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Title: Active-Site Flexibility and Substrate Specificity in a Bacterial Virulence Factor: Crystallographic Snapshots of an Epoxide Hydrolase

Authors:
; ; ; ; ; ;
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
1397653
Grant/Contract Number:
SC0012704; AC02-76SF00515
Resource Type:
Journal Article: Publisher's Accepted Manuscript
Journal Name:
Structure
Additional Journal Information:
Journal Volume: 25; Journal Issue: 5; Related Information: CHORUS Timestamp: 2017-10-04 21:48:56; Journal ID: ISSN 0969-2126
Publisher:
Elsevier
Country of Publication:
United Kingdom
Language:
English

Citation Formats

Hvorecny, Kelli L., Bahl, Christopher D., Kitamura, Seiya, Lee, Kin Sing Stephen, Hammock, Bruce D., Morisseau, Christophe, and Madden, Dean R. Active-Site Flexibility and Substrate Specificity in a Bacterial Virulence Factor: Crystallographic Snapshots of an Epoxide Hydrolase. United Kingdom: N. p., 2017. Web. doi:10.1016/j.str.2017.03.002.
Hvorecny, Kelli L., Bahl, Christopher D., Kitamura, Seiya, Lee, Kin Sing Stephen, Hammock, Bruce D., Morisseau, Christophe, & Madden, Dean R. Active-Site Flexibility and Substrate Specificity in a Bacterial Virulence Factor: Crystallographic Snapshots of an Epoxide Hydrolase. United Kingdom. doi:10.1016/j.str.2017.03.002.
Hvorecny, Kelli L., Bahl, Christopher D., Kitamura, Seiya, Lee, Kin Sing Stephen, Hammock, Bruce D., Morisseau, Christophe, and Madden, Dean R. 2017. "Active-Site Flexibility and Substrate Specificity in a Bacterial Virulence Factor: Crystallographic Snapshots of an Epoxide Hydrolase". United Kingdom. doi:10.1016/j.str.2017.03.002.
@article{osti_1397653,
title = {Active-Site Flexibility and Substrate Specificity in a Bacterial Virulence Factor: Crystallographic Snapshots of an Epoxide Hydrolase},
author = {Hvorecny, Kelli L. and Bahl, Christopher D. and Kitamura, Seiya and Lee, Kin Sing Stephen and Hammock, Bruce D. and Morisseau, Christophe and Madden, Dean R.},
abstractNote = {},
doi = {10.1016/j.str.2017.03.002},
journal = {Structure},
number = 5,
volume = 25,
place = {United Kingdom},
year = 2017,
month = 5
}

Journal Article:
Free Publicly Available Full Text
This content will become publicly available on May 2, 2018
Publisher's Accepted Manuscript

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  • Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is a virulence factor secreted by Pseudomonas aeruginosa that reduces the quantity of CFTR in the apical membrane of human airway epithelial cells. Initial sequence analysis suggested that Cif is an epoxide hydrolase (EH), but its sequence violates two strictly conserved EH motifs and also is compatible with other {alpha}/{beta} hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8-{angstrom} resolution by X-ray crystallography. The catalytic triad consists of residues Asp129, His297, and Glu153, which are conserved across themore » family of EHs. At other positions, sequence deviations from canonical EH active-site motifs are stereochemically conservative. Furthermore, detailed enzymatic analysis confirms that Cif catalyzes the hydrolysis of epoxide compounds, with specific activity against both epibromohydrin and cis-stilbene oxide, but with a relatively narrow range of substrate selectivity. Although closely related to two other classes of {alpha}/{beta} hydrolase in both sequence and structure, Cif does not exhibit activity as either a haloacetate dehalogenase or a haloalkane dehalogenase. A reassessment of the structural and functional consequences of the H269A mutation suggests that Cif's effect on host-cell CFTR expression requires the hydrolysis of an extended endogenous epoxide substrate.« less
  • Pseudomonas aeruginosa is an opportunist Gram-negative bacterial pathogen responsible for a wide range of infections in immunocompromized individuals and is a leading cause of mortality in cystic fibrosis patients. A number of secreted virulence factors, including various proteolytic enzymes, contribute to the establishment and maintenance of Pseudomonas infection. One such is LasA, an M23 metallopeptidase related to autolytic glycylglycine endopeptidases such as Staphylococcus aureus lysostaphin and LytM, and to DD-endopeptidases involved in entry of bacteriophage to host bacteria. LasA is implicated in a range of processes related to Pseudomonas virulence, including stimulating ectodomain shedding of the cell surface heparan sulphatemore » proteoglycan syndecan-1 and elastin degradation in connective tissue. Here we present crystal structures of active LasA as a complex with tartrate and in the uncomplexed form. While the overall fold resembles that of the other M23 family members, the LasA active site is less constricted and utilizes a different set of metal ligands. The active site of uncomplexed LasA contains a five-coordinate zinc ion with trigonal bipyramidal geometry and two metal-bound water molecules. Using these structures as a starting point, we propose a model for substrate binding by LasA that explains its activity against a wider range of substrates than those used by related lytic enzymes, and offer a catalytic mechanism for M23 metallopeptidases consistent with available structural and mutagenesis data. Our results highlight how LasA is a structurally distinct member of this endopeptidase family, consistent with its activity against a wider range of substrates and with its multiple roles in Pseudomonas virulence.« less