This project represents a continuing effort to determine the role of structure and dynamics in determining function of metalloproteins. In particular, mechanisms of molecular recognition and interprotein electron transfer in the camphor hydroxylase pathway from Pseudomonas putida are the targets of investigation. This system consists of three proteins, the heme-containing cytochrome P-450/cam (CYP101), the Fe2S2 ferredoxin putidaredoxin (Pdx) and the NADH-dependent flavoprotein putidaredoxin reductase(PdR). Cypo101 catalyzes the 5-exo-hydroxylation of camphor by molecular oxygen, a reaction that requires two electrons supplied to the cytochrome sequentially by Pdx. Pdx is also required as an effector for substrate turnover. The electrons are supplied to Pdx by PdR by oxidation of PdR-bound NADH. The camphor hydroxylase system is a good model for human P450 enzymes involved in steroid hormone biosynthesis and processing of xenobiotics, drug metabolism and carcinogenesis. Improved methods are sought for determining structure and dynamics of paramagnetic proteins such as Pdx using selective isotope labeling and multinuclear NMR methods. A model for Pdx CYP101 interactions has been developed and will be tested using protein engineering (mutagenesis) and NMR characterization of the solution complexes. Advances in NMR methodology, along with improved expression techniques in deuterated media, have allowed the first high resolution multi-dimensional NMR spectra of a uniformly 2H(13C), 15N- labeled CYP101 to be presented. Structure-activity relationships of a novel metalloenzyme, E2, are also under investigation. E2 is involved in the methionine salvage pathway, and produces different products from the same substrate depending on the metal bound in the E2 active site. We have determined the global fold of E2 by NMR and are investigating the differences in substrate binding between the different metal-containing isoforms of the enzyme. The methionine salvage pathway is important for feedback inhibition of polyamine biosynthesis, which are associated with tissue pathology in mammals.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BBCA (01))
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Wehrle, Janna P
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Brandeis University
Schools of Arts and Sciences
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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
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
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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