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

Publication Type:

Journal Article

Source:

J Inorg Biochem, Volume 166, p.26-33 (2017)

Keywords:

Amino Acid Substitution, Asparagine, Catalysis, Catalytic Domain, Crystallography, X-Ray, Humans, Hydroxylation, Mixed Function Oxygenases, Mutation, Missense, Oxygen, Repressor Proteins

Abstract:

<p>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 O via oxidative decarboxylation of α-ketoglutarate (αKG) to generate an enzyme-based oxidant which hydroxylates the Asn 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 O activation occurs after substrate binds. We tested the hypothesis that the facial triad carboxylate (Asp) of FIH linked substrate binding and O binding sites. Asp 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 O with a catalytic efficiency similar to that of wild-type (WT) FIH (kaK=0.17μMmin), but led to defects in the coupling of O activation to substrate hydroxylation. Steady-state kinetics showed similar catalytic efficiencies for hydroxylation by WT-FIH (k/K=0.42μMmin) and D201G (k/K=0.34μMmin); 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 O binding order. Our data support a model in which the facial triad carboxylate Asp provides both steric and polar contacts to favor O access to the Fe(II) only after substrate binds, leading to coupled turnover in FIH and other αKG oxygenases.</p>

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PILATUS
Beamline: 
24-ID-C