Structural basis of substrate selectivity of E. coli prolidase.

Jeremy Weaver,Hays S Rye,Tylan Watts,Pingwei Li,Annalisa Pastore

doi:10.1371/journal.pone.0111531

Abstract

Prolidases, metalloproteases that catalyze the cleavage of Xaa-Pro dipeptides, are conserved enzymes found in prokaryotes and eukaryotes. In humans, prolidase is crucial for the recycling of collagen. To further characterize the essential elements of this enzyme, we utilized the Escherichia coli prolidase, PepQ, which shares striking similarity with eukaryotic prolidases. Through structural and bioinformatic insights, we have extended previous characterizations of the prolidase active site, uncovering a key component for substrate specificity. Here we report the structure of E. coli PepQ, solved at 2.0 Å resolution. The structure shows an antiparallel, dimeric protein, with each subunit containing N-terminal and C-terminal domains. The C-terminal domain is formed by the pita-bread fold typical for this family of metalloproteases, with two Mg(II) ions coordinated by five amino-acid ligands. Comparison of the E. coli PepQ structure and sequence with homologous structures and sequences from a diversity of organisms reveals distinctions between prolidases from Gram-positive eubacteria and archaea, and those from Gram-negative eubacteria, including the presence of loop regions in the E. coli protein that are conserved in eukaryotes. One such loop contains a completely conserved arginine near the catalytic site. This conserved arginine is predicted by docking simulations to interact with the C-terminus of the substrate dipeptide. Kinetic analysis using both a charge-neutralized substrate and a charge-reversed variant of PepQ support this conclusion, and allow for the designation of a new role for this key region of the enzyme active site.

Highlights

Prolidases, known as Xaa-Pro dipeptidases, are metalloproteases that catalyze the hydrolysis of dipeptides containing a Cterminal proline residue
The E. coli sequence shows good coverage of the human gene, with only one region of 10–15 residues missing (Figure 1, between E. coli residues 120–125). These additional regions may be shifted in our alignment, in a previous alignment [5], four regions of at least ten residues appeared in E. coli and human prolidase, but did not appear in P. furiosus prolidase (E. coli residues 35–53, 303–321, 360–372 and 391–415)
The results presented here support a role for substrate length specificity in pita-bread fold enzymes through the positioning of an active site arginine

Summary

Introduction

Prolidases, known as Xaa-Pro dipeptidases, are metalloproteases that catalyze the hydrolysis of dipeptides containing a Cterminal proline residue. These enzymes are conserved in prokaryotes and eukaryotes, including single-celled organisms, such as yeast, and humans and higher plants [1,2,3,4,5,6,7,8]. Despite the importance of human prolidase and the disease states associated with various mutations of the gene, knockout and knockdown studies in several eukaryotic model organisms have yet to reveal an essential role for prolidase [14,15,16,17]. Further studies are required for insight into the role of prolidase in collagen metabolism and human health

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Results

Conclusion