Studies on the Substrate Selectivity of the Hypoxia‐Inducible Factor Prolyl Hydroxylase 2 Catalytic Domain

Abstract

In animals, the response to chronic hypoxia is mediated by upregulation of the α,β-heterodimeric hypoxia-inducible factors (HIFs). Levels of HIFα isoforms, but not HIFβ, are regulated by their post-translational modification as catalysed by prolyl hydroxylase domain enzymes (PHDs). Different roles for the human HIF-1/2α isoforms and their two oxygen-dependent degradation domains (ODDs) are proposed. We report kinetic and NMR analyses of the ODD selectivity of the catalytic domain of wild-type PHD2 (which is conserved in nearly all animals) and clinically observed variants. Studies using Ala scanning and "hybrid" ODD peptides imply that the relatively rigid conformation of the (hydroxylated) proline plays an important role in ODD binding. They also reveal differential roles in binding for the residues on the N- and C-terminal sides of the substrate proline. The overall results indicate how the PHDs achieve selectivity for HIFα ODDs and might be of use in identifying substrate-selective PHD inhibitors.

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

“…S6B ). As reported ( 60 , 61 ), HsPHD2 catalyzed little 2OG turnover (<10% in 1 h) in the presence (or absence) of any of the Skp1 proteins ( Fig. S6C ).…”

“…Notably, HsSkp1 E147P induced the most efficient 2OG turnover with TgPhyA ( Figure S6b). As reported (60,61), HsPHD2 catalyzed little 2OG turnover (< 10 % in an hour) in the presence (or absence) of any of the Skp1 proteins ( Figure S6c). By contrast with HsPHD2 and DdPhyA, TgPhyA exhibited substantial 'uncoupled' 2OG turnover in the absence of substrate, with > 80 % 2OG turnover being observed by NMR within an hour, in the presence of L-ascorbate ( Figure S6d).…”

“…1 H excitation sculpting suppression NMR spectra were obtained using 64 scans. Changes in integration of the peaks of interest (the 2OG methylene triplet at 2.22 ppm and succinate methylene singlet at 2.18 ppm) were monitored and plotted, as reported ( 60 , 61 ). Data were processed using Bruker 3.1 software.…”

“…Assay mixtures contained 50 μ m 2OG, in the presence or absence of 50 μ m Skp1 (as indicated in the Supporting information ) buffered with 50 m m Tris-D 11 , pH 7.5, 10% H 2 O, 90% (v/v) D 2 O. Data were recorded with a Bruker AVIII 700 MHz NMR spectrometer equipped with a 5-mm inverse cryoprobe using 5-mm NMR tubes ( 60 , 61 ).…”

Carbon Monoxide is an Inhibitor of HIF Prolyl Hydroxylase Domain 2

“…It has been shown that the hydroxylation of either CODD or NODD may lead to the degradation of HIF1α [39,40] . Although NODD hydroxylation is more sensitive to changes in oxygen concentration than CODD hydroxylation, [41] kinetics and binding studies showed that CODD is the preferred substrate and is a stronger binder of PHDs than NODD, which is also supported by structural studies [42–45] …”