Crystal structure of aminopeptidase N from human pathogen Neisseria meningitidis

Abstract

Aminopeptidases are ubiquitous hydrolases that cleave the N-terminal residues of proteins and peptides for maturation, activation, or degradation, and therefore are involved in numerous biological processes.1,2 They are broadly distributed throughout all kingdoms of life and are found in subcellular organelles, cytoplasm, and in membrane-bound fractions.3 Many aminopeptidases use a set of conserved residues within a structural scaffold to form an active site capable of binding either one or two divalent metal ions that aid catalysis. Zn+2, Co+2, and Mn+2 are being the most common metals found in the active site.4–8 One of more extensively studied members of the aminopeptidase family is aminopeptidase N (APN) [alternative names: alanine aminopeptidase; aminopeptidase M; microsomal aminopeptidase; GP150; CD13; (EC 3.4.11.2)]. The APN sequence family is large and broadly distributed and includes members found in bacteria and eukaryotes, including plants and mammals. PsiBlast search identified over 1000 APN family members. Typically APNs are monomeric or homodimeric. In higher eukaryotes these enzymes are expressed in many tissues, with the highest level found in the intestinal and kidney brush border membranes, brain, lung, blood vessels, and primary cultures of fibroblasts. The sequence analysis indicates that aminopeptidase N is a member of the M1 family of the MA clan of peptidases, also termed gluzincins.5 The amino acid sequence fingerprints of the M1 family of zinc-metallopeptidases are the HEXXH(X18)E (a zinc binding motif) and GXMEN (an exopeptidase motif).9 Prominent members of this family include mammalian membrane-bound aminopeptidases [P-LAP, aminopeptidase A (APA), thyrotropin-releasing hormone degrading enzyme (TRHDE)], cytosolic proteins [puromycin-sensitive aminopeptidase (PSA) and leukotriene A4 hydrolase (LTA4H)], and secretory proteins such as [adipocyte-derived leucine aminopeptidase (A-LAP) and aminopeptidase B (APB)].5,9 The APNs catalyze liberation of N-terminal amino acids from a broad spectrum of substrates including small peptides, amide, or arylamide. The N-terminal residue is a preferably neutral or basic amino acid, although it has been reported that an intact XPro dipeptide was released when the terminal hydrophobic residue was followed by a prolyl residue.10 The diversity of function that APNs play depends on their location and source tissue. Some APNs have been used commercially, such as the APN from Lactococcus lactis, which has been used in the food industry.11 Aminopeptidases N are also present in many pathogenic bacteria and represent potential drug targets.9 In this article, we report the crystal structure of APN from N. meningitides at 2.05-A resolution.