The facial triad in the α-ketoglutarate dependent oxygenase FIH: A role for sterics in linking substrate binding to O2 activation

Abstract

The factor inhibiting hypoxia inducible factor-1α (FIH) is a nonheme Fe(II)/αKG oxygenase using a 2-His-1-Asp facial triad. FIH activates O2 via oxidative decarboxylation of α-ketoglutarate (αKG) to generate an enzyme-based oxidant which hydroxylates the Asn803 residue within the C-terminal transactivation domain (CTAD) of HIF-1α. Tight coupling of these two sequential reactions requires a structural linkage between the Fe(II) and the substrate binding site to ensure that O2 activation occurs after substrate binds. We tested the hypothesis that the facial triad carboxylate (Asp201) of FIH linked substrate binding and O2 binding sites. Asp201 variants of FIH were constructed and thoroughly characterized in vitro using steady-state kinetics, crystallography, autohydroxylation, and coupling measurements. Our studies revealed each variant activated O2 with a catalytic efficiency similar to that of wild-type (WT) FIH (kcat/KM(O2) = 0.17 μM−1 min−1), but led to defects in the coupling of O2 activation to substrate hydroxylation. Steady-state kinetics showed similar catalytic efficiencies for hydroxylation by WT-FIH (kcat/KM(CTAD) = 0.42 μM−1 min−1) and D201G (kcat/KM(CTAD) = 0.34 μM−1 min−1); hydroxylation by D201E was greatly impaired, while hydroxylation by D201A was undetectable. Analysis of the crystal structure of the D201E variant revealed steric crowding near the diffusible ligand site supporting a role for sterics from the facial triad carboxylate in the O2 binding order. Our data support a model in which the facial triad carboxylate Asp201 provides both steric and polar contacts to favor O2 access to the Fe(II) only after substrate binds, leading to coupled turnover in FIH and other αKG oxygenases.

Born to sense: biophysical analyses of the oxygen sensing prolyl hydroxylase from the simplest animal <em>Trichoplax adhaerens</em>

“…In the Ta PHD.Mn structure without Ta ODD, the manganese ion is octahedrally coordinated by a conserved triad of residues (H209 Ta PHD , D211 Ta PHD , and H270 Ta PHD ), an acetate ion, and two water molecules. 48 , 51 Deep within the 2OG binding pocket an additional acetate ion forms a salt bridge with R279 Ta PHD , apparently mimicking 2OG C5 carboxylate binding ( Figure 3A ).…”

Chloride Supports O2 Activation in the D201G Facial Triad Variant of Factor-Inhibiting Hypoxia Inducible Factor, an α-Ketoglutarate Dependent Oxygenase

“…Notably, the conservative Asp201→ Glu variant, which bound the Fe by a His 2 Glu facial triad, exhibited anion independent hydroxylation (Figure 1A) albeit at roughly 10% the rate of WT-FIH. 20…”

O2 Activation by Nonheme FeII α-Ketoglutarate-Dependent Enzyme Variants: Elucidating the Role of the Facial Triad Carboxylate in FIH

“…From crystallography performed on this variant, the presence of the extra methyl group on the carboxylate affects its orientation relative to the Fe II center such that it has a steric clash between its unbound O and the position where the sixth water ligand would bind, resulting in the 5C site with a monodentate carboxylate or a 6C site with a very weakly bound distal O of the glutamate residue. 20 From computational studies on wt , a similar steric clash between asparagine on the CAD substrate and the water ligand bound to the Fe II center leads to H 2 O dissociation and an open coordination position for O 2 reactivity. 29 Coupled with the MCD results on the (Fe II /αKG)D201E site, the steric interaction with the sixth water ligand is an important factor along with the donation of the αKG in determining the 6C → 5C conversion in the FIH active site.…”

“…Previous studies of these enzyme variants focused on substrate hydroxylation and not coordination changes at the Fe II center. 20 However, these variants reveal perturbations that provide mechanistic insight into the role of the carboxylate in controlling the Fe II site, thus the timing of O 2 activation. In this study, we have used magnetic circular dichroism (MCD) spectroscopy to define the coordination number and geometry of the different D201X-Fe II variants and their interaction with αKG and CAD cosubstrates and correlated these structural changes with O 2 consumption kinetics to characterize the role of the facial triad carboxylate in O 2 activation for enzyme function.…”