Crystal Structure of Aminopeptidase N (Proteobacteria Alanyl Aminopeptidase) from Escherichia coli and Conformational Change of Methionine 260 Involved in Substrate Recognition

Itō, Kiyoshi; Nakajima, Yoshitaka; Onohara, Yuko; Takeo, M.; Nakashima, Kazunori; Matsubara, Futoshi; Ito, Takashi; Yoshimoto, Tadashi

doi:10.1074/jbc.m605203200

Cited by 131 publications

(164 citation statements)

References 37 publications

(3 reference statements)

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“…We determined the X-ray crystal structure rPfA-M1 to 2.1 Å (Table S2). The structure confirmed that rPfA-M1 adopts the bacterial aminopeptidase N-fold (20,22,23), and comprises 26 ␣-helices and 7 ␤-sheets divided into 4 domains ( Fig. 2A).…”

Section: Resultssupporting

confidence: 55%

“…2A). The catalytic domain II (residues 392-649) adopts a thermolysin-like fold and contains the active site, incorporating the zinc-binding motif H 496 EYFHX 17 KE 519 and the well-conserved G 490 AMEN motif involved in substrate recognition (20,22,23 Inspection of the molecular surface of PfA-M1 reveals 2 openings to the active site cavity. The first opening (N-terminal channel) comprises a shallow 8-Å-long groove at the junction of domains I and IV (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The active site of PfA-M1 is buried deep in the structure, providing a solution to the problem of controlling appropriate substrate entry, as well as preventing hydrolysis of unwanted substrates. By comparison with the available bacterial aminopeptidases structures (20,22,23), PfA-M1 can, thus, be considered to adopt a ''closed'' conformation. Interestingly, structural studies on the homologous tricorn-interacting factor 3, F3, reveals this protease undergoes a substantial conformational changes on substrate entry, where domain IV swings away from domain II, exposing the active site.…”

Section: Figmentioning

confidence: 99%

“…In E. coli aminopeptidase N protein Met 454 , positioned immediately preceding the GAMEN exopeptidase motif, is postulated to function as a cushion to accept substrates (20), altering the size of the active site pocket. This residue is conserved in bacterial aminopeptidases; however, the equivalent position (469) in eukaryote M1 aminopeptidases is commonly a smaller valine or alanine residue.…”

Section: Figmentioning

confidence: 99%

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Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase

McGowan

Porter

Lowther

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

142

278

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Plasmodium falciparum parasites are responsible for the major global disease malaria, which results in >2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase, PfA-M1, a validated drug target. The X-ray crystal structure of PfA-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH 2]Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite. drug design ͉ malaria ͉ structural biology ͉ protease T here are 300-500 million cases of clinical malaria annually, and 1.4-2.6 million deaths. Malaria is caused by apicomplexan parasites of the genus Plasmodium, with Plasmodium falciparum the most lethal of the 4 species that infect humans. Clinical manifestations begin when parasites enter erythrocytes, and most antimalaria drugs, such as chloroquine, exert their action by preventing the parasite development within these cells (1). As a result of the rapid spread of drug-resistant parasites, there is a constant need to identify and validate new antimalarial targets.Intraerythrocytic parasites have limited capacity for de novo amino acid synthesis and rely on degradation of host hemoglobin (Hb) to maintain protein metabolism and synthesis, and an osmotically stable environment within the erythrocyte (1-4). Within the erythrocytes, malaria parasites consume as much as 75% of the cellular Hb (1). Hb is initially degraded by the concerted action of cysteine-, aspartyl-, and metalloendoproteases, and a dipeptidase (cathepsin C) within a digestive vacuole (DV) to di-and tripeptide fragments (5, 6). These fragments are suggested to be exported to the parasite cytoplasm, where further hydrolysis to release free amino acids takes place [supporting information (SI) Fig. S1; see refs. 7 and 8].The release of amino acids involves 2 metallo-exopeptidases: an alanyl aminopeptidase, PfA-M1 (9, 10), and a leucine aminopeptidase, PfA-M17 (7,11,12). We have demonstrated that the aminopeptidase inhibitor bestatin, an antibiotic and natural analogue of the dipeptide Phe-Leu derived from the fungus Streptomyces olivoretticul, prevents P. falciparum malaria growth in culture (13,14). More recently, it was shown not only that synthetic phosphinate dipeptide analogues that inhibit metallo-aminopeptidases prevented the growth of wildty...

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Section: Resultssupporting

confidence: 55%

Section: Resultsmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

See 2 more Smart Citations

Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase

McGowan

Porter

Lowther

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

142

278

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“…Crystal structures of ePepN, hErPepN [not published, deposited in protein data bank (PDB) with ID: 2XDT], F3, LTA 4 H, PepN from Neisseria meningitidis (NmPepN) and from Plasmodium falciparum (PfPepN) have been reported. [4][5][6][7][8][9][10] Within the M1 class peptidases, all enzymes have four domains except, LTA 4 H which has only three domains. First two domains (Domains I and II) share highest sequence and structural homology among all these structures including the LTA 4 H. The LTA 4 H has a unique C-terminal domain instead of Domains III and IV, probably required to perform the leukotriene hydrolase activity.…”

Section: Introductionmentioning

confidence: 99%

Glu121‐Lys319 salt bridge between catalytic and N‐terminal domains is pivotal for the activity and stability of Escherichia coli aminopeptidase N

et al. 2012

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Escherichia coli aminopeptidase N (ePepN) belongs to the gluzincin family of M1 class metalloproteases that share a common primary structure with consensus zinc binding motif (HEXXH-(X18)-E) and an exopeptidase motif (GXMEN) in the active site. There is one amino acid, E121 in Domain I that blocks the extended active site grove of the thermolysin like catalytic domain (Domain II) limiting the substrate to S1 pocket. E121 forms a part of the S1 pocket, while making critical contact with the amino-terminus of the substrate. In addition, the carboxylate of E121 forms a salt bridge with K319 in Domain II. Both these residues are absolutely conserved in ePepN homologs. Analogous Glu-Asn pair in tricon interacting factor F3 (F3) and Gln-Asn pair in human leukotriene A 4 hydrolase (LTA 4 H) are also conserved in respective homologs. Mutation of either of these residues individually or together substantially reduced or entirely eliminated enzymatic activity. In addition, thermal denaturation studies suggest that the mutation at K319 destabilizes the protein as much as by 3.7°C, while E121 mutants were insensitive. Crystal structure of E121Q mutant reveals that the enzyme is inactive due to the reduced S1 subsite volume. Together, data presented here suggests that ePepN, F3, and LTA 4 H homologs adopted a divergent evolution that includes E121-K319 or its analogous pairs, and these cannot be interchanged.

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Medicinal Chemistry of α‐Hydroxy‐β‐Amino Acids

Ziora

Skwarczynski

Kiso

2011

Amino Acids, Peptides and Proteins in Organic Chemistry

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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 enzymebestatin complex were determined at 1.

Cited by 131 publications

References 37 publications

Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase

Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase

Glu121‐Lys319 salt bridge between catalytic and N‐terminal domains is pivotal for the activity and stability of Escherichia coli aminopeptidase N

Medicinal Chemistry of α‐Hydroxy‐β‐Amino Acids

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