How Are Substrate Binding and Catalysis Affected by Mutating Glu127 and Arg161 in Prolyl-4-hydroxylase? A QM/MM and MD Study

Abstract

Prolyl-4-hydroxylase is a vital enzyme for human physiology involved in the biosynthesis of 4-hydroxyproline, an essential component for collagen formation. The enzyme performs a unique stereo- and regioselective hydroxylation at the C4 position of proline despite the fact that the C5 hydrogen atoms should be thermodynamically easier to abstract. To gain insight into the mechanism and find the origin of this regioselectivity, we have done a quantum mechanics/molecular mechanics (QM/MM) study on wildtype and mutant structures. In a previous study (Timmins et al., 2017) we identified several active site residues critical for substrate binding and positioning. In particular, the Glu127 and Arg161 were shown to form multiple hydrogen bonding and ion-dipole interactions with substrate and could thereby affect the regio- and stereoselectivity of the reaction. In this work, we decided to test that hypothesis and report a QM/MM and molecular dynamics (MD) study on prolyl-4-hydroxylase and several active site mutants where Glu127 or Arg161 are mutated for Asp, Gln, or Lys. Thus, the R161D and R161Q mutants give very high barriers for hydrogen atom abstraction from any proline C–H bond and therefore will be inactive. The R161K mutant, by contrast, sees the regio- and stereoselectivity of the reaction change but still is expected to hydroxylate proline at room temperature. By contrast, the Glu127 mutants E127D and E127Q show possible changes in regioselectivity with the former being more probable to react compared to the latter.

Does Substrate Positioning Affect the Selectivity and Reactivity in the Hectochlorin Biosynthesis Halogenase?

“…Thus, due to tight substrate binding and positioning, the hydrogen atom abstraction will be selective from only one of the two hydrogen atoms from the ω-1 position of the substrate. Hence the hydrogen atom abstraction will guide the enantioselectivity as seen before on related enzymes (Karamzadeh et al, 2010 ; Pratter et al, 2013 ; Timmins and de Visser, 2017 ; Timmins et al, 2017 ). Nevertheless, in HctB the first halogenation of substrate is followed by a second halogen transfer through the binding of another molecule of O 2 and Cl − to repeat the cycle and the formation of the dihalogenated product.…”

“…Furthermore, the methods reproduced experimental product distributions of bifurcation processes well (Ji et al, 2015 ; Kaczmarek et al, 2018 ). Finally, for the nonheme iron dioxygenase prolyl-4-hydroxylase six hydrogen atom abstraction barriers from substrate were investigated and the QM/MM predicted the correct regioselectivity and therefore the methods are expected to predict regio- and chemoselectivities well (Karamzadeh et al, 2010 ; Pratter et al, 2013 ; Timmins and de Visser, 2017 ; Timmins et al, 2017 ).…”

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.…”

A Comparative Review on the Catalytic Mechanism of Nonheme Iron Hydroxylases and Halogenases

“…Specifically, most NHFeHas require their substrate be tethered to a phosphopantetheine arm (PPT) associated with an acyl-carrier protein (ACP) [136,137], whereas the hydroxylases tend to have free substrates, although they are often bound tightly in a substrate binding pocket. For example, the prolyl-4-hydroxylase from Clamydomonas reinhardtii tightly binds the substrate through selective hydrogen bonding involving amino acids in two loops (β 3 -β 4 loop and the β II -β III ) which cover the substrate [138,139]. The only halogenase that forms an exception to this rule appears to be WelO5, whose recent discovery revealed that it did not require a PPT arm to deliver its substrate into the active site.…”

“…As mentioned above, the catalytic cycle of NHFeHys and NHFeHas is largely similar; however, post-hydrogen atom abstraction from the substrate, the systems bifurcate. In the NHFeHys, a hydroxyl radical rebounds, forming the hydroxylated product [138,139,148,149], in contrast to NHFeHas, whereby, a chloride radical relay gives the halogenated product. The factors related to bifurcation have been the subject of study for many years, ever since the discovery of NHFeHas, and has created a number of controversies and mechanistic possibilities.…”