title: Substrate specificity of the Escherichia coli outer membrane protease OmpP.
abstract: Escherichia coli OmpP is an F episome-encoded outer membrane protease that exhibits 71% amino acid sequence identity with OmpT. These two enzymes cleave substrate polypeptides primarily between pairs of basic amino acids. We found that, like OmpT, purified OmpP is active only in the presence of lipopolysaccharide. With optimal peptide substrates, OmpP exhibits high catalytic efficiency (k(cat)/K(m) = 3.0 x 10(6) M(-1)s(-1)). Analysis of the extended amino acid specificity of OmpP by substrate phage revealed that both Arg and Lys are strongly preferred at the P1 and P1' sites of the enzyme. In addition, Thr, Arg, or Ala is preferred at P2; Leu, Ala, or Glu is preferred at P4; and Arg is preferred at P3'. Notable differences in OmpP and OmpT specificities include the greater ability of OmpP to accept Lys at the P1 or P1', site as well as the prominence of Ser at P3 in OmpP substrates. Likewise, the OmpP P1 site could better accommodate Ser; as a result, OmpP was able to cleave a peptide substrate between Ser-Arg about 120 times more efficiently than was OmpT. Interestingly, OmpP and OmpT cleave peptides with three consecutive Arg residues at different sites, a difference in specificity that might be important in the inactivation of cationic antimicrobial peptides. Accordingly, we show that the presence of an F' episome results in increased resistance to the antimicrobial peptide protamine both in ompT mutants and in wild-type E. coli cells.
authors.affiliation: Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712, USA.
authors.forename: Bum-Yeol
authors.initials: BY
authors.lastname: Hwang
authors.forename: Navin
authors.initials: N
authors.lastname: Varadarajan
authors.forename: Haixin
authors.initials: H
authors.lastname: Li
authors.forename: Sarah
authors.initials: S
authors.lastname: Rodriguez
authors.forename: Brent L
authors.initials: BL
authors.lastname: Iverson
authors.forename: George
authors.initials: G
authors.lastname: Georgiou
mesh_terms: D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides / Q000378:metabolism / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006867:Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D011479:Protamines / Q000378:metabolism / Q000494:pharmacology; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
chemical_list: D023181:Antimicrobial Cationic Peptides; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D019151:Peptide Library; D010455:Peptides; D011479:Protamines; C499588:ompT protein, E coli; C482905:OmpX protein, E coli; D006867:Hydrolases; D010447:Peptide Hydrolases