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The structure of Rv3717 reveals a novel amidase from Mycobacterium tuberculosis.

Kumar A, Kumar S, Kumar D, Mishra A, Dewangan RP, Shrivastava P, Ramachandran S, Taneja B - Acta Crystallogr. D Biol. Crystallogr. (2013)

Bottom Line: The structure reveals a short flexible hairpin turn that partially occludes the active site and may be involved in autoregulation.Rv3717 utilizes its net positive charge for substrate binding and exhibits activity towards a broad spectrum of substrate cell walls.The enzymatic activity of Rv3717 was confirmed by isolation and identification of its enzymatic products by LC/MS.

View Article: PubMed Central - HTML - PubMed

Affiliation: Structural Biology Unit, CSIR-IGIB, South Campus, Mathura Road, New Delhi 110 025, India.

ABSTRACT
Bacterial N-acetylmuramoyl-L-alanine amidases are cell-wall hydrolases that hydrolyze the bond between N-acetylmuramic acid and L-alanine in cell-wall glycopeptides. Rv3717 of Mycobacterium tuberculosis has been identified as a unique autolysin that lacks a cell-wall-binding domain (CBD) and its structure has been determined to 1.7 Å resolution by the Pt-SAD phasing method. Rv3717 possesses an α/β-fold and is a zinc-dependent hydrolase. The structure reveals a short flexible hairpin turn that partially occludes the active site and may be involved in autoregulation. This type of autoregulation of activity of PG hydrolases has been observed in Bartonella henselae amidase (AmiB) and may be a general mechanism used by some of the redundant amidases to regulate cell-wall hydrolase activity in bacteria. Rv3717 utilizes its net positive charge for substrate binding and exhibits activity towards a broad spectrum of substrate cell walls. The enzymatic activity of Rv3717 was confirmed by isolation and identification of its enzymatic products by LC/MS. These studies indicate that Rv3717, an N-acetylmuramoyl-L-alanine amidase from M. tuberculosis, represents a new family of lytic amidases that do not have a separate CBD and are regulated conformationally.

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Cell-wall hydrolase activity of Rv3717. (a) The cell-wall hydrolase activity of rRv3717-A was monitored by incubating live M. smegmatis cells with no (filled squares), 1 µg ml−1 (crosses), 25 µg ml−1 (filled triangles) or 50 µg ml−1 (filled circles) rRv3717-A or with BSA (filled diamonds). Error bars represent one standard deviation from the mean value of OD600 in triplicate experiments. The inset shows the mathematical fit for rRv3717-A (50 µg ml−1 reaction): log(ΔOD600) = 0.016x − 4.43 [general form log(ΔOD600) = ax − b, where the values of a and b are average values for each point]. (b) Zymograph analysis of rRv3717-A with cell suspensions of different heat-killed bacteria as substrates: (i) Paenibacillus sp., (ii) B. avium, (iii) E. coli DH5α, (iv) E. aerogenes, (v) L. acidophilus, (vi) B. thuringiensis, (vii) B. pumilus, (viii) B. subtilis and (ix) E. coli W311. Zymograph analysis was carried out in a dose-dependent manner; in (i) lane 1 contains 4 µg, lane 2 contains 2 µg and lane 3 contains 1 µg rRv3717-A. L, Lysozyme (4 µg); B, BSA (4 µg); and R, rRv3717-A (4 µg). Lower doses are not indicated in the other panels for clarity. The zone of clearance indicates cell-wall hydrolysis. A molecular-weight marker (lane M) is included in each panel and is labelled in kDa.
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fig4: Cell-wall hydrolase activity of Rv3717. (a) The cell-wall hydrolase activity of rRv3717-A was monitored by incubating live M. smegmatis cells with no (filled squares), 1 µg ml−1 (crosses), 25 µg ml−1 (filled triangles) or 50 µg ml−1 (filled circles) rRv3717-A or with BSA (filled diamonds). Error bars represent one standard deviation from the mean value of OD600 in triplicate experiments. The inset shows the mathematical fit for rRv3717-A (50 µg ml−1 reaction): log(ΔOD600) = 0.016x − 4.43 [general form log(ΔOD600) = ax − b, where the values of a and b are average values for each point]. (b) Zymograph analysis of rRv3717-A with cell suspensions of different heat-killed bacteria as substrates: (i) Paenibacillus sp., (ii) B. avium, (iii) E. coli DH5α, (iv) E. aerogenes, (v) L. acidophilus, (vi) B. thuringiensis, (vii) B. pumilus, (viii) B. subtilis and (ix) E. coli W311. Zymograph analysis was carried out in a dose-dependent manner; in (i) lane 1 contains 4 µg, lane 2 contains 2 µg and lane 3 contains 1 µg rRv3717-A. L, Lysozyme (4 µg); B, BSA (4 µg); and R, rRv3717-A (4 µg). Lower doses are not indicated in the other panels for clarity. The zone of clearance indicates cell-wall hydrolysis. A molecular-weight marker (lane M) is included in each panel and is labelled in kDa.

Mentions: The activity of the single catalytic domain of rRv3717-A was tested by monitoring the decrease in turbidity of a live M. smegmatis suspension as a substrate. Significant lytic activity was observed at 25 and 50 µg rRv3717-A per millilitre of culture (Fig. 4 ▶a). In comparison, buffer control (no protein) or incubation with BSA resulted in no hydrolysis. The activity of lysozyme towards live M. smegmatis culture was also calculated. The activity of rRv3717-A obtained from this assay was 20.3583 × 10−4 AU nmol−1 min−1 and was found to be nearly 2.5-fold higher than that of lysozyme (8.1714 × 10−4 AU nmol−1 min−1).


