Structure and mechanism of a proline-specific aminopeptidase from Escherichia coli

Abstract

The structure of the proline-specific aminopeptidase (EC 3.4.11.9) from Escherichia coli has been solved and refined for crystals of the native enzyme at a 2.0-Å resolution, for a dipeptide-inhibited complex at 2.3-Å resolution, and for a low-pH inactive form at 2.7-Å resolution. The protein crystallizes as a tetramer, more correctly a dimer of dimers, at both high and low pH, consistent with observations from analytical ultracentrifuge studies that show that the protein is a tetramer under physiological conditions. The monomer folds into two domains. The active site, in the larger C-terminal domain, contains a dinuclear manganese center in which a bridging water molecule or hydroxide ion appears poised to act as the nucleophile in the attack on the scissile peptide bond of Xaa-Pro. The metal-binding residues are located in a single subunit, but the residues surrounding the active site are contributed by three subunits. The fold of the protein resembles that of creatine amidinohydrolase (creatinase, not a metalloenzyme). The C-terminal catalytic domain is also similar to the single-domain enzyme methionine aminopeptidase that has a dinuclear cobalt center.Proline-specific peptidases have been described in a wide variety of organisms and specifically cleave either the amide bond after a proline residue or the imide bond that precedes it (1). Proline aminopeptidases (EC 3.4.11.9) specifically release the N-terminal residue from a peptide where the penultimate residue is proline. These enzymes have significant sequence homology with (i) some other amino peptidases (e.g., methionine aminopeptidase, AMPM) and (ii) the Xaa-Pro dipeptidases, prolidases, that specifically cleave Xaa-Pro dipeptides. All these enzymes are activated in the presence of divalent metal ions, though the biologically active metal is not always certain. In vitro the prolyl peptidases are most active in the presence of Mn 2ϩ . Structural comparisons between AMPM and creatine amidinohydrolase (creatinase) and sequence comparisons between these proteins and aminopeptidase P (AMPP) and prolidase have suggested that the catalytic domains of all four enzymes have a common fold (2). This work presents the crystal structure of AMPP, the product of the pepP gene in Escherichia coli. The structures of a complex of AMPP with a dipeptide inhibitor and a low-pH inactive form are used to devise a plausible mechanism for peptide hydrolysis. EXPERIMENTAL PROCEDURESOverexpression and Purification of AMPP. AMPP was originally isolated and characterized in E. coli (3) and was subsequently cloned and overexpressed (4). The details of the overexpression and purification of AMPP used in this work will be published elsewhere. Briefly, AMPP was overexpressed in the E. coli AN1459͞pPL670. Lysed cells were subject to (NH 4 ) 2 SO 4 precipitation at 4°C followed by centrifugation. The pellet was resuspended and applied to a DEAE-Fractogel column (Merck). The AMPP-containing fraction was then passed over a ceramic hydroxyapatite column (Bio-Rad). This simple purifi...

Biochemical studies on Helicobacter pylori arginase: Insight into the difference in activity compared to other arginases

“…The pK a 6.3 in our case may correspond to a histidine residue as the pK a of free histidine in water is 6.3. Metalbridging hydroxide ion has been found to act as a nucleophile in a number hydrolytic manganese metalloenzymes such as inorganic pyrophosphatase (33), serine/threonine phosphatase-1 (34) and proline specific aminopeptidase from E. coli (35). Our data show that there is a variation in the pH dependence of the H. pylori enzyme with the metal ions; Co 21 showed a single ionization, whereas Mn 21 showed double ionization.…”

Structure of creatine amidinohydrolase from Actinobacillus

“…The present work provides additional structural data on the striking similarities between creatinases, proline aminopeptidases (AMPPs; Wilce et al, 1998) and methionine aminopeptidases (AMPMs; Roderick & Matthews, 1993;Tahirov et al, 1998;Liu et al, 1998). The catalytic C-terminal domains of these three protein classes all have the same pitta-bread fold (Bazan et al, 1994).…”

“…creatinase, AMPP, AMPM and prolidases were compared, we found only 13 conserved residues (data not shown). Despite the fact that the overall tertiary structures of creatinase and AMPP are similar, the oligomerization of creatinase (dimer) differs from the AMPP oligomerization (tetramer) (Wilce et al, 1998).…”

“…Other than creatinases, many other amidinohydrolases are found; namely, proline aminopeptidases (AMPPs; Wilce et al, 1998) and methionine aminopeptidases (AMPMs; Roderick & Matthews, 1993;Tahirov et al, 1998;Liu et al, 1998). Structural comparisons between creatinase and AMPM and AMPP hydrolases and sequence comparisons between these proteins have suggested that the catalytic domain of all these enzymes shares a common pitta-bread fold (Bazan et al, 1998).…”

Structural Analysis and Bioengineering of Thermostable Pyrococcus furiosus Prolidase for the Optimization of Organophosphorus Nerve Agent Detoxification

“…Modified from Ghosh, 1998. amino acid residues are conserved among all of the prolidase enzymes as well as Alteromonas OPAA, and therefore were predicted to function as the metal-binding residues in prolidases, a prediction that has since been confirmed by X-ray crystal structure analysis of P. furiosus prolidase (Maher et al 2004). An equivalent type metal center is also observed in the enzyme aminopeptidase from E. coli, which has a dinuclear metal center containing Mn 2+ ions (Wilce et al 1998) involving the five amino acids residues Asp-260, Asp-271, His-354, Glu-383, and Glu-406 ( Figure 6). This comparison is also particularly relevant as a number of other characterized prolidases such as those isolated from L. casei, X. maltophilia, human, and Alteromonas OPAA are Mn 2+ -dependent, rather than Co 2+ -dependent, and thus likely contain a dinuclear Mn center instead.…”