7T8Q

CRYSTAL STRUCTURE OF T151G CAO1

Summary for 7T8Q

• Entry DOI: 10.2210/pdb7t8q/pdb
• Descriptor: Carotenoid oxygenase 1, FE (II) ION, CHLORIDE ION, ... (4 entities in total)
• Functional Keywords: non-heme iron, beta propeller, stilbene, dioxygenase, oxidoreductase
• Biological source: Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987)
• Total number of polymer chains: 4
• Total formula weight: 238393.46

Authors

Daruwalla, A.,Kiser, P.D. (deposition date: 2021-12-16, release date: 2023-07-05, 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 (2.15 Å)