The lipolytic activity of LipJ, a stress-induced enzyme, is regulated by its C-terminal adenylate cyclase domain

2021 ◽  
Author(s):  
Bandana Kumari ◽  
Jashandeep Kaur ◽  
Pratibha Maan ◽  
Arbind Kumar ◽  
Jagdeep Kaur
Keyword(s):  
Escherichia Coli ◽  
Adenylate Cyclase ◽  
Mycobacterium Smegmatis ◽  
Specific Activity ◽  
Lipolytic Activity ◽  
Cell Surface Properties ◽  
Cyclase Domain ◽  
N Terminus ◽  
Enzyme Specific Activity

Aim: The confirmation of lipolytic activity and role of Rv1900c in the mycobacterium physiology Methods: rv1900c/N-terminus domain ( rv1900NT) were cloned in pET28a/ Escherichia coli, purified by affinity chromatography and characterized. Results: A zone of clearance on tributyrin-agar and activity with pNP-decanoate confirmed the lipolytic activity of Rv1900c. The Rv1900NT demonstrated higher enzyme specific activity, Vmax and kcat, but Rv1900c was more thermostable. The lipolytic activity of Rv1900c decreased in presence of ATP. Mycobacterium smegmatis expressing rv1900c/ rv1900NT altered colony morphology, growth, cell surface properties and survival under stress conditions. The effect was more prominent with Rv1900NT as compared with Rv1900c. Conclusion: The study confirmed the lipolytic activity of Rv1900c and suggested its regulation by the adenylate cyclase domain and role in intracellular survival of bacteria.

scholarly journals A Monoacylglycerol Lipase from Mycobacterium smegmatis Involved in Bacterial Cell Interaction

2010 ◽  
Vol 192 (18) ◽  
pp. 4776-4785 ◽  
Author(s):  
Rabeb Dhouib ◽  
Françoise Laval ◽  
Frédéric Carrière ◽  
Mamadou Daffé ◽  
Stéphane Canaan
Keyword(s):  
Mycobacterium Smegmatis ◽  
Wild Type Strain ◽  
Specific Activity ◽  
Cell Interaction ◽  
Monoacylglycerol Lipase ◽  
Homologous Proteins ◽  
Dependent Activity ◽  
Hydrolysis Of

ABSTRACT MSMEG_0220 from Mycobacterium smegmatis, the ortholog of the Rv0183 gene from M. tuberculosis, recently identified and characterized as encoding a monoacylglycerol lipase, was cloned and expressed in Escherichia coli. The recombinant protein (rMSMEG_0220), which exhibits 68% amino acid sequence identity with Rv0183, showed the same substrate specificity and similar patterns of pH-dependent activity and stability as the M. tuberculosis enzyme. rMSMEG_0220 was found to hydrolyze long-chain monoacylglycerol with a specific activity of 143 ± 6 U mg−1. Like Rv0183 in M. tuberculosis, MSMEG_0220 was found to be located in the cell wall. To assess the in vivo role of the homologous proteins, an MSMEG_0220 disrupted mutant of M. smegmatis (MsΔ0220) was produced. An intriguing change in the colony morphology and in the cell interaction, which were partly restored in the complemented mutant containing either an active (ComMsΔ0220) or an inactive (ComMsΔ0220S111A) enzyme, was observed. Growth studies performed in media supplemented with monoolein showed that the ability of both MsΔ0220 and ComMsΔ0220S111A to grow in the presence of this lipid was impaired. Moreover, studies of the antimicrobial susceptibility of the MsΔ0220 strain showed that this mutant is more sensitive to rifampin and more resistant to isoniazid than the wild-type strain, pointing to a critical structural role of this enzyme in mycobacterial physiology, in addition to its function in the hydrolysis of exogenous lipids.

Protective role of Mycobacterium leprae small heat-shock protein in heterologous hosts, Escherichia coli and Mycobacterium smegmatis, grown under stress

2013 ◽  
Vol 62 (7) ◽  
pp. 959-967 ◽  
Author(s):  
Jayapal Jeya Maheshwari ◽  
Kuppamuthu Dharmalingam
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Mycobacterium Smegmatis ◽  
Small Heat Shock Protein ◽  
Mycobacterium Leprae ◽  
Protective Role ◽  
Low Oxygen