The structure of Rv3717 reveals a novel amidase from Mycobacterium tuberculosis.

Kumar A, Kumar S, Kumar D, Mishra A, Dewangan RP, Shrivastava P, Ramachandran S, Taneja B - Acta Crystallogr. D Biol. Crystallogr. (2013)

Cell-wall hydrolase activity of Rv3717. (a) The cell-wall hydrolase activity of rRv3717-A was monitored by incubating live M. smegmatis cells with no (filled squares), 1 µg ml−1 (crosses), 25 µg ml−1 (filled triangles) or 50 µg ml−1 (filled circles) rRv3717-A or with BSA (filled diamonds). Error bars represent one standard deviation from the mean value of OD600 in triplicate experiments. The inset shows the mathematical fit for rRv3717-A (50 µg ml−1 reaction): log(ΔOD600) = 0.016x − 4.43 [general form log(ΔOD600) = ax − b, where the values of a and b are average values for each point]. (b) Zymograph analysis of rRv3717-A with cell suspensions of different heat-killed bacteria as substrates: (i) Paenibacillus sp., (ii) B. avium, (iii) E. coli DH5α, (iv) E. aerogenes, (v) L. acidophilus, (vi) B. thuringiensis, (vii) B. pumilus, (viii) B. subtilis and (ix) E. coli W311. Zymograph analysis was carried out in a dose-dependent manner; in (i) lane 1 contains 4 µg, lane 2 contains 2 µg and lane 3 contains 1 µg rRv3717-A. L, Lysozyme (4 µg); B, BSA (4 µg); and R, rRv3717-A (4 µg). Lower doses are not indicated in the other panels for clarity. The zone of clearance indicates cell-wall hydrolysis. A molecular-weight marker (lane M) is included in each panel and is labelled in kDa.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852659&req=5

fig4: Cell-wall hydrolase activity of Rv3717. (a) The cell-wall hydrolase activity of rRv3717-A was monitored by incubating live M. smegmatis cells with no (filled squares), 1 µg ml−1 (crosses), 25 µg ml−1 (filled triangles) or 50 µg ml−1 (filled circles) rRv3717-A or with BSA (filled diamonds). Error bars represent one standard deviation from the mean value of OD600 in triplicate experiments. The inset shows the mathematical fit for rRv3717-A (50 µg ml−1 reaction): log(ΔOD600) = 0.016x − 4.43 [general form log(ΔOD600) = ax − b, where the values of a and b are average values for each point]. (b) Zymograph analysis of rRv3717-A with cell suspensions of different heat-killed bacteria as substrates: (i) Paenibacillus sp., (ii) B. avium, (iii) E. coli DH5α, (iv) E. aerogenes, (v) L. acidophilus, (vi) B. thuringiensis, (vii) B. pumilus, (viii) B. subtilis and (ix) E. coli W311. Zymograph analysis was carried out in a dose-dependent manner; in (i) lane 1 contains 4 µg, lane 2 contains 2 µg and lane 3 contains 1 µg rRv3717-A. L, Lysozyme (4 µg); B, BSA (4 µg); and R, rRv3717-A (4 µg). Lower doses are not indicated in the other panels for clarity. The zone of clearance indicates cell-wall hydrolysis. A molecular-weight marker (lane M) is included in each panel and is labelled in kDa.
Mentions: The activity of the single catalytic domain of rRv3717-A was tested by monitoring the decrease in turbidity of a live M. smegmatis suspension as a substrate. Significant lytic activity was observed at 25 and 50 µg rRv3717-A per millilitre of culture (Fig. 4 ▶a). In comparison, buffer control (no protein) or incubation with BSA resulted in no hydrolysis. The activity of lysozyme towards live M. smegmatis culture was also calculated. The activity of rRv3717-A obtained from this assay was 20.3583 × 10−4 AU nmol−1 min−1 and was found to be nearly 2.5-fold higher than that of lysozyme (8.1714 × 10−4 AU nmol−1 min−1).

Bottom Line: The structure reveals a short flexible hairpin turn that partially occludes the active site and may be involved in autoregulation.Rv3717 utilizes its net positive charge for substrate binding and exhibits activity towards a broad spectrum of substrate cell walls.The enzymatic activity of Rv3717 was confirmed by isolation and identification of its enzymatic products by LC/MS.

View Article: PubMed Central - HTML - PubMed

Affiliation: Structural Biology Unit, CSIR-IGIB, South Campus, Mathura Road, New Delhi 110 025, India.

ABSTRACT
Bacterial N-acetylmuramoyl-L-alanine amidases are cell-wall hydrolases that hydrolyze the bond between N-acetylmuramic acid and L-alanine in cell-wall glycopeptides. Rv3717 of Mycobacterium tuberculosis has been identified as a unique autolysin that lacks a cell-wall-binding domain (CBD) and its structure has been determined to 1.7 Å resolution by the Pt-SAD phasing method. Rv3717 possesses an α/β-fold and is a zinc-dependent hydrolase. The structure reveals a short flexible hairpin turn that partially occludes the active site and may be involved in autoregulation. This type of autoregulation of activity of PG hydrolases has been observed in Bartonella henselae amidase (AmiB) and may be a general mechanism used by some of the redundant amidases to regulate cell-wall hydrolase activity in bacteria. Rv3717 utilizes its net positive charge for substrate binding and exhibits activity towards a broad spectrum of substrate cell walls. The enzymatic activity of Rv3717 was confirmed by isolation and identification of its enzymatic products by LC/MS. These studies indicate that Rv3717, an N-acetylmuramoyl-L-alanine amidase from M. tuberculosis, represents a new family of lytic amidases that do not have a separate CBD and are regulated conformationally.

Show MeSH
Related in: MedlinePlus