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Title: Bacillus anthracis Prolyl 4-Hydroxylase Interacts with and Modifies Elongation Factor Tu

Abstract

Prolyl hydroxylation is a very common post-translational modification and plays many roles in eukaryotes such as collagen stabilization, hypoxia sensing, and controlling protein transcription and translation. There is a growing body of evidence that suggests that prokaryotes contain prolyl 4-hydroxylases (P4Hs) homologous to the hypoxia-inducible factor (HIF) prolyl hydroxylase domain (PHD) enzymes that act on elongation factor Tu (EFTu) and are likely involved in the regulation of bacterial translation. Recent biochemical and structural studies with a PHD from Pseudomonas putida (PPHD) determined that it forms a complex with EFTu and hydroxylates a prolyl residue of EFTu. Moreover, while animal, plant, and viral P4Hs act on peptidyl proline, most prokaryotic P4Hs have been known to target free l-proline; the exceptions include PPHD and a P4H from Bacillus anthracis (BaP4H) that modifies collagen-like proline-rich peptides. Here we use biophysical and mass spectrometric methods to demonstrate that BaP4H recognizes full-length BaEFTu and a BaEFTu 9-mer peptide for site-specific proline hydroxylation. Using size-exclusion chromatography coupled small-angle X-ray scattering (SEC–SAXS) and binding studies, we determined that BaP4H forms a 1:1 heterodimeric complex with BaEFTu. The SEC–SAXS studies reveal dissociation of BaP4H dimeric subunits upon interaction with BaEFTu. While BaP4H is unusual within bacteria in thatmore » it is structurally and functionally similar to the animal PHDs and collagen P4Hs, respectively, this work provides further evidence of its promiscuous substrate recognition. It is possible that the enzyme might have evolved to hydroxylate a universally conserved protein in prokaryotes, similar to the PHDs, and implies a functional role in B. anthracis.« less

Authors:
 [1];  [1];  [2];  [3]; ORCiD logo [1]
  1. Department of Chemistry, The University of Iowa, Iowa City, Iowa 52242, United States
  2. Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, United States
  3. Biophysics Collaborative Access Team, Argonne National Laboratory, Argonne, Illinois 60439, United States
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH)
OSTI Identifier:
1418029
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 56; Journal Issue: 43
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Schnicker, Nicholas J., Razzaghi, Mortezaali, Guha Thakurta, Sanjukta, Chakravarthy, Srinivas, and Dey, Mishtu. Bacillus anthracis Prolyl 4-Hydroxylase Interacts with and Modifies Elongation Factor Tu. United States: N. p., 2017. Web. doi:10.1021/acs.biochem.7b00601.
Schnicker, Nicholas J., Razzaghi, Mortezaali, Guha Thakurta, Sanjukta, Chakravarthy, Srinivas, & Dey, Mishtu. Bacillus anthracis Prolyl 4-Hydroxylase Interacts with and Modifies Elongation Factor Tu. United States. doi:10.1021/acs.biochem.7b00601.
Schnicker, Nicholas J., Razzaghi, Mortezaali, Guha Thakurta, Sanjukta, Chakravarthy, Srinivas, and Dey, Mishtu. Tue . "Bacillus anthracis Prolyl 4-Hydroxylase Interacts with and Modifies Elongation Factor Tu". United States. doi:10.1021/acs.biochem.7b00601.
@article{osti_1418029,
title = {Bacillus anthracis Prolyl 4-Hydroxylase Interacts with and Modifies Elongation Factor Tu},
author = {Schnicker, Nicholas J. and Razzaghi, Mortezaali and Guha Thakurta, Sanjukta and Chakravarthy, Srinivas and Dey, Mishtu},
abstractNote = {Prolyl hydroxylation is a very common post-translational modification and plays many roles in eukaryotes such as collagen stabilization, hypoxia sensing, and controlling protein transcription and translation. There is a growing body of evidence that suggests that prokaryotes contain prolyl 4-hydroxylases (P4Hs) homologous to the hypoxia-inducible factor (HIF) prolyl hydroxylase domain (PHD) enzymes that act on elongation factor Tu (EFTu) and are likely involved in the regulation of bacterial translation. Recent biochemical and structural studies with a PHD from Pseudomonas putida (PPHD) determined that it forms a complex with EFTu and hydroxylates a prolyl residue of EFTu. Moreover, while animal, plant, and viral P4Hs act on peptidyl proline, most prokaryotic P4Hs have been known to target free l-proline; the exceptions include PPHD and a P4H from Bacillus anthracis (BaP4H) that modifies collagen-like proline-rich peptides. Here we use biophysical and mass spectrometric methods to demonstrate that BaP4H recognizes full-length BaEFTu and a BaEFTu 9-mer peptide for site-specific proline hydroxylation. Using size-exclusion chromatography coupled small-angle X-ray scattering (SEC–SAXS) and binding studies, we determined that BaP4H forms a 1:1 heterodimeric complex with BaEFTu. The SEC–SAXS studies reveal dissociation of BaP4H dimeric subunits upon interaction with BaEFTu. While BaP4H is unusual within bacteria in that it is structurally and functionally similar to the animal PHDs and collagen P4Hs, respectively, this work provides further evidence of its promiscuous substrate recognition. It is possible that the enzyme might have evolved to hydroxylate a universally conserved protein in prokaryotes, similar to the PHDs, and implies a functional role in B. anthracis.},
doi = {10.1021/acs.biochem.7b00601},
journal = {Biochemistry},
number = 43,
volume = 56,
place = {United States},
year = {Tue Oct 17 00:00:00 EDT 2017},
month = {Tue Oct 17 00:00:00 EDT 2017}
}