pochapskyThomas C. Pochapsky, Ph.D.
Professor of Chemistry
Protein Structure

Ph.D., University of Illinois at Urbana-Champaign

Contact Information
Pochapsky Lab Home Page

Our group is interested in how biomolecules such as enzymes and proteins do their jobs.  Nature has had a very long time (4 billion years!) to come up with just the right combinations of sequence and fold to accomplish all of the biochemical tasks required by a living organism.  We don't have quite that long, so we use a variety of biophysical and molecular biological methods to try and tease out the details of protein structure and function.  Our aim is to be able to apply what we learn to such things as rational drug design and protein engineering.
Cytochrome P450 monooxygenases catalyze the selective oxidation of unactivated C-H and C-C bonds by molecular oxygen, and this activity is critical for diverse biological functions including steroid hormone biosynthesis, drug and xenobiotic metabolism and clearance, and pro-drug activation.  We are the first group to apply high-resolution NMR methods to understanding structure-activity relationships in the P450 superfamily.  NMR offers a unique perspective on these enzymes, as it allows atomic-resolution detail of the enzymes as they exist in solution.  The P450 reaction cycle is complicated, requiring multiple substrate binding, electron and proton transfer steps, with interactions between redox partners and effector molecules leading to poorly understood conformational changes.  We have identified critical (and previously unrecognized) conformational changes that occur during the course of the reaction cycle, and have pinpointed particular residues that are involved in those changes using NMR and mutagenesis.  We are currently using a combination of enzyme activity assays, site-directed mutagenesis and NMR to “evolve”  new P450 enzymes based on the well-understood cytochrome P450cam (Figure 1).  Our aim is to produce new enzymes that combine the efficiency and selectivity of the wild-type enzyme with modified substrate selectivity/product specificity.  We envision a family of enzymes, each with its own particular substrate/product combination, that can be used for environmentally friendly manufacturing of fine chemicals.

figure 1
Figure 1. Cytochrome P450cam

Selected Publications:

ACI-reductone dioxygenase 1 (ARD1) is an effector of the heterotrimeric G protein beta subunit in Arabidopsis. Friedman EJ, Wang HX, Jiang K, Perovic I, Deshpande A, Pochapsky TC, Temple BR, Hicks SN, Harden TK, Jones AM.J Biol Chem. 2011 Jun 28. Epub ahead of print. [abstract]

Experimentally restrained molecular dynamics simulations for characterizing the open states of cytochrome P450cam. Asciutto EK, Dang M, Pochapsky SS, Madura JD, Pochapsky TC.Biochemistry. 2011 Mar 15;50(10):1664-71. Epub 2011 Feb 8. [abstract]

Spring-loading the active site of cytochrome P450cam. Dang M, Pochapsky SS, Pochapsky TC.Metallomics. 2011 Apr 1;3(4):339-43. Epub 2010 Dec 24. [abstract]

Conformational plasticity and structure/function relationships in cytochromes P450. Pochapsky TC, Kazanis S, Dang M. Antioxid Redox Signal. 2010 Oct;13(8):1273-96. Review. [abstract] [article]

Redox-dependent dynamics in cytochrome P450cam. Pochapsky SS, Dang M, OuYang B, Simorellis AK, Pochapsky TC. Biochemistry. 2009 May 26;48(20):4254-61. [abstract]

Structural and dynamic implications of an effector-induced backbone amide cis-trans isomerization in cytochrome P450cam. Asciutto EK, Madura JD, Pochapsky SS, OuYang B, Pochapsky TC. J Mol Biol. 2009 May 15;388(4):801-14. [abstract]

Solution NMR structure of putidaredoxin-cytochrome P450cam complex via a combined residual dipolar coupling-spin labeling approach suggests a role for Trp106 of putidaredoxin in complex formation.  Zhang W, Pochapsky SS, Pochapsky TC, Jain NU. J Mol Biol. 2008 Dec 12;384(2):349-63. [abstract]

A Functional Proline Switch in Cytochrome P450cam. Bo OuYang, Susan Sondej Pochapsky, Marina Dang, and Thomas C. Pochapsky (2008) Structure. 2008 May 7; 16(5). [abstract]

Specific effects of potassium ion binding on wild-type and L358P cytochrome P450cam. OuYang B, Pochapsky SS, Pagani GM, Pochapsky TC. (2006) Biochemistry. 2006 Dec 5;45(48):14379-88. [abstract]

One protein, two enzymes revisited: a structural entropy switch interconverts the two isoforms of acireductone dioxygenase. Ju T, Goldsmith RB, Chai SC, Maroney MJ, Pochapsky SS, Pochapsky TC. (2006) J Mol Biol. 2006 Nov 3;363(4):823-34. [abstract]

Comparison of the complexes formed by cytochrome P450cam with cytochrome b5 and putidaredoxin, two effectors of camphor hydroxylase activity. Rui L, Pochapsky SS, Pochapsky TC. (2006) Biochemistry. 2006 Mar 28;45(12):3887-97. [abstract]

A refined model for the structure of acireductone dioxygenase from Klebsiella ATCC 8724 incorporating residual dipolar couplings. Pochapsky TC, Pochapsky SS, Ju T, Hoefler C, Liang J. (2006) J Biomol NMR. 2006 Feb;34(2):117-27. [abstract]

The immediate-early ethylene response gene OsARD1 encodes an acireductone dioxygenase involved in recycling of the ethylene precursor S-adenosylmethionine. Sauter M, Lorbiecke R, Ouyang B, Pochapsky TC, Rzewuski G. (2005) Plant J. 2005 Dec;44(5):718-29. [abstract]

Detection of a High-Barrier Conformational Change in the Active Site of Cytochrome P450cam upon Binding of Putidaredoxin. Wei J.Y., Pochapsky T.C., and Pochapsky S.S. (2005) Journal of the American Chemical Society, 127: 6974-6976.

