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Journal of Bacteriology, September 2004, p. 5919-5925, Vol. 186, No. 17
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.17.5919-5925.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Substrate Specificity of the Escherichia coli Outer Membrane Protease OmpT

John D. McCarter,1,{dagger} Daren Stephens,2 Kevin Shoemaker,3 Steve Rosenberg,3 Jack F. Kirsch,1 and George Georgiou2*

Department of Molecular and Cell Biology, University of California, Berkeley, and Center for Advanced Materials, Lawrence Berkeley Laboratory, Berkeley,1 Chiron Corporation, Emeryville, California,3 Department of Chemical Engineering and Institute for Cell and Molecular Biology, University of Texas, Austin, Texas2

Received 18 March 2004/ Accepted 7 June 2004

OmpT is a surface protease of gram-negative bacteria that has been shown to cleave antimicrobial peptides, activate human plasminogen, and degrade some recombinant heterologous proteins. We have analyzed the substrate specificity of OmpT by two complementary substrate filamentous phage display methods: (i) in situ cleavage of phage that display protease-susceptible peptides by Escherichia coli expressing OmpT and (ii) in vitro cleavage of phage-displayed peptides using purified enzyme. Consistent with previous reports, OmpT was found to exhibit a virtual requirement for Arg in the P1 position and a slightly less stringent preference for this residue in the P1' position (P1 and P1' are the residues immediately prior to and following the scissile bond). Lys, Gly, and Val were also found in the P1' position. The most common residues in the P2' position were Val or Ala, and the P3 and P4 positions exhibited a preference for Trp or Arg. Synthetic peptides based upon sequences selected by bacteriophage display were cleaved very efficiently, with kcat/Km values up to 7.3 x 106 M–1 s–1. In contrast, a peptide corresponding to the cleavage site of human plasminogen was hydrolyzed with a kcat/Km almost 106-fold lower. Overall, the results presented in this work indicate that in addition to the P1 and P1' positions, additional amino acids within a six-residue window (between P4 and P2') contribute to the binding of substrate polypeptides to the OmpT binding site.

* Corresponding author. Mailing address: Department of Chemical Engineering and Institute for Cell and Molecular Biology, University of Texas, Austin, TX 78705. Phone: (512) 471-6975. Fax: (512) 471-7963. E-mail: gg{at}che.utexas.edu.

{dagger} Present address: Amgen, Inc., Thousand Oaks, CA 91320-1799.

Journal of Bacteriology, September 2004, p. 5919-5925, Vol. 186, No. 17
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.17.5919-5925.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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