title: 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-A resolution, respectively. The enzyme is composed of four domains: an N-terminal beta-domain (Met(1)-Asp(193)), a catalytic domain (Phe(194)-Gly(444)), a middle beta-domain (Thr(445)-Trp(546)), and a C-terminal alpha-domain (Ser(547)-Ala(870)). The structure of the catalytic domain exhibits similarity to thermolysin, and a metal-binding motif (HEXXHX(18)E) is found in the domain. The zinc ion is coordinated by His(297), His(301), Glu(320), and a water molecule. The groove on the catalytic domain that contains the active site is covered by the C-terminal alpha-domain, and a large cavity is formed inside the protein. However, there exists a small hole at the center of the C-terminal alpha-domain. The N terminus of bestatin is recognized by Glu(121) and Glu(264), which are located in the N-terminal and catalytic domains, respectively. Glu(298) and Tyr(381), 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 Met(260) functions as a cushion to accept substrates with different N-terminal residue sizes, resulting in the broad substrate specificity of this enzyme.
authors.affiliation: Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Ito
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Nakajima
authors.forename: Yuko
authors.initials: Y
authors.lastname: Onohara
authors.forename: Masahide
authors.initials: M
authors.lastname: Takeo
authors.forename: Kanako
authors.initials: K
authors.lastname: Nakashima
authors.forename: Futoshi
authors.initials: F
authors.lastname: Matsubara
authors.forename: Takashi
authors.initials: T
authors.lastname: Ito
authors.forename: Tadashi
authors.initials: T
authors.lastname: Yoshimoto
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018826:CD13 Antigens / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007930:Leucine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D008715:Methionine / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013379:Substrate Specificity
chemical_list: D021122:Protein Subunits; D008715:Methionine; D018826:CD13 Antigens; D007930:Leucine; C012211:ubenimex