Substrate preference of the HIF-prolyl hydroxylase-2 (PHD2) and substrate-induced conformational change

Abstract

HIF prolyl-4-hydroxylase 2 (PHD2) is a non-heme Fe, 2-oxoglutarate (2OG) dependent dioxygenase that regulates the hypoxia inducible transcription factor (HIF) by hydroxylating two conserved prolyl residues in N-terminal oxygen degradation domain (NODD) and C-terminal oxygen degradation domain (CODD) of HIF-1α. Prior studies have suggested that the substrate preference of PHD2 arises from binding contacts with the β2β3 loop of PHD2. In this study we tested the substrate selectivity of PHD2 by kinetic competition assays, varied ionic strength, and global protein flexibility using amide H/D exchange (HDX). Our results revealed that PHD2 preferred CODD by 20-fold over NODD and that electrostatics influenced this effect. Global HDX monitored by mass spectrometry indicated that binding of Fe(II) and 2OG stabilized the overall protein structure but the saturating concentrations of either NODD or CODD caused an identical change in protein flexibility. These observations imply that both substrates stabilize the β2β3 loop to the same extent. Under unsaturated substrate conditions NODD led to a higher HDX rate than CODD due to its lower binding affinity to PHD2. Our results suggest that loop closure is the dominant contributor to substrate selectivity in PHD2.

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

“…To test the effect of this contact on steps between 2OG binding and ODD binding, initial rate data were measured as a function of varied [2OG] using saturating concentrations of Fe 2+ (10 µM), CODD (10 µM), and ascorbate (1 mM); [O 2 ] was fixed at ambient levels (217 uM). WT-PHD2 exhibited simple saturation kinetics over the range of 1 – 50 µM 2OG, with values of k cat (2.3 min −1 ) and k cat /K M(2OG) (2.7 µM −1 min −1 ) similar to those previously reported 14,18,22–24 . Similarly, the conservative Thr 387 →Asn variant exhibited saturation kinetics over the tested concentration range, with modestly decreased rate constants relative to WT-PHD2 (Table 1).…”

Kinetic Investigations of the Role of Factor Inhibiting Hypoxia-inducible Factor (FIH) as an Oxygen Sensor

“…We also investigated the HIF substrate dependence of O 2 kinetics for both PHD2 and FIH. PHD2 has been shown to express a preference for hydroxylation of the CODD site, compared with NODD ( 46 , 61 ). However, we found in both pre-steady-state and steady-state experiments that O 2 -initiated reaction rates are independent of the nature of the prime substrate.…”

Oxygen sensing strategies in mammals and bacteria

“…Hydroxylation of either of two proline residues (Pro402, Pro564) in the ODD by PHD2 is a prerequisite of HIF-1α recognition by the von Hippel-Lindau protein (pVHL) (Fig. 4) [59], with hydroxylation at Pro564 being ten-fold faster than at Pro402 [60-62]. pVHL is the recognition component of an E3 ubiquitin-protein ligase which targets HIF-1α for rapid proteasomal degradation after successive rounds of ubiquitinylation [63].…”