Understanding How Prolyl-4-hydroxylase Structure Steers a Ferryl Oxidant toward Scission of a Strong C−H Bond

Amy Timmins, Maud Saint-Andre, Samuel De Visser

    Research output: Contribution to journalArticlepeer-review


    Prolyl-4-hydroxylase (P4H) is a nonheme iron hydroxylase that regio- and stereospecifically hydroxylates proline residues in a peptide chain into R-4-hydroxyproline, which is essential for collagen crosslinking purposes in the human body. Surprisingly, in P4H a strong aliphatic C–H bond is activated, while thermodynamically much weaker aliphatic C–H groups, i.e. at the C3 and C5 positions, are untouched. Little is known on the origins of the high regio- and stereoselectivity of P4H and many nonheme and heme enzymes in general and insight into this matter may be relevant to Biotechnology as well as Drug Development. The active site of the protein contains two aromatic residues (Tyr140 and Trp243) we expected to be crucial for guiding the regioselectivity of the reaction. We performed a detailed quantum mechanics/molecular me-chanics (QM/MM) and molecular dynamics (MD) study on wild-type and mutant structures. The work shows that, Trp243 is involved in key protein loop-loop interactions that affect the shape and size of the substrate binding pocket and its mutation has major long-range effects. By contrast, the Tyr140 residue guides the regio- and stereoselectivity by holding the substrate and ferryl oxidant in a specific orientation through hydrogen bonding and -stacking interactions. Compelling evidence is found that the Tyr140 residue is involved in expelling the product from the binding pocket after the reaction is complete. It is shown that mutations where the hydrogen bonding network that involves the Tyr140 and Trp243 residues is disrupted leads to major changes in folding of the protein and the size and shape of the substrate binding pocket. Specifically, the Trp243 resi-due positions the amino acid side chains of Arg161 and Glu127 in specific orientations with substrate. As such, the P4H en-zyme is a carefully designed protein with a subtle and rigid secondary structure that enables the binding of substrate, guides the regioselectivity and expels product efficiently.
    Original languageEnglish
    Pages (from-to)9855-9866
    Number of pages12
    JournalJournal of the American Chemical Society
    Early online date28 Jun 2017
    Publication statusPublished - 26 Jul 2017

    Research Beacons, Institutes and Platforms

    • Manchester Institute of Biotechnology


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