Expression and characterization of the peptidase domain of Streptococcus pneumoniae ComA, a bifunctional ATP-binding cassette transporter involved in quorum sensing pathway.
ComA, a member of the bacteriocin ATP-binding cassette transporters, is postulated to be responsible for both the processing of the propeptide ComC and secretion of the mature competence-stimulating peptide, which regulates the competence and subsequent genetic transformation in Streptococcus pneumoniae. A recombinant N-terminal peptidase domain of ComA, designated PEP, was expressed as a soluble protein in Escherichia coli, purified to homogeneity, and characterized. Gel-filtration analysis revealed that PEP functions as a monomer. The purified PEP exhibited an efficient proteolytic activity for the substrate ComC, which was cleaved after the double glycine site. The stability of PEP was examined by circular dichroism analyses. A convenient method for analyzing the proteolytic activity of PEP was developed, and the kinetic parameters for ComC were determined (k(cat) = 1.5 +/- 0.083 min(-1) and Km = 62 +/- 9.0 microM). Replacements of Cys17 of PEP with Ser or Ala and His96 with Ala resulted in complete loss of activity, indicating that both Cys17 and His96 are essential for the catalysis. Together with information from a protease data base, the N-terminal domain of ComA was concluded to belong to the same clan as the papain-like cysteine proteases. Mutant substrates, in which each of the double glycines was replaced with Ala, were cleaved very poorly by PEP. The mechanism of this strict substrate specificity is discussed on the basis of the sequence alignment with other cysteine proteases.
Ishii
Seiji
S
Department of Biochemistry, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan.
Yano
Takato
T
Hayashi
Hideyuki
H
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D010450:Endopeptidases / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005998:Glycine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D010447:Peptide Hydrolases / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013296:Streptococcus pneumoniae / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics
D001426:Bacterial Proteins; C061262:ComA protein, Bacteria; D004268:DNA-Binding Proteins; D026901:Membrane Transport Proteins; D000255:Adenosine Triphosphate; D010450:Endopeptidases; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D005998:Glycine