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Literature summary extracted from

  • Chandran, A.; Srikalaivani, R.; Paul, A.; Vijayan, M.
    Biochemical characterization of Mycobacterium tuberculosis LexA and structural studies of its C-terminal segment (2019), Acta Crystallogr. Sect. D, 75, 41-55 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.4.21.88 gene lexA, recombinant expression of His-tagged wild-type LexA and mutant enzymes S160A, K197A, and G126D, the isolated C-terminal segment and the N-domain in Escherichia coli strain BL21(DE3) pLysS Mycobacterium tuberculosis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.4.21.88 purified recombinant His-tagged wild-type LexA and mutant enzymes S160A, K197A, and G126D, the isolated C-terminal segment and the N-domain, wild-type LexA diffraction-quality crystals grow by vapour diffusion method in about three months from a drop consisting of 0.002 ml of 6 mg/ml protein solution and 0.002 ml of 100 mM bis-Tris, pH 6.5 or pH 8.5, 100 mM NaCl or 200 mM MgCl2, 5% glycerol, 25% PEG 3350 (form I and form II, respectively). The microbatch-under-oil method is used for crystallization of the C-domain by mixing of 0.002 ml of 10 mg/ml protein solution with 0.002 ml 100 mM Tris-HCl, pH 7.5, 25% glycerol, and 40% v/v pentaerythritol ethoxylate (15/4 EO/OH). Diffraction-quality crystals of the S160A mutant are obtained by microbatch-under-oil method and mixing of 0.002 ml 10 mg/ml protein and 0.002 ml 100 mM bis-Tris, pH 6.5, 200 mM MgCl2, and 25% w/v PEG 3350 (form III), and of mutant K197A by mixing of 0.002 ml 10 mg/ml protein solution with 0.002 ml crystallization solution containing 100 mM HEPES, pH 7.5, 20 mM MgCl2, and 22% w/v polyacrylic acid sodium salt (form IV), while crystals of the G126D mutant are grown from a solution consisting of 0.002 ml of 10 mg/ml protein with 0.002 ml of 100 mM Tris-HCl, pH 8.5, 100 mM sodium acetate trihydrate, and 30% w/v PEG 4000 (form V), mutant crystals grow about 2 months, X-ray diffraction structure determination and analysis at 1.48-2.25 A resolution Mycobacterium tuberculosis

Protein Variants

EC Number Protein Variants Comment Organism
3.4.21.88 G126D site-directed mutagenesis, cleavage site mutant Mycobacterium tuberculosis
3.4.21.88 K197A site-directed mutagenesis, active site mutant Mycobacterium tuberculosis
3.4.21.88 S160A site-directed mutagenesis, active site mutant Mycobacterium tuberculosis

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.4.21.88 NaCl variation of the sodium chloride concentration from 25 mM to 1.5 M do not show any significant change in the autoproteolysis of MtLexA Mycobacterium tuberculosis

Organism

EC Number Organism UniProt Comment Textmining
3.4.21.88 Mycobacterium tuberculosis P9WHR7
-
-
3.4.21.88 Mycobacterium tuberculosis ATCC 25618 P9WHR7
-
-
3.4.21.88 Mycobacterium tuberculosis H37Rv P9WHR7
-
-

Posttranslational Modification

EC Number Posttranslational Modification Comment Organism
3.4.21.88 proteolytic modification the autocatalytic cleavage of MtLexA and the mutant proteins is analyzed, effects of pH and temperature, the enzyme is autocatalytically active at pH 9.5-11.0 and at up to 55°C Mycobacterium tuberculosis

Purification (Commentary)

EC Number Purification (Comment) Organism
3.4.21.88 recombinant His-tagged wild-type and mutant enzymes, as well as enzyme fragments from Escherichia coli strain BL21(DE3) pLysS by nickel affinity chromatography and gel filtration, to over 95% purity Mycobacterium tuberculosis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.4.21.88 additional information qualitative analysis of DNA-binding activities of MtLexA and its mutants, MtLexA binds to dsDNA with the consensus sequence GAAC-N4-GTTT/C. Modeling of the MtLexA-DNA complex, overview. The length of the flanking sequences of bound DNA does not affect DNA binding by LexA. Analysis of autocatalytic cleavage of MtLexA and enzyme mutants Mycobacterium tuberculosis ?
-
 ?
3.4.21.88 additional information qualitative analysis of DNA-binding activities of MtLexA and its mutants, MtLexA binds to dsDNA with the consensus sequence GAAC-N4-GTTT/C. Modeling of the MtLexA-DNA complex, overview. The length of the flanking sequences of bound DNA does not affect DNA binding by LexA. Analysis of autocatalytic cleavage of MtLexA and enzyme mutants Mycobacterium tuberculosis H37Rv ?
-
 ?
3.4.21.88 additional information qualitative analysis of DNA-binding activities of MtLexA and its mutants, MtLexA binds to dsDNA with the consensus sequence GAAC-N4-GTTT/C. Modeling of the MtLexA-DNA complex, overview. The length of the flanking sequences of bound DNA does not affect DNA binding by LexA. Analysis of autocatalytic cleavage of MtLexA and enzyme mutants Mycobacterium tuberculosis ATCC 25618 ?
-
 ?

Subunits

EC Number Subunits Comment Organism
3.4.21.88 homodimer 2 * 12000, C-terminal catalytic core, SDS-PAGE Mycobacterium tuberculosis

Synonyms

EC Number Synonyms Comment Organism
3.4.21.88 MtLexA
-
 Mycobacterium tuberculosis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.4.21.88 37
-
 assay at Mycobacterium tuberculosis

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
3.4.21.88 37 55 self-cleavage activity at Mycobacterium tuberculosis

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
3.4.21.88 55
-
 above, melting temperature of wild-type and mutant enzymes Mycobacterium tuberculosis

pH Range

EC Number pH Minimum pH Maximum Comment Organism
3.4.21.88 9.5 11 self-cleavage activity at Mycobacterium tuberculosis

General Information

EC Number General Information Comment Organism
3.4.21.88 additional information a complex of Mycobacterium tuberculosis LexA and the cognate SOS box is modeled in which the mutual orientation of the two N-terminal domains differs from that in the Escherichia coli LexA-DNA complex, complex structure analysis, overview Mycobacterium tuberculosis
3.4.21.88 physiological function LexA is a critical protein involved in the bacterial SOS response, which consists of the coordinated activation of a network of genes required for DNA repair and mutagenesis in response to DNA damage. The clinical relevance of the SOS response in bacteria can be attributed not only to its involvement in virulence and mutagenesis, but also in the spread of antibiotic resistance Mycobacterium tuberculosis