ABSTRACT: The removal of N-terminal translation initiator Met by methionine aminopeptidase (MetAP) is often crucial for the function and stability of proteins. On the basis of crystal structure and sequence alignment of MetAPs, we have engineered Escherichia coli MetAP by the mutation of three residues, Y168G, M206T, Q233G, in the substrate-binding pocket. Our engineered MetAPs are able to remove the Met from bulky or acidic penultimate residues, such as Met, His, Asp, Asn, Glu, Gln, Leu, Ile, Tyr, and Trp, as well as from small residues. The penultimate residue, the second residue after Met, was further removed if the antepenultimate residue, the third residue after Met, was small. By the coexpression of engineered MetAP in E. coli through the same or a separate vector, we have successfully produce
