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

Abstract

Prolyl-4-hydroxylase (P4H) is a non-heme iron hydroxylase that regio- and stereospecifically hydroxylates proline residues in a peptide chain into R-4-hydroxyproline, which is essential for collagen cross-linking purposes in the human body. Surprisingly, in P4H, a strong aliphatic C-H bond is activated, while thermodynamically much weaker aliphatic C-H groups, that is, at the C and C positions, are untouched. Little is known on the origins of the high regio- and stereoselectivity of P4H and many non-heme 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 (Tyr and Trp) that we expected to be crucial for guiding the regioselectivity of the reaction. We performed a detailed quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) study on wild-type and mutant structures. The work shows that Trp 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 Tyr residue is shown to guide the regio- and stereoselectivity by holding the substrate and ferryl oxidant in a specific orientation through hydrogen bonding and π-stacking interactions. Compelling evidence is found that the Tyr 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 Tyr and Trp residues is disrupted lead to major changes in folding of the protein and the size and shape of the substrate binding pocket. Specifically, the Trp residue positions the amino acid side chains of Arg and Glu in specific orientations with substrate. As such, the P4H enzyme 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.

Catalytic Mechanism of Nogalamycin Monoxygenase: How Does Nature Synthesize Antibiotics without a Metal Cofactor?

“…46,47 Thereafter, dioxygen was placed in a position near the active site Asn residues. A standard procedure was followed for the subsequent set-up of the system, as described in more detail elsewhere, [38][39][40][48][49][50][51] and briefly summarized here. The enzyme model was set up using procedures proposed by Thiel et al 52,53 The substrate bound enzyme structure with dioxygen bound was protonated to pH 7 with the PROPKA method using the PDB2PQR web service, 54 whereby all Glu and Asp side chains were deprotonated and all Arg and Lys side chains protonated.…”

Selective Formation of an FeIVO or an FeIIIOOH Intermediate From Iron(II) and H2O2: Controlled Heterolytic versus Homolytic Oxygen–Oxygen Bond Cleavage by the Second Coordination Sphere

“…With all methods, apart from BP86, the triplet 1 IV -O is the ground state in agreement with Mçssbauer data reported above.D espite these fluctuations in spin-state energies,p revious studies of ours;h owever,s howed that changing the density functional method generally does not change regio-and stereo-selectivities of ac hemical reaction but only affects the relative energies. [38,39] In the triplet and quintet spin states, 1 II -H 2 O 2 -a and 1 II -H 2 O 2 -b are close in energy with as mall preference of the latter at B3LYP level of theory.W ell higher in energy than these quintet spin H 2 O 2 complexes are the triplet spin states., Therefore,the triplet spin 3 1 II -H 2 O 2 will not play arole in the reaction mechanism leading to the formation of the Fe IV -oxo.…”

Second‐Coordination Sphere Effects on Selectivity and Specificity of Heme and Nonheme Iron Enzymes

“…For example, prolyl‐4‐hydroxylase is an α‐ketoglutarate dependent nonheme iron dioxygenase that catalyzes the biosynthesis of (2 S ,4 R )‐4‐hydroxyproline from proline; an important component of collagen in the skin . Computational modeling showed that thermodynamically C 5 ‐hydroxylation of proline should be the energetically favorable pathway, yet this product is not formed by this enzyme . Similarly, the nonheme iron dioxygenase AsqJ performs two steps in the biosynthesis process of 4′‐methoxy‐viridicatin; one involving a desaturation and the other an epoxidation reaction .…”

“…Thermodynamically, however, the weakest C−H bond of proline is at the C 5 position and hence, the question is how the protein can block a low energy hydrogen atom abstraction pathway from the C 5 ‐H position and drive the reaction to the thermodynamically unfavorable C 4 ‐hydroxylation pathway instead. To understand how P4H avoids hydrogen atom abstraction from the C 5 ‐H position in favor of C 4 ‐H hydroxylation, a series of detailed computational studies were performed …”