The chemistry catalyzed by cytochromes P450 (activation of molecular oxygen to react with organic molecules) is inherently hazardous to the organism, and must be tightly regulated or take place in a controlled environment. Structural and dynamic changes that take place in the course of the catalytic cycle of the P450 enzyme are part of this regulation, and help determine enzyme specificity and efficiency. While crystal structures have been determined for many P450s, and provide an important starting point for understanding structure-function relationships, methodology is lacking for monitoring changes in structure and dynamics as a function of substrate and effector binding or for rapid characterization of active sites of P450s and their interactions with substrates and inhibitors. We apply multidimensional nuclear magnetic resonance (NMR) and tandem mass spectrometry (MS-MS) to P450 enzymes to fill this need. Extensive sequential 1H, 15N and 13C resonance assignments have been made in cytochrome P450cam (CYP101). A discrete conformational change occurs upon binding of effector to CYP101 that reorients the substrate appropriately for chemistry, and details of this conformational change have been elucidated by a combination of NMR, mutagenesis and simulations. MS-MS has been used to localize redox-dependent changes in local protein dynamics in CYP101 by hydrogen/deuterium exchange. During the next project period, NMR assignments of CYP101 will be extended to regions of slow amide exchange and paramagnetic broadening. MS-MS and NMR will be used to identify dynamic """"""""hot-spots"""""""" important for substrate binding. The structure of effector-bound CYP101 will be determined using residual dipolar couplings. NMR resonances in the active sites of P450s can be easily distinguished by comparison of paramagnetic and diamagnetic forms. The binding of substrates and inhibitors in the active sites of mammalian P450 enzymes with CYP2B4 and CYP3A4 will be characterized. Methodology developed during the current period for NMR structural characterization of paramagnetic metalloproteins will be further refined.

Public Health Relevance

Project Narrative Cytochrome P450 monooxygenases are critical in many human physiological processes, including drug metabolism and activation, steroid and arachadonic acid biosynthesis, and """"""""toxic waste disposal"""""""", that is, degradation of foreign compounds prior to excretion. Understanding the activity of these enzymes is important for predicting the behavior of inhibitors and substrates as a part of drug design. This project is aimed at understanding these enzymes to aid in the design of more effective pharmaceuticals.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function A Study Section (MSFA)
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Wehrle, Janna P
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Brandeis University
Schools of Arts and Sciences
United States
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Pochapsky, Thomas C; Wong, Nathan; Zhuang, Yihao et al. (2018) NADH reduction of nitroaromatics as a probe for residual ferric form high-spin in a cytochrome P450. Biochim Biophys Acta Proteins Proteom 1866:126-133
Asciutto, Eliana K; Pochapsky, Thomas C (2018) Some Surprising Implications of NMR-directed Simulations of Substrate Recognition and Binding by Cytochrome P450cam (CYP101A1). J Mol Biol 430:1295-1310
Tietz, Drew R; Podust, Larissa M; Sherman, David H et al. (2017) Solution Conformations and Dynamics of Substrate-Bound Cytochrome P450 MycG. Biochemistry 56:2701-2714
Tietz, Drew R; Colthart, Allison M; Sondej Pochapsky, Susan et al. (2017) Substrate recognition by two different P450s: Evidence for conserved roles in a common fold. Sci Rep 7:13581
Deshpande, Aditi R; Pochapsky, Thomas C; Ringe, Dagmar (2017) The Metal Drives the Chemistry: Dual Functions of Acireductone Dioxygenase. Chem Rev 117:10474-10501
Colthart, Allison M; Tietz, Drew R; Ni, Yuhua et al. (2016) Detection of substrate-dependent conformational changes in the P450 fold by nuclear magnetic resonance. Sci Rep 6:22035
Pochapsky, Thomas C (2014) Examining how enzymes self-organize in a membrane. Proc Natl Acad Sci U S A 111:3659-60
Li, Shengying; Tietz, Drew R; Rutaganira, Florentine U et al. (2012) Substrate recognition by the multifunctional cytochrome P450 MycG in mycinamicin hydroxylation and epoxidation reactions. J Biol Chem 287:37880-90
Asciutto, Eliana K; Young, Matthew J; Madura, Jeffry et al. (2012) Solution structural ensembles of substrate-free cytochrome P450(cam). Biochemistry 51:3383-93
Friedman, Erin J; Wang, Helen X; Jiang, Kun et al. (2011) Acireductone dioxygenase 1 (ARD1) is an effector of the heterotrimeric G protein beta subunit in Arabidopsis. J Biol Chem 286:30107-18

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