title: Identification of active site serine and histidine residues in Escherichia coli outer membrane protease OmpT.
abstract: Escherichia coli outer membrane protease OmpT has been characterised as a serine protease based on its inhibitor profile, but serine protease consensus sequences are absent. By site-directed mutagenesis we substituted all conserved serines and histidines. Substitution of His(101) and His(212) by Ala, Asn or Gln resulted in variant enzymes with 0.01 and 9-20% residual enzymatic activity towards a fluorogenic pentapeptide substrate, respectively. The mutations S140A and S201A did not decrease activity, while variants S40A and S99A yielded 0.5 and 0.2% residual activities, respectively. When measured with a dipeptide substrate the variant S40A demonstrated full activity, whereas variant S99A displayed at least 500-fold reduced activity. We conclude that Ser(99) and His(212) are essential active site residues. We propose that OmpT is a novel serine protease with Ser(99) as the active site nucleophile and His(212) as general base.
authors.affiliation: Department of Enzymology, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
authors.forename: R A
authors.initials: RA
authors.lastname: Kramer
authors.forename: N
authors.initials: N
authors.lastname: Dekker
authors.forename: M R
authors.initials: MR
authors.lastname: Egmond
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006639:Histidine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D012694:Serine; D006639:Histidine; D012697:Serine Endopeptidases; C057840:omptin outer membrane protease