The aim of this study is to examine the in vivo role of a small heat-shock protein (sHsp18) from Mycobacterium leprae in the survival of heterologous recombinant hosts carrying the gene encoding this protein under different environmental conditions that are normally encountered by M. leprae during its infection of the human host. Using an Escherichia coli system where shsp18 expression is controlled by its native promoter, we show that expression of shsp18 is induced under low oxygen tension, nutrient depletion and oxidative stress, all of which reflect the natural internal environment of the granulomas where the pathogen resides for long periods. We demonstrate the in vivo chaperone activity of sHsp18 through its ability to confer survival advantage to recombinant E. coli at heat-shock temperatures. Additional evidence for the protective role of sHsp18 was obtained when Mycobacterium smegmatis harbouring a copy of shsp18 was found to multiply better in human macrophages. Furthermore, the autokinase activity of sHsp18 protein demonstrated for what is believed to be the first time in this study implies that some of the functions of sHsp18 might be controlled by the phosphorylation state of this protein. Results from this study suggest that shsp18 might be one of the factors that facilitate the survival and persistence of M. leprae under stress and autophosphorylation of sHsp18 protein could be a mechanism used by this protein to sense changes in the external environment.

scholarly journals The role of cysteine residues 129 and 329 in Escherichia coli K1 CMP-NeuAc synthase

1993 ◽  
Vol 295 (2) ◽  
pp. 485-491 ◽  
Author(s):  
G Zapata ◽  
P P Roller ◽  
J Crowley ◽  
W F Vann
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Acetylneuraminic Acid ◽  
Denatured States ◽  
Escherichia Coli K1

N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Escherichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catalytic activity and structure of the protein has been investigated by site-directed mutagenesis and chemical modification. The enzyme is inactivated by the thiol-specific reagent dithiodipyridine. Inactivation by this reagent is decreased in the presence of the nucleotide substrate CTP, suggesting that a thiol residue is at or near the active site. Site-directed mutagenesis of either residue Cys-129 to serine or Cys-329 to selected amino acids has minor effects on the specific activity of the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. The limited reactivity of the enzyme to other thiol-blocking reagents suggests that its cysteine residues are partially exposed. The accessibility and role of the cysteine residues in enzyme structure were investigated by fluorescence, c.d. and denaturation studies of wild-type and mutant enzymes. The mutation of Cys-129 to serine makes the enzyme more sensitive to heat and chemical denaturation, but does not cause gross changes in the protein structure as judged by the c.d. spectrum. The mutant containing Ser-129 instead of Cys-129 had a complex denaturation pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthase consisting of several partially denatured states. Cys-329 reacts more readily with N-[14C]ethylmaleimide when the enzyme is in a heat-induced relaxed state. Cys-129 is less reactive and is probably a buried residue.

Expression in Escherichia coli of fragments of the coiled-coil rod domain of rabbit myosin: influence of different regions of the molecule on aggregation and paracrystal formation

1991 ◽  
Vol 99 (4) ◽  
pp. 823-836
Author(s):  
S.J. Atkinson ◽  
M. Stewart
Keyword(s):  
Escherichia Coli ◽  
Ionic Strength ◽  
Coiled Coil ◽  
Periodic Variation ◽  
Heptad Repeat ◽  
C Terminus ◽  
N Terminus ◽  
Low Ionic Strength ◽  
Myosin Rod

We have expressed in Escherichia coli a cDNA clone corresponding broadly to rabbit light meromyosin (LMM) together with a number of modified polypeptides and have used this material to investigate the role of different aspects of molecular structure on the solubility properties of LMM. The expressed material was characterized biochemically and structurally to ensure that it retained the coiled-coil conformation of the native molecule. Full-length recombinant LMM retained the general solubility properties of myosin and, although soluble at high ionic strength, precipitated when the ionic strength was reduced below 0.3 M. Constructs in which the ‘skip’ residues (that disrupt the coiled-coil heptad repeat) were deleted had solubility properties indistinguishable from the wild type, which indicated that the skip residues did not play a major role in determining the molecular interactions involved in assembly. Deletions from the N terminus of LMM did not alter the solubility properties of the expressed material, but deletion of 92 residues from the C terminus caused a large increase in solubility at low ionic strength, indicating that a determinant important for interaction between LMM molecules was located in this region. The failure of deletions from the molecule's N terminus to alter its solubility radically suggested that the periodic variation of charge along the myosin rod may not be as important as proposed for determining the strength of binding between molecules and thus the solubility of myosin.

Purified postheparin plasma lipoprotein lipase in primary hyperlipoproteinemias

1975 ◽  
Vol 39 (6) ◽  
pp. 1022-1033 ◽  
Author(s):  
D. Ganesan ◽  
R. H. Bradford ◽  
G. Ganesan ◽  
W. J. McConathy ◽  
P. Alaupovic ◽  
...  
Keyword(s):  
Lipoprotein Lipase ◽  
Specific Activity ◽  
Lipolytic Activity ◽  
Plasma Lipoprotein ◽  
Type I ◽  
Activity Type ◽  
Type Iii ◽  
Protein Peak ◽  
Enzyme Specific Activity ◽  
Hydrolysis Of

