5KJD

Synechocystis apocarotenoid oxygenase (ACO) mutant - Glu150Gln

5KJD の概要

• エントリーDOI: 10.2210/pdb5kjd/pdb
• 関連するPDBエントリー: 5KJA 5KJB 5KK0
• 分子名称: Apocarotenoid-15,15'-oxygenase, FE (II) ION (3 entities in total)
• 機能のキーワード: iron-coordination, active site, carotenoid binding, ligand interaction, non-heme iron, mutagenesis, oxidoreductase
• 由来する生物種: Synechocystis sp. (strain PCC 6803 / Kazusa)
• タンパク質・核酸の鎖数: 5
• 化学式量合計: 271995.26

構造登録者

Sui, X.,Kiser, P.D.,Palczewski, K. (登録日: 2016-06-18, 公開日: 2016-08-03, 最終更新日: 2023-09-27)

主引用文献

Sui, X.,Zhang, J.,Golczak, M.,Palczewski, K.,Kiser, P.D.
Key Residues for Catalytic Function and Metal Coordination in a Carotenoid Cleavage Dioxygenase.
J.Biol.Chem., 291:19401-19412, 2016

PubMed Abstract: Carotenoid cleavage dioxygenases (CCDs) are non-heme iron-containing enzymes found in all domains of life that generate biologically important apocarotenoids. Prior studies have revealed a critical role for a conserved 4-His motif in forming the CCD iron center. By contrast, the roles of other active site residues in catalytic function, including maintenance of the stringent regio- and stereo-selective cleavage activity, typically exhibited by these enzymes have not been thoroughly investigated. Here, we examined the functional and structural importance of active site residues in an apocarotenoid-cleaving oxygenase (ACO) from Synechocystis Most active site substitutions variably lowered maximal catalytic activity without markedly affecting the Km value for the all-trans-8'-apocarotenol substrate. Native C15-C15' cleavage activity was retained in all ACO variants examined suggesting that multiple active site residues contribute to the enzyme's regioselectivity. Crystallographic analysis of a nearly inactive W149A-substituted ACO revealed marked disruption of the active site structure, including loss of iron coordination by His-238 apparently from an altered conformation of the conserved second sphere Glu-150 residue. Gln- and Asp-150-substituted versions of ACO further confirmed the structural/functional requirement for a Glu side chain at this position, which is homologous to Glu-148 in RPE65, a site in which substitution to Asp has been associated with loss of enzymatic function in Leber congenital amaurosis. The novel links shown here between ACO active site structure and catalytic activity could be broadly applicable to other CCD members and provide insights into the molecular pathogenesis of vision loss associated with an RPE65 point mutation.

PubMed: 27453555
DOI: 10.1074/jbc.M116.744912

実験手法

X-RAY DIFFRACTION (2.75 Å)