Structural basis of the promiscuity of the unusual Fe(II) and 2-oxoglutarate dependent human aspartate/asparagine-β-hydroxylase
- Mariska de Munnik
- Amelia Brasnett
- Tiankun Zhou
- William Myers
- Yicheng Wang
- Kuntal Chatterjee
- Anthony Tumber
- Stephen A. Marshall
- Philipp S. Simon
- Pierre Aller
- Anastasiia Shilova
- Danny Axford
- Hiroki Makita
- Daniel W. Paley
- Vandana Tiwari
- Alexander T. Stead
- Sebastian Dehe
- Humberto Sanchez
- Daniel J. Rosenberg
- Roberto Alonso-Mori
- Asmit Bhowmick
- Junko Yano
- Vittal K. Yachandra
- Jaehyun Park
- Sehan Park
- Allen M. Orville
- Lennart Brewitz
- Jan F. Kern
- Christopher J. Schofield
- Patrick Rabe
2026-02-25
Protein-hydroxylation catalysed by Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases is an important regulatory mechanism in human biology. Such oxygenases typically coordinate their Fe(II) cofactor via a conserved triad of an aspartate- or glutamate- and two histidine-residues. By contrast, aspartate/asparagine β-hydroxylase (AspH), which catalyses asparagine/aspartate-residue oxidation in epidermal growth factor-like domains (EGFDs), has only two histidine-residues (H679, H725), with a water occupying the site normally occupied by an aspartate- or glutamate-residue. We describe mechanistic studies with catalytically active AspH crystals. Turnover studies with single crystals under cryogenic conditions give (3 R )-hydroxylated EGFDs with the product alcohol coordinating Fe(II) trans to H725. Time-resolved serial crystallography of microcrystals using an acoustic droplet ejection system, coupled to X-ray emission analyses, demonstrate turnover within 1.5 s, giving a product complex in which Fe(II) is regenerated. Solution and crystallographic studies with the O 2 surrogate nitric oxide imply O 2 binds to Fe(II) trans to H725. The additional Fe-chelating water is maintained throughout AspH catalysis and is not directly involved in substrate hydroxylation, because O 2 is the sole oxygen source in alcohol products, as shown by 18 O labelling studies. The results reveal how AspH accommodates both aspartate- and asparagine-substrates and will assist in efforts targeting AspH for cancer treatment.