Key Residues for Catalytic Function and Metal Coordination in a Carotenoid Cleavage Dioxygenase
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 associatedmore »
- Authors:
-
- Case Western Reserve Univ., Cleveland, OH (United States). Dept. of Pharmacology. School of Medicine
- Case Western Reserve Univ., Cleveland, OH (United States). Dept. of Pharmacology. School of Medicine; Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, OH (United States). Research Service
- Publication Date:
- Research Org.:
- Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
- Sponsoring Org.:
- USDOE Office of Science (SC)
- OSTI Identifier:
- 1376249
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Journal of Biological Chemistry
- Additional Journal Information:
- Journal Volume: 291; Journal Issue: 37; Journal ID: ISSN 0021-9258
- Publisher:
- American Society for Biochemistry and Molecular Biology
- Country of Publication:
- United States
- Language:
- ENGLISH
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; enzyme mutation; metalloenzyme; rhodopsin; site-directed mutagenesis; x-ray crystallography; Leber congenital amaurosis; RPE65; non-heme iron; regioselectivity; retinal
Citation Formats
Sui, Xuewu, Zhang, Jianye, Golczak, Marcin, Palczewski, Krzysztof, and Kiser, Philip D. Key Residues for Catalytic Function and Metal Coordination in a Carotenoid Cleavage Dioxygenase. United States: N. p., 2016.
Web. doi:10.1074/jbc.m116.744912.
Sui, Xuewu, Zhang, Jianye, Golczak, Marcin, Palczewski, Krzysztof, & Kiser, Philip D. Key Residues for Catalytic Function and Metal Coordination in a Carotenoid Cleavage Dioxygenase. United States. https://doi.org/10.1074/jbc.m116.744912
Sui, Xuewu, Zhang, Jianye, Golczak, Marcin, Palczewski, Krzysztof, and Kiser, Philip D. Sun .
"Key Residues for Catalytic Function and Metal Coordination in a Carotenoid Cleavage Dioxygenase". United States. https://doi.org/10.1074/jbc.m116.744912. https://www.osti.gov/servlets/purl/1376249.
@article{osti_1376249,
title = {Key Residues for Catalytic Function and Metal Coordination in a Carotenoid Cleavage Dioxygenase},
author = {Sui, Xuewu and Zhang, Jianye and Golczak, Marcin and Palczewski, Krzysztof and Kiser, Philip D.},
abstractNote = {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.},
doi = {10.1074/jbc.m116.744912},
journal = {Journal of Biological Chemistry},
number = 37,
volume = 291,
place = {United States},
year = {Sun Jul 24 00:00:00 EDT 2016},
month = {Sun Jul 24 00:00:00 EDT 2016}
}
Web of Science
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