Crystal Structure and Substrate Recognition of Aminopeptidase N

Graduate School of Biomedical Sciences, Nagasaki University
â—‹Yoshitaka Nakajima Yuko Onohara Kiyoshi Ito Yue Xu Kanako Nakashima Futoshi Matsubara Tadashi Yoshimoto

Aminopeptidase N (APN; EC from Escherichia coli is a zinc peptidase, member of peptidase family M1. This enzyme possesses broad-substrate specificity and prefers substrates of N-terminal Arg, Lys, and Ala residues. It is noteworthy that the enzyme shows a little activity against peptides with an N-terminal Pro residue. In order to clarify catalytic and substrate-recognition mechanism of APN, we investigated this enzyme using X-ray crystallography.
The structure of ligand-free form and enzyme-bestatin, -amastatin, -L-leucine complex were determined at 1.50, 1.60, 1.65, and 1.55 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 (Thr445-Trp546), and a C-terminal α-domain (Ser547-Ala870). The structure of the catalytic domain exhibits similarity to thermolysin, and a metal binding motif (HEXXHX18E) is found in the domain. The zinc ion is coordinated by His297, 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 existed a small hole at the center of the C-terminal α-domain. The amino terminus of bestatin, amastatin, and L-leucine was recognized by residues 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 among the ligand-free form and three complexes indicated that Met260 functions as a cushion to accept substrates with different amino terminal sizes, resulting in the broad substrate specificity of this enzyme.