Crystal structure of aminopeptidase N (Proteobacteria alanyl aminopeptidase) from Escherichia coli and conformational change of methionine 260 involved in substrate recognition

Abstract

Aminopeptidase N from Escherichia coli is a broad specificity zinc exopeptidase belonging to aminopeptidase clan MA, family M1. The structures of the ligand-free form and the enzyme-bestatin complex were determined at 1.5- and 1.6-Å resolution, respectively. The enzyme is composed of four domains: an N-terminal β-domain (Met1-Asp193), a catalytic domain (Phe194-Gly444), a middle β-domain (Thr 445-Trp546), and a C-terminal α-domain (Ser 547-Ala870). The structure of the catalytic domain exhibits similarity to thermolysin, and a metal-binding motif (HEXXHX 18E) is found in the domain. The zinc ion is coordinated by His 297, His301, Glu320, and a water molecule. The groove on the catalytic domain that contains the active site is covered by the C-terminal α-domain, and a large cavity is formed inside the protein. However, there exists a small hole at the center of the C-terminal α-domain. The N terminus of bestatin is recognized by Glu121 and Glu264, which are located in the N-terminal and catalytic domains, respectively. Glu298 and Tyr381, located near the zinc ion, are considered to be involved in peptide cleavage. A difference revealed between the ligand-free form and the enzyme-bestatin complex indicated that Met260 functions as a cushion to accept substrates with different N-terminal residue sizes, resulting in the broad substrate specificity of this enzyme. © 2006 by The American Society for Biochemistry and Molecular Biology.