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Journal of Bacteriology, January 2007, p. 522-530, Vol. 189, No. 2
0021-9193/07/$08.00+0     doi:10.1128/JB.01493-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Substrate Specificity of the Escherichia coli Outer Membrane Protease OmpP{triangledown}

Bum-Yeol Hwang,1,2 Navin Varadarajan,1,3 Haixin Li,1,5 Sarah Rodriguez,1,3 Brent L. Iverson,1,3 and George Georgiou1,2,4,5*

Institute for Cellular and Molecular Biology,1 Departments of Chemical Engineering,2 Chemistry and Biochemistry,3 Biomedical Engineering,4 Section of Molecular Genetics and Microbiology, University of Texas, Austin, Texas 787125

Received 21 September 2006/ Accepted 27 October 2006

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 (kcat/Km = 3.0 x 106 M–1s–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.

* Corresponding author. Mailing address: Department of Chemical Engineering, University of Texas, Austin, TX 78712. Phone: (512) 471-6975. Fax: (512) 471-7963. E-mail: gg{at}che.utexas.edu.

{triangledown} Published ahead of print on 3 November 2006.

Journal of Bacteriology, January 2007, p. 522-530, Vol. 189, No. 2
0021-9193/07/$08.00+0     doi:10.1128/JB.01493-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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