Cargando…

Structural Basis of Substrate Selectivity of E. coli Prolidase

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 pro...

Descripción completa

Detalles Bibliográficos
Autores principales: Weaver, Jeremy, Watts, Tylan, Li, Pingwei, Rye, Hays S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4213023/
https://www.ncbi.nlm.nih.gov/pubmed/25354344
http://dx.doi.org/10.1371/journal.pone.0111531
_version_ 1782341786973241344
author Weaver, Jeremy
Watts, Tylan
Li, Pingwei
Rye, Hays S.
author_facet Weaver, Jeremy
Watts, Tylan
Li, Pingwei
Rye, Hays S.
author_sort Weaver, Jeremy
collection PubMed
description 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.
format Online
Article
Text
id pubmed-4213023
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-42130232014-11-05 Structural Basis of Substrate Selectivity of E. coli Prolidase Weaver, Jeremy Watts, Tylan Li, Pingwei Rye, Hays S. PLoS One Research Article 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. Public Library of Science 2014-10-29 /pmc/articles/PMC4213023/ /pubmed/25354344 http://dx.doi.org/10.1371/journal.pone.0111531 Text en © 2014 Weaver et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Weaver, Jeremy
Watts, Tylan
Li, Pingwei
Rye, Hays S.
Structural Basis of Substrate Selectivity of E. coli Prolidase
title Structural Basis of Substrate Selectivity of E. coli Prolidase
title_full Structural Basis of Substrate Selectivity of E. coli Prolidase
title_fullStr Structural Basis of Substrate Selectivity of E. coli Prolidase
title_full_unstemmed Structural Basis of Substrate Selectivity of E. coli Prolidase
title_short Structural Basis of Substrate Selectivity of E. coli Prolidase
title_sort structural basis of substrate selectivity of e. coli prolidase
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4213023/
https://www.ncbi.nlm.nih.gov/pubmed/25354344
http://dx.doi.org/10.1371/journal.pone.0111531
work_keys_str_mv AT weaverjeremy structuralbasisofsubstrateselectivityofecoliprolidase
AT wattstylan structuralbasisofsubstrateselectivityofecoliprolidase
AT lipingwei structuralbasisofsubstrateselectivityofecoliprolidase
AT ryehayss structuralbasisofsubstrateselectivityofecoliprolidase