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

ABSTRACT: The factor inhibiting hypoxia inducible factor-1? (FIH) is a nonheme Fe(II)/?KG oxygenase using a 2-His-1-Asp facial triad. FIH activates O2 via oxidative decarboxylation of ?-ketoglutarate (?KG) to generate an enzyme-based oxidant which hydroxylates the Asn803 residue within the C-terminal transactivation domain (CTAD) of HIF-1?. 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 (kcataKM(O2)=0.17?M-1min-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?M-1min-1) and D201G (kcat/KM(CTAD)=0.34?M-1min-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 ?KG oxygenases.

SUBMITTER: Hangasky JA 

PROVIDER: S-EPMC5161613 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature