bi7b00601_si_001.pdf (1.93 MB)
Bacillus anthracis Prolyl 4‑Hydroxylase Interacts with and Modifies Elongation Factor Tu
journal contribution
posted on 2017-10-05, 00:00 authored by Nicholas
J. Schnicker, Mortezaali Razzaghi, Sanjukta Guha Thakurta, Srinivas Chakravarthy, Mishtu DeyProlyl 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.
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collagen P 4HsBaP 4Helongation factor TuP 4Hs actPHDPPHDBaP 4H dimeric subunitsModifies Elongation Factor Tu Prolyl hydroxylationSECcollagen-like proline-rich peptidesprolyl 4- hydroxylasesBaP 4H formssite-specific proline hydroxylationHIFprolyl hydroxylase domainprokaryotic P 4HsBaEFTu 9- mer peptide
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