Analogs of 1-phosphonooxy-2,2-dihydroxy-3-oxo-5-(methylthio)pentane, an acyclic intermediate in the methionine salvage pathway: a new preparation and characterization of activity with E1 enolase/phosphatase from Klebsiella oxytoca. Zhang Y., Heinsen M.H., Kostic M., Pagani G.M., Riera T.V., Perovic .I, Hedstrom L., Snider B.B., Pochapsky T.C. (2004) Bioorg Med Chem. 12:3847-55. [abstract]

A conserved histidine in vertebrate-type ferredoxins is critical for redox-dependent dynamics. Kostic M., Bernhardt R, Pochapsky TC. (2003) Biochemistry. 42:8171-82. [abstract]

A model for effector activity in a highly specific biological electron transfer complex: the cytochrome P450(cam)-putidaredoxin couple. Pochapsky SS, Pochapsky TC, Wei JW. (2003) Biochemistry. 42:5649-56. [abstract]

Modeling and experiment yields the structure of acireductone dioxygenase from Klebsiella pneumoniae. Pochapsky TC, Pochapsky SS, Ju T, Mo H, Al-Mjeni F, Maroney MJ. (2002) Nat Struct Biol. 9:966-72 [abstract].

Rapid Recycle 13C, 15N and 13C, 13C' Heteronuclear and Homonuclear Multiple Quantum Coherence Detection for Resonance Assignments in Paramagnetic Proteins: Example of Ni+2-Containing Acireductone Dioxygenase (ARD). Kostic M, Pochapsky TC, Pochapsky SS. (2002) J. Am. Chem. Soc., 124(31):9054-9055 [abstract].

Comparison of functional domains in vertebrate-type ferredoxins. Kostic M, Pochapsky SS, Pochapsky TC, Obenauer J, Mo H, Pagani GM and Pejchal R. (2002) Biochemistry 41: 5978-5989. [abstract]

XAS Investigation of the Structure and Function of Ni in Acireductone Dioxygenase. Al-Mjeni F, Ju T, Pochapsky TC, and Maroney MJ. (2002) Biochemistry 41: 6761-6769.

A Molecular Level Study of Complex Formation between Putidaredoxin and Cytochrome P450 by Scanning Tunneling Microscopy. Mukhopadhyay R, Wong LL, Lo KK, Pochapsky T and Hill HA. (2002) Physical Chemistry Chemical Physics 641-646.

Nuclear magnetic resonance as a tool in drug discovery, metabolism and disposition. Pochapsky TC and Pochapsky SS. (2001) Curr. Top. Med. Chem. 1: 427-441. [abstract]

Redox-dependent conformational selection in a Cys4Fe2S2 ferredoxin. Pochapsky TC, Kostic M, Jain N, Pejchal R. (2001) Biochemistry 19: 5602-5614. [abstract].

Mechanistic studies of two dioxygenases in the methionine salvage pathway of Klebsiella pneumoniae. Dai Y, Pochapsky TC and Abeles RH. (2001) Biochemistry, 40: 6379-6387. [abstract]

BF4- as a probe for ion pair solution structure using interionic one- and two-dimensional 19F{1H} NOEs. Pochapsky TC and Hofstetter C. (2000) Magn. Reson. Chem. 38: 90-94.

Pereira de Araujo AF, Pochapsky TC, Joughin B. (1999) Thermodynamics of interactions between amino acid side chains: experimental differentiation of aromatic-aromatic, aromatic-aliphatic, and aliphatic-aliphatic side-chain interactions in water. Biophys J. 76: 2319-28. [abstract]

A model for the solution structure of oxidized terpredoxin, a Fe2S2 ferredoxin from Pseudomonas. Mo H, Pochapsky SS, Pochapsky TC. (1999) Biochemistry. 38: 5666-75. PDF version. [abstract]

A refined model for the solution structure of oxidized putidaredoxin. Pochapsky TC, Jain NU, Kuti M, Lyons TA, Heymont J. (1999)Biochemistry. 38: 4681-90. PDF version. [abstract]

A new assignment strategy for the hyperfine-shifted 13C and 15N resonances in Fe2S2 ferredoxins. Jain NU, Pochapsky TC. (1999) Biochem Biophys Res Commun. 258: 54-9. [abstract]

Solution structure and dynamics of a serpin reactive site loop using interleukin 1beta as a presentation scaffold. Arico-Muendel CC, Patera A, Pochapsky TC, Kuti M, Wolfson AJ. (1999) Protein Eng. 12:189-202. [abstract]

1H, 13C and 15N NMR assignments for a carbon monoxide generating metalloenzyme from Klebsiella pneumoniae. Mo H, Dai Y, Pochapsky SS, Pochapsky TC. (1999) J Biomol NMR. 14: 287-8.

Designed molecular recognition: A commentary on possible design elements. Pochapsky TC. (1999) . Enantiomer 4: 437-444.

NMR structure determination of ion pairs derived from quinine: A model for templating in asymmetric phase transfer reductions by BH4- with implications for rational design of phase transfer catalysts. Pochapsky TC, Hofstetter C and Wilkinson PS. (1999) J. Org. Chem. 64: 8794-8800.

 


Last update: August 17, 2011.
 
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