Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine |
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Authors: | Caroline Aldag, Igor A. Gromov, In s Garcí a-Rubio, Konstanze von Koenig, Ilme Schlichting, Bernhard Jaun, Donald Hilvert |
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Affiliation: | Caroline Aldag, Igor A. Gromov, Inés García-Rubio, Konstanze von Koenig, Ilme Schlichting, Bernhard Jaun, and Donald Hilvert |
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Abstract: | The unique monooxygenase activity of cytochrome P450cam has been attributed to coordination of a cysteine thiolate to the heme cofactor. To investigate this interaction, we replaced cysteine with the more electron-donating selenocysteine. Good yields of the selenoenzyme were obtained by bacterial expression of an engineered gene containing the requisite UGA codon for selenocysteine and a simplified yet functional selenocysteine insertion sequence (SECIS). The sulfur-to-selenium substitution subtly modulates the structural, electronic, and catalytic properties of the enzyme. Catalytic activity decreases only 2-fold, whereas substrate oxidation becomes partially uncoupled from electron transfer, implying a more complex role for the axial ligand than generally assumed. |
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Keywords: | protein engineering selenocysteine insertion sequence element selenoenzyme stop codon suppression X-ray crystallography |
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