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Identification of active site serine and histidine residues in Escherichia coli outer membrane protease OmpT
Author(s) -
Kramer R.Arjen,
Dekker Niek,
Egmond Maarten R
Publication year - 2000
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(00)01231-x
Subject(s) - serine protease , protease , active site , biochemistry , escherichia coli , masp1 , serine , bacterial outer membrane , chemistry , histidine , pentapeptide repeat , site directed mutagenesis , proteases , enzyme , tmprss6 , biology , microbiology and biotechnology , peptide , mutant , gene
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.