Increased Turnover at Limiting O₂ Concentrations by the Thr³⁸⁷ → Ala Variant of HIF-Prolyl Hydroxylase PHD2

Abstract

PHD2 is a 2-oxoglutarate, non-heme Fe2+ dependent oxygenase that senses O2 levels in human cells by hydroxylating two prolyl residues in the oxygen dependent degradation domain (ODD) of HIF1α. Identifying the active site contacts that determine the rate of reaction under limiting O2 is crucial for understanding how these enzymes sense pO2, and may suggest methods for chemically altering hypoxia responses. A hydrogen bonding network extends from the Fe(II) cofactor through ordered waters to the Thr387 residue in the second coordination sphere. Here we tested the impact of the sidechain of Thr387 on the reactivity of PHD2 toward O2 through a combination of point mutagenesis, steady state kinetic experiments and {FeNO}7 EPR spectroscopy. The steady state kinetic parameters for Thr387→Asn were very similar to those of WT-PHD2, but kcat and kcat/KM(O2) for Thr387→Ala were increased by roughly 15-fold. X-band EPR spectroscopy of the {FeNO}7 centers of the (Fe+NO+2OG) enzyme forms showed the presence of a more rhombic line shape in Thr387→Ala than seen for WT-PHD2, indicating an altered conformation for bound gas in this variant. Here we show that the sidechain of residue Thr387 plays a significant role in determining the rate of turnover by PHD2 at low [O2].

Biochemical and biophysical analyses of hypoxia sensing prolyl hydroxylases from Dictyostelium discoideum and Toxoplasma gondii

“…The presence of electron density for a well-defined water molecule ligating trans to the proximal histidine (His 191 ) of the metal binding triad is significant. The unusually slow reaction of PHD2 and other animal PHDs including TaPHD, with dioxygen is proposed to enable their hypoxia sensing roles ( 56 , 64 , 65 , 66 , 67 ). Controlled reaction of the PHDs with dioxygen is proposed to be achieved via tightly regulated translocation of dioxygen from the protein surface to the Fe(II), leading to displacement of a metal ligating water ( 65 ).…”

Pharmacological targeting of the HIF hydroxylases – A new field in medicine development

“…Notably, in the case of the PHDs, the Fe (IV) =O intermediate has not been observed spectroscopically, consistent with hydroxylation of the HIF-α substrate by this intermediate not being rate limiting in PHD catalysis (Flashman et al, 2010;Tarhonskaya et al, 2015). In contrast for PHD2, there is evidence that binding of oxygen to the 2OG substrate complex coupled to displacement of a metal bound water in the enzyme-substrate complex is rate limiting in catalysis (Flagg et al, 2012;Flashman et al, 2010;Pektas et al, 2015;Tarhonskaya et al, 2014a;Tarhonskaya et al, 2015), a property consistent with the oxygen sensing role of the PHDs. Another distinguishing feature of PHD2 is that it forms an unusually stable enzyme Fe(II) 2OG complex (McNeill et al, 2005b).…”

Gas tunnel engineering of prolyl hydroxylase reprograms hypoxia signaling in cells

“…[40] Lastly, T387 was also excluded as it forms an H-bond with iron’s aqua ligand and mutations to this residue can change the modality of O 2 binding to iron. [41]…”

“…Previous studies have reported the K M(O2) of PHD2 to lie in the 400-600 μM range, 6,33 which is much higher than analogues enzymes ( Table S2 ). PHD2’s K M(O2) is tuned to such high values so the enzyme can differentiate between physoxia and hypoxia.…”