8FU2

Crystal structure of T151G CAO1 in complex with piceatannol

Summary for 8FU2

• Entry DOI: 10.2210/pdb8fu2/pdb
• Related: 7T8Q
• Descriptor: Carotenoid oxygenase, FE (II) ION, PICEATANNOL, ... (5 entities in total)
• Functional Keywords: non-heme iron, beta propeller, stilbene, dioxygenase, oxidoreductase, oxidoreductase-substrate complex, oxidoreductase/substrate
• Biological source: Neurospora crassa
• Total number of polymer chains: 4
• Total formula weight: 240725.60

Authors

Kiser, P.D. (deposition date: 2023-01-16, release date: 2024-08-07, Last modification date: 2025-04-09)

Primary citation

DeWeese, D.E.,Everett, M.P.,Babicz Jr., J.T.,Daruwalla, A.,Solomon, E.I.,Kiser, P.D.
Spectroscopy and crystallography define carotenoid oxygenases as a new subclass of mononuclear non-heme Fe II enzymes.
J.Biol.Chem., :108444-108444, 2025

PubMed Abstract: Carotenoid cleavage dioxygenases (CCDs) are non-heme Fe enzymes that catalyze the oxidative cleavage of alkene bonds in carotenoids, stilbenoids, and related compounds. How these enzymes control the reaction of O with their alkene substrates is unclear. Here, we apply spectroscopy in conjunction with X-ray crystallography to define the iron coordination geometry of a model CCD, CAO1, in its resting state and following substrate binding and coordination sphere substitutions. Resting CAO1 exhibits a five-coordinate (5C), square pyramidal Fe center that undergoes steric distortion towards a trigonal bipyramidal geometry in the presence of piceatannol. Titrations with the O-analog, nitric oxide (NO), show a >100-fold increase in iron-NO affinity upon substrate binding, defining a crucial role for the substrate in activating the Fe site for O reactivity. The importance of the 5C Fe structure for reactivity was probed through mutagenesis of the second-sphere Thr151 residue of CAO1, which occludes ligand binding at the sixth coordination position. A T151G substitution resulted in the conversion of the iron center to a six-coordinate (6C) state and a 135-fold reduction in apparent catalytic efficiency towards piceatannol compared to the wild-type enzyme. Substrate complexation resulted in partial 6C to 5C conversion, indicating solvent dissociation from the iron center. Additional substitutions at this site demonstrated a general functional importance of the occluding residue within the CCD superfamily. Taken together, these data suggest an ordered mechanism of CCD catalysis occurring via substrate-promoted solvent replacement by O. CCDs thus represent a new class of mononuclear non-heme Fe enzymes.

PubMed: 40147775
DOI: 10.1016/j.jbc.2025.108444

Experimental method

X-RAY DIFFRACTION (1.9 Å)