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 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.

AToxoplasmaProlyl Hydroxylase Mediates Oxygen Stress Responses by Regulating Translation Elongation

“…1). The tree was rooted with a clade of bacterial PHDs (group C) that prolyl hydroxylate elongation factor Tu (12, 19), because their more divergent sequences are potentially ancestral to the other genes. The sequences organize into two broad additional groups, A and B, which likely evolved from a primordial gene duplication since some species are represented in both groups.…”

Routes towards Novel Collagen-Like Biomaterials

“…Nevertheless, E. coli has no intrinsic apparatus to catalyze the aforementioned secondary modifications, which therefore result in unstable products, mostly due to the lack of 4-hydroxyproline. A newly discovered bacterial P4H found in Bacillus anthracis [56,57], might be able to overcome this limitation in future experiments. In addition, recent advances have been made in coexpressing eukaryotic proline-4-hydroxylase in origami-type E. coli [58], which provides conditions similar to that of the endoplasmic reticulum (ER) within its cytosol and therefore allow the activity of the transgenic P4H-complex, as well as the yeast P. pastoris [59,60] which, being an eukaryote, has the necessary organelles for early-stage collagen assembly and the ability to produce high MW proteins, although only with poor yields and varying degrees of hydroxylation [61,62].…”

The Hydroxyproline Proteome of HeLa Cells with Emphasis on the Active Sites of Protein Disulfide Isomerases

“…Post-translational modification (PTM) of prolines in proteins is known to occur in bacteria 1 and eukarya, but not archaea. 2 All known proline modifications are catalyzed by 2oxoglutarate dioxygenases (OG dioxygenases).…”