Some Surprising Implications of NMR-directed Simulations of Substrate Recognition and Binding by Cytochrome P450cam (CYP101A1)

• Autores: Asciutto, Eliana Karina; Pochapsky, Thomas C.
• Año de publicación: 2018
• Idioma: inglés
• Tipo de recurso: artículo
• Materia: ANISOTROPIC NETWORK MODEL; CYTOCHROME P450 STEREOSPECIFICITY; PERTURBATION RESPONSE SCANNING; RESIDUAL DIPOLAR COUPLINGS; SOLUTION CONFORMATIONAL ENSEMBLES
• Institución: Consejo Nacional de Investigaciones Científicas y Técnicas

Cytochrome P450cam (CYP101A1) catalyzes the stereospecific 5-exo hydroxylation of d-camphor by molecular oxygen. Previously, residual dipolar couplings measured for backbone amide 1H–15N correlations in both substrate-free and bound forms of CYP101A1 were used as restraints in soft annealing molecular dynamic simulations in order to identify average conformations of the enzyme with and without substrate bound. Multiple substrate-dependent conformational changes remote from the enzyme active site were identified, and site-directed mutagenesis and activity assays confirmed the importance of these changes in substrate recognition. The current work makes use of perturbation response scanning (PRS) and umbrella sampling molecular dynamic of the residual dipolar coupling-derived CYP101A1 structures to probe the roles of remote structural features in enforcing the regio- and stereospecific nature of the hydroxylation reaction catalyzed by CYP101A1. An improper dihedral angle Ψ was defined and used to maintain substrate orientation in the CYP101A1 active site, and it was observed that different values of Ψ result in different PRS response maps. Umbrella sampling methods show that the free energy of the system is sensitive to Ψ and bound substrate forms an important mechanical link in the transmission of mechanical coupling through the enzyme structure. Finally, a qualitative approach to interpreting PRS maps in terms of the roles of secondary structural features is proposed.
Fil: Asciutto, Eliana Karina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Pochapsky, Thomas C.. Universidad Brandeis; Estados Unidos