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

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Prolyl hydroxylation is a key post-translational modification in eukaryotes, crucial for collagen stabilization and hypoxia sensing.
• Prokaryotic prolyl 4-hydroxylases (P4Hs) homologous to human HIF-PHDs are increasingly identified, targeting elongation factor Tu (EFTu) and potentially regulating bacterial translation.
• While most bacterial P4Hs modify free proline, some, like PPHD and BaP4H, act on peptides, indicating diverse substrate specificities.

Purpose of the Study:

• To investigate the substrate recognition and complex formation of Bacillus anthracis prolyl 4-hydroxylase (BaP4H) with elongation factor Tu (EFTu).
• To characterize the structural and functional similarities between BaP4H and eukaryotic P4Hs.
• To provide evidence for the promiscuous substrate recognition of BaP4H and its potential role in bacterial translation regulation.

Main Methods:

• Biophysical techniques and mass spectrometry were employed to study BaP4H-BaEFTu interactions.
• Size-exclusion chromatography coupled with small-angle X-ray scattering (SEC-SAXS) was used to analyze complex formation and structural changes.
• Binding studies were conducted to confirm substrate recognition.

Main Results:

• BaP4H was demonstrated to recognize and specifically hydroxylate both full-length BaEFTu and a BaEFTu-derived peptide.
• BaP4H forms a 1:1 heterodimeric complex with BaEFTu, with dissociation of BaP4H subunits observed upon binding.
• Structural and functional similarities between BaP4H and eukaryotic P4Hs were further supported, highlighting promiscuous substrate recognition.

Conclusions:

• BaP4H exhibits unusual substrate specificity within bacteria, acting on a universally conserved protein (EFTu) and showing structural/functional parallels with animal PHDs and collagen P4Hs.
• The findings suggest a potential conserved role for BaP4H in regulating bacterial translation, similar to the function of PHDs in eukaryotes.
• This study advances the understanding of bacterial post-translational modifications and enzyme evolution.