2026-10-09 スタンフォード大学
Structure of the human enzyme AspH: The enlarged view shows the enzyme’s iron-containing active site (orange sphere) bound to its protein substrate. Using time resolved X-ray crystallography, the researchers observed how AspH activates oxygen and hydroxylates proteins, providing new insight into a family of enzymes involved in oxygen sensing, development and cancer. | Patrick Rabe / University of Oxford and Greg Stewart / SLAC National Accelerator Laboratory
＜関連情報＞
- https://news.stanford.edu/stories/2026/10/x-rays-moelcular-movies-enzymes-cancer-penicillin
- https://www.nature.com/articles/s41929-026-01618-4
- https://www.nature.com/articles/s41467-026-69425-w
時間分解X線自由電子レーザー研究により、イソペニシリンNシンターゼ触媒反応中に予期せぬ中間体が同定された Unanticipated intermediates during isopenicillin N synthase catalysis identified by time-resolved X-ray free-electron laser studies
Patrick Rabe, Hiroki Makita, Rebecca Southwart, Midhun George Thomas, Mihails Kazaks, Shyam Basak, Tiankun Zhou, William K. Myers, Margaret D. Doyle, Philipp S. Simon, Shusaku Hayama, J. Frederick W. Mosselmans, Alexander T. Stead, Abraham O. Oluwole, Malkit Sami, Ian Clifton, Pierre Aller, Asmit Bhowmick, Anthony Tumber, Jos J. A. G. Kamps, Karina Calvopiña, Agata Butryn, Robert Bosman, Arjun Cheema, … Christopher J. Schofield
Nature Catalysis Published:09 October 2026
DOI:https://doi.org/10.1038/s41929-026-01618-4
Abstract
Biosynthesis of penicillin and cephalosporin antibiotics involves the oxidation of the tripeptide δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) to give the conformationally strained bicyclic penicillin ring system, as catalysed by the Fe(II)-dependent oxidase isopenicillin N synthase (IPNS). The structures of the precursors for the sequential β-lactam and thiazolidine ring forming steps in IPNS have remained elusive. Here we studied the reaction of anaerobic IPNS:Fe(II):ACV microcrystals with O2 using time-resolved serial femtosecond crystallography and X-ray emission spectroscopy. High-resolution structures reveal that ACV cysteinyl oxidation produces a thioaldehyde that unexpectedly does not coordinate the iron centre. The use of deuterium-labelled ACV enabled the observation of a monocyclic β-lactam with restoration of the Fe–S bond. Together with mutagenesis, spectroscopic and computational studies, the results support Fe(IV)-mediated penicillin formation involving active-site water molecules. These findings provide structural insight into IPNS catalysis, inform on the wider Fe(II) oxygenase superfamily and highlight the power of X-ray free-electron laser methods for resolving transient enzymatic intermediates.
特異なFe(II)および2-オキソグルタル酸依存性ヒトアスパラギン酸/アスパラギン-β-ヒドロキシラーゼの多機能性の構造的基盤 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, … Patrick Rabe
Nature Communications Published:25 February 2026
DOI:https://doi.org/10.1038/s41467-026-69425-w
Abstract
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.
