Abstract
Proline hydroxylation is the most prevalent post-translational modification in collagen. The resulting product trans-4-hydroxyproline (Hyp) is of critical importance for the stability and thus function of collagen, with defects leading to several diseases. Prolyl 4-hydroxylases (P4Hs) are mononuclear non-heme iron α-ketoglutarate (αKG)-dependent dioxygenases that catalyze Hyp formation. Although animal and plant P4Hs target peptidyl proline, prokaryotes have been known to use free l-proline as a precursor to form Hyp. The P4H from Bacillus anthracis (BaP4H) has been postulated to act on peptidyl proline in collagen peptides, making it unusual within the bacterial clade, but its true physiological substrate remains enigmatic. Here we use mass spectrometry, fluorescence binding, x-ray crystallography, and docking experiments to confirm that BaP4H recognizes and acts on peptidyl substrates but not free l-proline, using elements characteristic of an Fe(II)/αKG-dependent dioxygenases. We further show that BaP4H can hydroxylate unique peptidyl proline sites in collagen-derived peptides with asymmetric hydroxylation patterns. The cofactor-bound crystal structures of BaP4H reveal active site conformational changes that define open and closed forms and mimic "ready" and "product-released" states of the enzyme in the catalytic cycle. These results help to clarify the role of BaP4H as well as provide broader insights into human collagen P4H and proteins with poly-l-proline type II helices.
Highlights
13360 JOURNAL OF BIOLOGICAL CHEMISTRY alents for these enzymes to reduce O2 to a hydroxyl group [2]
The high-resolution MS results demonstrate for the first time that Bacillus anthracis P4H (BaP4H) can hydroxylate proline residues in both X and Y positions of collagen-like (P-P-G)5 and (P-P-G)10 peptides
Preferential hydroxylation of a proline in the X position of (P-P-G)10 has been observed with CrP4H-1A [32], At-Prolyl 4-hydroxylases (P4Hs)-1 acts preferentially but not exclusively on proline in the Y position [31]
Summary
13360 JOURNAL OF BIOLOGICAL CHEMISTRY alents for these enzymes to reduce O2 to a hydroxyl group [2]. Attempts toward determining the crystal structure of a substrate-bound form of BaP4H has not been fully successful, molecular docking was used to simulate the binding mode of a collagen-like proline-rich (P-PG)5 peptide.
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