Human Oxygenase Variants Employing a Single Protein FeII Ligand Are Catalytically Active

Abstract

Aspartate/asparagine-β-hydroxylase (AspH) is a human 2-oxoglutarate (2OG) and Fe^II oxygenase that catalyses C3 hydroxylations of aspartate/asparagine residues of epidermal growth factor-like domains (EGFDs). Unusually, AspH employs two histidine residues to chelate Fe^II rather than the typical triad of two histidine and one glutamate/aspartate residue. We report kinetic, inhibition, and crystallographic studies concerning human AspH variants in which either of its Fe^II binding histidine residues are substituted for alanine. Both the H725A and, in particular, the H679A AspH variants retain substantial catalytic activity. Crystal structures clearly reveal metal-ligation by only a single protein histidine ligand. The results have implications for the functional assignment of 2OG oxygenases and for the design of non-protein biomimetic catalysts.

Keywords: 2-oxoglutarate dependent oxygenase; aspartate/asparagine-β-hydroxylase; biomimetic catalysis; facial triad; metallo-enzymes.

Figure 1

AspH catalyses a typical 2OG oxygenase reaction but has an atypical Fe^II binding mode, employing two rather than the typical three protein residues. a) The AspH reaction; b) view of the AspH Fe^II binding site (H679 and H725 complex the active site metal ion with Mn substituting for Fe; PDB: 6YYW) ^[8] compared with that of c) a human 2OG oxygenase with an Fe^II binding triad (i.e. H199, H279, and D201; PDB: 1H2L), ^[9] that is, the asparagine residue hydroxylase, factor inhibiting hypoxia‐inducible transcription factor HIF‐α (FIH).

Figure 2

Crystal structure views of the H679A and H725A AspH variants. Colors: grey: His₆‐AspH_315‐758 variants; yellow: carbon‐backbone of N‐oxalylglycine (NOG); green: carbon‐backbone of acetate; orange: carbon‐backbone of formate; slate blue: carbon‐backbone of propionate; violet: Mn; magenta: carbon‐backbone of the hFX‐EGFD1_86‐124‐4Ser peptide (Supporting Figure S2a); red: oxygen; blue: nitrogen. w: water. a–d) Representative OMIT electron density maps (mF_o‐DF_c) contoured to 3.0σ around key residues and the a) acetate in the H679A AspH:acetate structure, b) formate in the H679A AspH:hFX‐EGFD1_86‐124‐4Ser structure, c) propionate in the H725A AspH:hFX‐EGFD1_86‐124‐4Ser structure (note: propionate adopts two conformations and the orientation of its carboxylate differs from the orientations of acetate and formate in a,b), and d) NOG in the H679A AspH:Mn:NOG:hFX‐EGFD1_86‐124‐4Ser structure; e) superimposition of views of the H679A AspH:hFX‐EGFD1_86‐124‐4Ser and H725A AspH:hFX‐EGFD1_86‐124‐4Ser structures (colours: pale yellow: H725A AspH; dark green: carbon‐backbone of the hFX‐EGFD1_86‐124‐4Ser peptide); f) superimposition of views of the H679A AspH:Mn:NOG:hFX‐EGFD1_86‐124‐4Ser structure and the reported wt AspH:Mn:NOG:hFX‐EGFD1_86‐124‐4Ser structure (colours: bronze: wt AspH; lavender blue: Mn; maroon: carbon‐backbone of NOG; cyan: carbon‐backbone of the hFX‐EGFD1_86‐124‐4Ser peptide; PDB ID: 5JQY). ^[7]

Figure 3

A single protein‐bound ligand (i.e. H725) supports the metal ion in the H679A AspH variant. Colors: red: oxygen; blue: nitrogen. w: water. a) Active site view of the H679A AspH:Mn:NOG:hFX‐EGFD1_86‐124‐4Ser structure (colors: grey: H679A AspH; violet: Mn; yellow: carbon‐backbone of NOG; magenta: carbon‐backbone of the hFX‐EGFD1_86‐124‐4Ser peptide); b) active site view of the reported wt AspH:Mn:NOG:hFX‐EGFD1_86‐124‐4Ser structure (colours: bronze: wt AspH; lavender blue: Mn; maroon: carbon‐backbone of NOG; cyan: carbon‐backbone of the hFX‐EGFD1_86‐124‐4Ser peptide; PDB ID: 5JQY). ^[7]