Abstract
Aspartate/asparagine‐β‐hydroxylase (AspH) is a human 2‐oxoglutarate (2OG) and FeII oxygenase that catalyses C3 hydroxylations of aspartate/asparagine residues of epidermal growth factor‐like domains (EGFDs). Unusually, AspH employs two histidine residues to chelate FeII 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 FeII 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.
Highlights
Amelia Brasnett+, Inga Pfeffer+, Lennart Brewitz+, Rasheduzzaman Chowdhury, Yu Nakashima, Anthony Tumber, Michael A
It is related to the 2OG dependent halogenases, where the absence of the Asp/Glu residue of the HXD/E...H motif is used to enable FeII halide binding.[1b,4] Here we report the unexpected observation that AspH variants with a single FeII histidine ligand retain substantial catalytic activity
Apparent turnover numbers, apparent Michaelis constants (Kampp), and specificity constants of wt, H679A, and H725A His6-AspH315-758 determined for FeII, 2OG, and the hFX-EGFD186-124-4Ser substrate peptide.[a,b]
Summary
2-Oxoglutarate (2OG) and FeII dependent oxygenase superfamily enzymes are widely distributed and catalyse a range of oxidations, typically hydroxylations, though they catalyse other reactions including hydrogenations, desaturations, fragmentations, and ring formations.[1] In humans, they have important physiological roles, for example in the hypoxic response and epigenetics.[2] Most, but not all, of their reactions comprise two electron substrate oxidations coupled to conversion of 2OG to succinate and CO2. They have a conserved distorted double-stranded b-helix core fold which supports an active site with a single FeII. Apparent turnover numbers (kacaptp), apparent Michaelis constants (Kampp), and specificity constants (kcat/Km) of wt, H679A, and H725A His6-AspH315-758 determined for FeII, 2OG, and the hFX-EGFD186-124-4Ser substrate peptide.[a,b]
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