Abstract
A 2-His-1-carboxylate triad of iron binding residues is present in many non-heme iron oxygenases including the Fe(II) and 2-oxoglutarate (2OG)-dependent dioxygenases. Three variants (D201A, D201E, and D201G) of the iron binding Asp-201 residue of an asparaginyl hydroxylase, factor inhibiting HIF (FIH), were made and analyzed. FIH-D201A and FIH-D201E did not catalyze asparaginyl hydroxylation, but in the presence of a reducing agent, they displayed enhanced 2OG turnover when compared with wild-type FIH. Turnover of 2OG by FIH-D201A was significantly stimulated by the addition of HIF-1alpha(786-826) peptide. Like FIH-D201A and D201E, the D201G variant enhanced 2OG turnover but rather unexpectedly catalyzed asparaginyl hydroxylation. Crystal structures of the FIH-D201A and D201G variants in complex with Fe(II)/Zn(II), 2OG, and HIF-1alpha(786-826/788-806) implied that only two FIH-based residues (His-199 and His-279) are required for metal binding. The results indicate that variation of 2OG-dependent dioxygenase iron-ligating residues as a means of functional assignment should be treated with caution. The results are of mechanistic interest in the light of recent biochemical and structural analyses of non-heme iron and 2OG-dependent halogenases that are similar to the FIH-D201A/G variants in that they use only two His-residues to ligate iron.
Highlights
Hydroxylation is the most common reaction catalyzed by the 2OG oxygenases, but family members catalyze other oxidative reactions including demethylations, desaturations, epoxidations, and rearrangements
A structure of dimeric factor inhibiting hypoxiainducible factor (HIF) (FIH) in complex with Fe(II), 2OG, and HIF-1␣786–826 revealed that the sidechain carboxylate of FIH Asp-201 coordinates the iron via one oxygen, but that its other oxygen is positioned to accept a hydrogen bond from an Fe(II)-bound water molecule; upon substrate binding, the interaction of the water is apparently weakened because Asp-201 is repositioned to form a hydrogen bond with the backbone amide nitrogen of HIF-1␣ Asn-803 as observed in the crystal structures (Fig. 2) [17]
We report here that FIH-D201A and FIH-D201E do not catalyze HIF-1␣ hydroxylation, under appropriate conditions, these enzymes do catalyze enhanced 2OG turnover when compared with the wild-type FIH
Summary
The p300/CREB-binding protein co-activators [11,12,13] (for review, see Refs. 14 and 15). Studies on the iron cofactor binding residues of FIH have supported the assignment of FIH as a 2OG oxygenase and enabled identification of the iron binding residues as His-199, Asp-201, and His-279. Lando et al [12] demonstrated that substitution of either His-199 or Asp-201 to alanine residues ablated FIH activity in cells. Crystallographic analyses confirmed the assignment of the iron binding residues [16,17,18]. We report here that FIH-D201A and FIH-D201E do not catalyze HIF-1␣ hydroxylation, under appropriate conditions, these enzymes do catalyze enhanced 2OG turnover when compared with the wild-type FIH. The FIHD201G mutant catalyzes asparaginyl hydroxylation at comparable levels to wild-type FIH. Crystal structures of FIHD201A1⁄7Fe(II)1⁄72OG1⁄7HIF-1␣786–826 and D201G1⁄7Zn(II)1⁄72OG1⁄7 HIF-1␣788–806 reveal that the only FIH-based ligands required to bind iron are His-199 and His-279. Together with the recent studies on 2OG halogenases [6, 7] and spectroscopic analyses [4], our results indicate that there is more flexibility in the iron coordination chemistry of 2OG oxygenases than previously thought
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