Structure
Research ArticleCrystal structure of Aeromonas proteolytica aminopeptidase: a prototypical member of the co-catalytic zinc enzyme family
Introduction
In recent years much attention has been focused on the structure and mechanism of zinc-dependent proteases, many of which are implicated in the metabolism of a variety of important peptides including hormones, neurotransmitters and dietary peptides [1]. Thermolysin (an endopeptidase) and carboxypeptidase A (an exo- peptidase) both have one zinc ion in their active site which is essential for catalysis [2], [3], [4]. For structural and mechanistic studies, these enzymes are viewed as archetypal representatives of their protease classes. The aminopeptidases are widely distributed in nature and constitute an important subgroup of zinc-dependent proteases. These enzymes catalyze the hydrolysis of the amino-terminal peptide bond in polypeptides [5]. Among the aminopeptidase family, the zinc metalloenzymes contain either one or two metal ions per monomer. Early proposals assigned a catalytic role for only one zinc ion, attributing a vague structural or regulatory function to the second [6]. Recently, however, the possibility that the ‘two-zinc’ aminopeptidases may represent, from a mechanistic point of view, a new class of metallopeptidases, has given rise to a renewed interest in these enzymes [7].
In contrast to a number of mammalian aminopeptidases which are oligomeric structures of considerable size [8], the two-zinc containing aminopeptidase of the bacterial species Aeromonas proteolytica (EC 3.4.11.10) is a single peptide of M r 32 000 [9]. It is also an unusually stable extracellular enzyme that can be isolated in high yields from culture filtrates [10]. It thus appears to be a prototypical two-zinc aminopeptidase, ideally suited for structural studies designed to elucidate the catalytic mechanism of this class of enzyme. As the first step of such a study, we have determined the three-dimensional structure of A. proteolytica aminopeptidase (AAP).
While this work was in progress, the first crystallographic data on two other aminopeptidases was reported, firstly for the bovine lens leucine aminopeptidase (LAP), another two-zinc peptidase [11], and more recently for the methionine aminopeptidase (MAP) from Escherichia coli, which contains two cobalt ions in its active site [12]. Here, together with the general structural features of the A. proteolytica aminopeptidase, we report detailed structural information pertaining to the enzyme active site and the arrangement of the two zinc ions. In addition, we compare this three-dimensional structure with the two other known aminopeptidases.
Section snippets
Polypeptide chain fold
AAP folds into a single α/β globular domain with approximate maximal dimensions of 35 ×40 ×48 å 3. Using the program DSSP [13] to assign the secondary structure elements reveals that 35 % of the amino acid residues are involved in α-helices and 18 % in α-strands (Figure 1a). A ribbon drawing of the Cα backbone with the two zinc ions is shown in Figure 1b.
A centrally located, twisted β-sheet, sandwiched between α- helices, constitutes the hydrophobic core of the protein. This mixed β-sheet is
Biological implications
The aminopeptidase from the bacterial species Aeromonas proteolytica (AAP), the smallest zinc-dependent aminopeptidase thus far described, appears to be a prototype for a new class of zinc-dependent proteolytic enzymes with co-catalytic metal centers [7]. AAP therefore represents an interesting model for structural and mechanistic studies of this class of enzyme.
An important observation from this high-resolution study is the equivalent structural environment of the two zinc ions in AAP. This
Crystal data
Aeromonas proteolytica aminopeptidase was purified as previously described and crystallized following a slight modification of the published procedure [9]. Thus crystals used were obtained at pH 7.0 by the vapor diffusion technique in hanging drops with HEPES replacing Tris at the same concentration. Crystals diffract beyond 1.8 å resolution and belong to the hexagonal space group P6 1 22, with cell parameters a=111.0 å and c=92.1 å. The correct enantiomorphic space group, P6 1 22, was assigned
Acknowledgement
We are grateful to Dr JM Prescott for providing the subculture of Aeromonas proteolytica.
Bernard Chevrier (corresponding author) and Dino Moras, Laboratoire de Biologie Structurale, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg cedex, France.
Céline Schalk, Hugues D’Orchymont, Jean-Michel Rondeau and Céline Tarnus (corresponding author), Marion Merrell Dow Research Institute, 16 rue d’Ankara, 67080 Strasbourg cedex, France.
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Bernard Chevrier (corresponding author) and Dino Moras, Laboratoire de Biologie Structurale, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg cedex, France.
Céline Schalk, Hugues D’Orchymont, Jean-Michel Rondeau and Céline Tarnus (corresponding author), Marion Merrell Dow Research Institute, 16 rue d’Ankara, 67080 Strasbourg cedex, France.