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Structure‐function relationships for enzymatic activity of β‐carotene 9′,10′‐oxygenase (CMO2)
Author(s) -
Doerner Charles,
Gong Xiaoming,
Hardy Leon,
Rubin Lewis P
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.32.8
Subject(s) - mutant , enzyme , carotenoid , cleavage (geology) , chemistry , biochemistry , in vitro , in vivo , biology , gene , genetics , paleontology , fracture (geology)
Structure‐activity relationships of the carotenoid cleavage enzyme CMO2 have not been elucidated. Consequently, we have performed molecular simulations to determine stable 3D structures of CMO2 and correlation with protein function. Full‐length CMO2 was mutated at predicted critical residues (H226, H286, H357, E465, H574) and the mutated proteins were expressed in vitro . Functional activity was measured by quantitative lycopene metabolism in E. coli and HEK293 cells. CMO2 and mutant structures were predicted using protein threading (RaptorX) and scalable molecular dynamics simulation (NAMD) programs. RaptorX produced 10 possible 3D CMO2 structures. The best data fit was used for further modeling and simulations describing a structure containing four α‐helices and several β‐sheets. The interior of CMO2 features an opening/cleft that conforms to substrate binding/cleavage site criteria. Mutations of the selected residues to Ala(s) decreased the root mean squares deviation (RMSD) function c/w decreased CMO2 structure movement and stabilization. The bioinformatics results correspond with our finding that the expressed CMO2 mutants inhibit carotenoid metabolism. In conclusion, these results localize the catalytic region of CMO2. This work should facilitate further investigations of metabolism of specific carotenoids in vitro and in vivo.