Structural basis of the promiscuity of the unusual Fe(II) and 2-oxoglutarate dependent human aspartate/asparagine-β-hydroxylase
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<sub>2</sub> surrogate nitric oxide imply O<sub>2</sub> 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<sub>2</sub> is the sole oxygen source in alcohol products, as shown by <sup>18</sup>O labelling studies. The results reveal how AspH accommodates both aspartate- and asparagine-substrates and will assist in efforts targeting AspH for cancer treatment.
Authors
- Tiankun Zhou (ORCID: https://orcid.org/0000-0002-6116-7483)
- Patrick Rabe (ORCID: https://orcid.org/0000-0002-3448-9559)
- Hiroki Makita (ORCID: https://orcid.org/0000-0002-4968-914X)
- Amelia Brasnett
- Allen Milster Orville (ORCID: https://orcid.org/0000-0002-7803-1777)
- Humberto S. Sanchez (ORCID: https://orcid.org/0000-0003-3298-6348)
- Daniel J. Rosenberg (ORCID: https://orcid.org/0000-0001-8017-8156)
- Lennart Brewitz (ORCID: https://orcid.org/0000-0002-9465-777X)
- Pierre Aller (ORCID: https://orcid.org/0000-0002-1793-7030)
- Stephen Marshall (ORCID: https://orcid.org/0000-0001-7678-4989)
- Sehan Park (ORCID: https://orcid.org/0000-0002-8627-9078)
- Vittal K. Yachandra (ORCID: https://orcid.org/0000-0002-3983-7858)
- Mariska de Munnik (ORCID: https://orcid.org/0000-0003-0350-395X)
- Vandana Tiwari (ORCID: https://orcid.org/0000-0002-6865-1649)
- Danny Axford (ORCID: https://orcid.org/0000-0001-7694-8525)
- Tumber Anthony
- Roberto Alonso‐Mori (ORCID: https://orcid.org/0000-0002-5357-0934)
- Philipp S. Simon (ORCID: https://orcid.org/0000-0002-2859-4475)
- Asmit Bhowmick (ORCID: https://orcid.org/0000-0002-6154-6635)
- Daniel W. Paley⧓ (ORCID: https://orcid.org/0000-0003-1161-5142)
- Jaehyun Park (ORCID: https://orcid.org/0000-0002-9007-4892)
- Jan Kern
- Sebastian Dehe
- Junko Yano
- Alexander T. Stead (ORCID: https://orcid.org/0000-0001-9784-8216)
- Kuntal Chatterjee
- Yicheng Wang
- Christopher J. Schofield
Institutions
- Pohang University of Science and Technology (KR)
- Lawrence Berkeley National Laboratory (US)
- SLAC National Accelerator Laboratory (US)
- Diamond Light Source (GB)
- University of Oxford (GB)
- Research Complex at Harwell (GB)
- Linac Coherent Light Source (US)
- Lam Research (United States) (US)
Publication Details
- Journal
- Nature Communications
- Published
- 2026-02-25
- DOI
- https://doi.org/10.1038/s41467-026-69425-w
- Primary Topic
- Metal-Catalyzed Oxygenation Mechanisms
- Type
- article
- Field-Weighted Citation Impact
- 0.00