in the utilization of stored TG. We also show that in the lipY-deficient mutant, TG utilization was drastically decreased under nutrientdeprived condition. Thus, LIPY may be responsible for the utilization of stored TG during dormancy and reactivation of the pathogen.Tuberculosis caused by the organism Mycobacterium tuberculosis is one of the major public health threats in the world. The emergence of multidrug-resistant strains poses serious threats to the control of this disease due to the complex nature of second line drug treatment (1). Upon infection, the bacterium goes through an initial replicative phase inside the alveolar macrophages, after which it enters a nonreplicative, drug-resistant state of dormancy. This state of dormancy is probably induced by the environmental stress exerted upon the pathogen by the host's immune response. The bacterium is able to survive in this dormant state for decades until the host's immune system is weakened when it reactivates and causes the infectious disease (2). The current antimycobacterial drugs are able to kill only the actively replicating mycobacteria and do not clear the latent bacteria from the host (3). Thus, latency is a major problem in tuberculosis control. One-third of the world population is infected with the latent microorganism, and nearly 2 million deaths occur annually (4, 5). Individuals carrying a latent infection are estimated to harbor a 2-23% lifetime risk of reactivation (6).There is strong evidence that fatty acids may be the energy source for the mycobacterium during persistence (7-10). However, the source of these fatty acids is not known. We proposed that M. tuberculosis stores energy in the form of triacylglyce...

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“…Two intracellular lipolytic enzymes which may participate in the degradation of intracellular TAG have been identified and purified so far in mycobacteria. The first of these is a native enzyme which was purified from Mycobacterium phlei [19] and characterized. The second lipolytic enzyme was the expression product of the Rv3097c gene from the strain M. tuberculosis H37Rv in Escherichia coli [20].…”

Section: Introductionmentioning

confidence: 99%

Characterization of an exported monoglyceride lipase from Mycobacterium tuberculosis possibly involved in the metabolism of host cell membrane lipids

Côtes

Dhouib

Douchet

et al. 2007

Biochemical Journal

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The Rv0183 gene of the Mycobacterium tuberculosis H37Rv strain, which has been implicated as a lysophospholipase, was cloned and expressed in Escherichia coli. The purified Rv0183 protein did not show any activity when lysophospholipid substrates were used, but preferentially hydrolysed monoacylglycerol substrates with a specific activity of 290 units x mg(-1) at 37 degrees C. Rv0183 hydrolyses both long chain di- and triacylglycerols, as determined using the monomolecular film technique, although the turnover was lower than with MAG (monoacyl-glycerol). The enzyme shows an optimum activity at pH values ranging from 7.5 to 9.0 using mono-olein as substrate and is inactivated by serine esterase inhibitors such as E600, PMSF and tetrahydrolipstatin. The catalytic triad is composed of Ser110, Asp226 and His256 residues, as confirmed by the results of site-directed mutagenesis. Rv0183 shows 35% sequence identity with the human and mouse monoglyceride lipases and well below 15% with the other bacterial lipases characterized so far. Homologues of Rv0183 can be identified in other mycobacterial genomes such as Mycobacterium bovis, Mycobacterium smegmatis, and even Mycobacterium leprae, which is known to contain a low number of genes involved in the replication process within the host cells. The results of immunolocalization studies performed with polyclonal antibodies raised against the purified recombinant Rv0183 suggested that the enzyme was present only in the cell wall and culture medium of M. tuberculosis. Our results identify Rv0183 as the first exported lipolytic enzyme to be characterized in M. tuberculosis and suggest that Rv0183 may be involved in the degradation of the host cell lipids.

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Purification and properties of a triacylglycerol lipase from Mycobacterium phlei

Cited by 8 publications

References 14 publications

Physicochemical Characterization of Tween 80‐Hydrolyzing Esterases Produced by Rapidly Growing Mycobacteria

Physicochemical Characterization of Tween 80‐Hydrolyzing Esterases Produced by Rapidly Growing Mycobacteria

A Novel Lipase Belonging to the Hormone-sensitive Lipase Family Induced under Starvation to Utilize Stored Triacylglycerol in Mycobacterium tuberculosis

Characterization of an exported monoglyceride lipase from Mycobacterium tuberculosis possibly involved in the metabolism of host cell membrane lipids

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