Purified postheparin plasma lipoprotein lipase (LPL) of normolipidemic and primary hyperlipoproteinemic subjects was characterized by lipoprotein C polypeptide activation and specificity for triglycerides in chylomicrons and VLDL. Chromatography of normal LPL on Sephadex G-100 resulted in two protein peaks, LPLC-1 (activated by C-I but not C-II) and LPLC-II (activated by C-II but not C-I). LPL from type I hyperlipoproteinemic subjects was not activated by C-I and C-II activation was reduced to 40% of control. Hydrolysis of chylomicron and VLDL triglycerides was severely impaired. Although chromatography of type I LPL resulted in two protein peaks, the protein peak corresponding to LPLC-I did not exhibit lipolytic activity and LPLC-II was reduced to 50% of control in protein and enzyme specific activity. Type III LPL was normal in respect to LPLC-I while LPLC-II averaged 40% of control. Hydrolysis of chylomicron and VLDL was reduced to 50% and 10% of control, respectively. An etiological implication for LPLC-I and/or LPLC-II in type I and III hyperlipoproteinemias is suggested.

scholarly journals Identification and Analysis of “Extended −10” Promoters from Mycobacteria

1998 ◽  
Vol 180 (9) ◽  
pp. 2568-2573 ◽  
Author(s):  
Murali D. Bashyam ◽  
Anil K. Tyagi
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Sigma Factor ◽  
Mycobacterium Smegmatis ◽  
Important Determinant ◽  
Comparative Assessment ◽  
Binding Domain ◽  
Site Specific ◽  
E Coli

ABSTRACT Earlier studies from our laboratory on randomly isolated transcriptional signals of mycobacteria had revealed that the −10 region of mycobacterial promoters and the corresponding binding domain in the major sigma factor are highly similar to their Escherichia coli counterparts. In contrast, the sequences in −35 regions of mycobacterial promoters and the corresponding binding domain in the major sigma factor are vastly different from their E. colicounterparts (M. D. Bashyam, D. Kaushal, S. K. Dasgupta, and A. K. Tyagi, J. Bacteriol. 178:4847–4853, 1996). We have now analyzed the role of the TGN motif present immediately upstream of the −10 region of mycobacterial promoters. Sequence analysis and site-specific mutagenesis of a Mycobacterium tuberculosispromoter and a Mycobacterium smegmatis promoter reveal that the TGN motif is an important determinant of transcriptional strength in mycobacteria. We show that mutation in the TGN motif can drastically reduce the transcriptional strength of a mycobacterial promoter. The influence of the TGN motif on transcriptional strength is also modulated by the sequences in the −35 region. Comparative assessment of these extended −10 promoters in mycobacteria and E. coli suggests that functioning of the TGN motif in promoters of these two species is similar.

scholarly journals Role of the rfaG and rfaP genes in determining the lipopolysaccharide core structure and cell surface properties of Escherichia coli K-12.

1992 ◽  
Vol 174 (8) ◽  
pp. 2525-2538 ◽  
Author(s):  
C T Parker ◽  
A W Kloser ◽  
C A Schnaitman ◽  
M A Stein ◽  
S Gottesman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Surface Properties ◽  
Core Structure ◽  
Cell Surface Properties ◽  
K 12

scholarly journals Effect of directional pulling on mechanical protein degradation by ATP-dependent proteolytic machines

2017 ◽  
Vol 114 (31) ◽  
pp. E6306-E6313 ◽  
Author(s):  
Adrian O. Olivares ◽  
Hema Chandra Kotamarthi ◽  
Benjamin J. Stein ◽  
Robert T. Sauer ◽  
Tania A. Baker
Keyword(s):  
Escherichia Coli ◽  
Protein Degradation ◽  
Single Molecule ◽  
Wild Type ◽  
N Terminus ◽  
Multiple Copies ◽  
Rate Limiting ◽  
Hydrolysis Of ◽  
Translocation Speed

AAA+ proteases and remodeling machines couple hydrolysis of ATP to mechanical unfolding and translocation of proteins following recognition of sequence tags called degrons. Here, we use single-molecule optical trapping to determine the mechanochemistry of two AAA+ proteases, Escherichia coli ClpXP and ClpAP, as they unfold and translocate substrates containing multiple copies of the titinI27 domain during degradation initiated from the N terminus. Previous studies characterized degradation of related substrates with C-terminal degrons. We find that ClpXP and ClpAP unfold the wild-type titinI27 domain and a destabilized variant far more rapidly when pulling from the N terminus, whereas translocation speed is reduced only modestly in the N-to-C direction. These measurements establish the role of directionality in mechanical protein degradation, show that degron placement can change whether unfolding or translocation is rate limiting, and establish that one or a few power strokes are sufficient to unfold some protein domains.

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