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<p>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 <i>R</i>)-hydroxylated EGFDs with the product alcohol coordinating Fe(II) <i>trans</i> 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) <i>trans</i> 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.</p>

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Structural basis of the promiscuity of the unusual Fe(II) and 2-oxoglutarate dependent human aspartate/asparagine-β-hydroxylase

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月25日

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  • 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

摘要

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 O2 surrogate nitric oxide imply O2 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 O2 is the sole oxygen source in alcohol products, as shown by 18O labelling studies. The results reveal how AspH accommodates both aspartate- and asparagine-substrates and will assist in efforts targeting AspH for cancer treatment.