Identification of essential acidic residues of outer membrane protease OmpT supports a novel active site

Escherichia coli outer membrane protease OmpT has previously been classifie d as a serine protease with Ser(99) and His(212) as active site residues. T he recently solved X-ray structure of the enzyme was inconsistent with this classification, and the involvement of a nucleophilic water molecule was p roposed. Here, we substituted all conserved aspartate and glutamate residue s by alanines and measured the residual enzymatic activities of the variant s. Our results support the involvement of a nucleophilic water molecule tha t is activated by the Asp(210)/His(212) catalytic dyad. Activity is also st rongly dependent on Asp(83) and Asp(85). Both may function in binding of th e water molecule and/or oxyanion stabilization. The proposed mechanism impl ies a novel proteolytic catalytic site. (C) 2001 Federation of European Bio chemical Societies. Published by Elsevier Science B.V. All rights reserved.