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Hydroxylation Regiochemistry Is Robust to Active Site Mutations in Cytochrome P450cam (CYP101A1).
Alvarez, Guadalupe; Le, Thu; Wong, Nathan; Echave, Julian; Pochapsky, Thomas C; Asciutto, Eliana K.
Afiliación
  • Alvarez G; Instituto de Ciencias Físicas, Universidad Nacional de San Martín & CONICET, Campus Migueletes, 25 de Mayo y Francia, Buenos Aires 1650, Argentina.
  • Le T; Department of Biochemistry, Brandeis University, 415 South Street, Waltham, Massachusetts 02454, United States.
  • Wong N; Department of Biochemistry, Brandeis University, 415 South Street, Waltham, Massachusetts 02454, United States.
  • Echave J; Instituto de Ciencias Físicas, Universidad Nacional de San Martín & CONICET, Campus Migueletes, 25 de Mayo y Francia, Buenos Aires 1650, Argentina.
  • Pochapsky TC; Department of Biochemistry, Brandeis University, 415 South Street, Waltham, Massachusetts 02454, United States.
  • Asciutto EK; Department of Chemistry and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, 415 South Street, Waltham, Massachusetts 02454, United States.
Biochemistry ; 61(17): 1790-1800, 2022 09 06.
Article en En | MEDLINE | ID: mdl-35960510
ABSTRACT
Cytochrome P450cam (CYP101A1) catalyzes the hydroxylation of d-camphor by molecular oxygen. The enzyme-catalyzed hydroxylation exhibits a high degree of regioselectivity and stereoselectivity, with a single major product, d-5-exo-hydroxycamphor, suggesting that the substrate is oriented to facilitate this specificity. In previous work, we used an elastic network model and perturbation response scanning to show that normal deformation modes of the enzyme structure are highly responsive not only to the presence of a substrate but also to the substrate orientation. This work examines the effects of mutations near the active site on substrate localization and orientation. The investigated mutations were designed to promote a change in substrate orientation and/or location that might give rise to different hydroxylation products, while maintaining the same carbon and oxygen atom balances as in the wild type (WT) enzyme. Computational experiments and parallel in vitro site-directed mutations of CYP101A1 were used to examine reaction products and enzyme activity. 1H-15N TROSY-HSQC correlation maps were used to compare the computational results with detectable perturbations in the enzyme structure and dynamics. We found that all of the mutant enzymes retained the same regio- and stereospecificity of hydroxylation as the WT enzyme, with varying degrees of efficiency, which suggests that large portions of the enzyme have been subjected to evolutionary pressure to arrive at the appropriate sequence-structure combination for efficient 5-exo hydroxylation of camphor.
Asunto(s)

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Alcanfor / Alcanfor 5-Monooxigenasa Idioma: En Revista: Biochemistry Año: 2022 Tipo del documento: Article País de afiliación: Argentina

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Alcanfor / Alcanfor 5-Monooxigenasa Idioma: En Revista: Biochemistry Año: 2022 Tipo del documento: Article País de afiliación: Argentina