Using directed evolution to improve the solubility of the Cterminal domain of Escherichia coli aminopeptidaseP

The Escherichia coli aminopeptidase P (AMPP) is a protease with subunits that consist of two domains. Solution studies have shown that the activity of AMPP is manganese-dependent [1], and structural studies have shown that its active site contains two metals that are coordinated by residues from the C-terminal domain [2]. AMPP has a structure that is similar to that of prolidase and creatinase, but it is a tetramer, whereas both prolidase and creatinase are dimers [3]. Creatinase is a metal-independent enzyme that has an active site in a similar location to that of AMPP, whereas prolidase requires two metals that are coordinated to the protein via residues homologous to those found in AMPP. Methionine aminopeptidase is a monomeric protein that consists of a single domain that has structural similarity to the C-terminal domain of AMPP. Like prolidase, methionine aminopeptidase requires two metals that are coordinated via residues homologous to those of AMPP. These observations indicate that the C-terminal domain of AMPP, with its ‘pita-bread’ fold, is both stable and capable of being utilized for a number of catalytic functions. For this reason, we isolated the section of the AMPP gene that codes for the C-terminal domain and expressed it in E. coli. Surprisingly, this catalytic domain proved to be insoluble. Initially, it was thought that the change in solubility was due to the