6N20
Structure of L509V CAO1 - growth condition 2
Summary for 6N20
| Entry DOI | 10.2210/pdb6n20/pdb |
| Descriptor | Carotenoid oxygenase, FE (II) ION, CHLORIDE ION, ... (4 entities in total) |
| Functional Keywords | dioxygenase, non-heme iron protein, beta-propeller, oxidoreductase |
| Biological source | Neurospora crassa |
| Total number of polymer chains | 4 |
| Total formula weight | 238300.83 |
| Authors | Khadka, N.,Shi, W.,Kiser, P.D. (deposition date: 2018-11-12, release date: 2019-06-05, Last modification date: 2023-10-11) |
| Primary citation | Khadka, N.,Farquhar, E.R.,Hill, H.E.,Shi, W.,von Lintig, J.,Kiser, P.D. Evidence for distinct rate-limiting steps in the cleavage of alkenes by carotenoid cleavage dioxygenases. J.Biol.Chem., 294:10596-10606, 2019 Cited by PubMed Abstract: Carotenoid cleavage dioxygenases (CCDs) use a nonheme Fe(II) cofactor to split alkene bonds of carotenoid and stilbenoid substrates. The iron centers of CCDs are typically five-coordinate in their resting states, with solvent occupying an exchangeable site. The involvement of this iron-bound solvent in CCD catalysis has not been experimentally addressed, but computational studies suggest two possible roles. 1) Solvent dissociation provides a coordination site for O, or 2) solvent remains bound to iron but changes its equilibrium position to allow O binding and potentially acts as a proton source. To test these predictions, we investigated isotope effects (HO DO) on two stilbenoid-cleaving CCDs, oxygenase 2 (NOV2) and carotenoid oxygenase 1 (CAO1), using piceatannol as a substrate. NOV2 exhibited an inverse isotope effect (/ ∼ 0.6) in an air-saturated buffer, suggesting that solvent dissociates from iron during the catalytic cycle. By contrast, CAO1 displayed a normal isotope effect (/ ∼ 1.7), suggesting proton transfer in the rate-limiting step. X-ray absorption spectroscopy on NOV2 and CAO1 indicated that the protonation states of the iron ligands are unchanged within pH 6.5-8.5 and that the Fe(II)-aquo bond is minimally altered by substrate binding. We pinpointed the origin of the differential kinetic behaviors of NOV2 and CAO1 to a single amino acid difference near the solvent-binding site of iron, and X-ray crystallography revealed that the substitution alters binding of diffusible ligands to the iron center. We conclude that solvent-iron dissociation and proton transfer are both associated with the CCD catalytic mechanism. PubMed: 31138651DOI: 10.1074/jbc.RA119.007535 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.95 Å) |
Structure validation
Download full validation report
