Developing hybrid cytochrome P450 enzyme from CYP101 and CYP119

1. Developing hybrid cytochrome P450 enzyme from CYP101 and CYP119 Abhijeet P. Borole and Choo Y. Hamilton * Life Sciences Division, P.O. Box 2008, Oak Ridge National Laboratory, Oak Ridge TN 37931-6226 Phone: (865)576-7421; Fax: (865)574-6442; E-mail: borolea@ornl.gov * Joint Institute for the Energy and the Environment, The University of Tennessee, Knoxville 26th Symposium on Biotechnology for Fuels and Chemicals May 9-12, 2004

2. Abstract • Cytochrome P450 is one of the most versatile enzymes for oxygen activation of hydrophobic molecules such as polyaromatic hydrocarbons and alkanes. Industrial application of these enzymes has been limited by their low stability and requirement for a cofactor. In an attempt to increase thermostability of these enzymes, we are investigating properties of hybrid P450s combining a mesostable and a thermostable enzyme. CYP101 is our model hydrocarbon oxidizing enzyme and CYP119 is the model thermostable enzyme. A hybrid generated from these two proteins show retention of the temperature stability but alteration in substrate specificity. Preliminary results on the rational design of a thermostable protein combining the two enzymes are discussed.

3. CYP101, cytochrome P450cam CYP119, Thermostable P450 • From Pseudomonas putida • Capable of camphor hydroxylation • Mutants capable of alkane (C4-C6) hydroxylation • Requires putidaredoxin and NADH • 414 residues • Mol wt 46524 • From Sulfolobus solfataricus • Melting T = 91oC • Capable of hydroxylating lauric acid, styrene (non-natural substrates) • Natural substrates unknown • Requires cofactors • 368 residues • Mol wt 42850

4. CYP101 Poulos et.al., 1987, 195, 687-700 PDB structure 1AKD

5. CYP119 Structural similarity between CYP101 and CYP119 suggests potential for successful protein hybrid formation. Yano et.al., JBC, 2000, 275 31086-31092

6. Cloning of hybrid P450 • Primers: • cypF1B : 5’-gaattctgacgactgaaaccata-3’ • cypR1 : 5’-gctagcttgcgggcgcaggctctc-3’ • cypF2B : 5’-gctagcgacgacatagtgaag-3’ • cypR2B : 5’-ttcattactcttcaacctgaccac-3’ • Produce pcr products – digest with NheI and EcoRI (1), NheI and SpeI (2) • Ligate and transform into DH5a Plasmid vector used to clone hybrid (EcoRI-SpeI)

7. CYP119 CYP101-119 hybrid Obtained using computational software PROSPECT

8. Purification of hybrid • Grow cells to OD600 = 1.5 • Wash with Tris buffer pH 7.0, 50 mM • Lyse cells by sonication • Heat supernatant to 75oC for 15 minutes – centrifuge • 1st step: Ion exchange on HTQ column • 2nd step: Chromatofocussing using PBE118 column and exchange buffer 74

9. 8.14.03 A1 0.3 0.3 0.3 A2 0.2 0.2 0.2 0.1 0.1 0.1 A3 0 0 0 250 250 250 350 350 350 450 450 450 550 550 550 650 650 650 Wavelength (nm) Wavelength (nm) Wavelength (nm) A1-B2 in 50mM Tris pH7.0+ 0.1MKCl A2-B2 (A1)+N2 + DT(P) A3-B2 (A2) +CO Properties of hybrid P450 CYP119 Hybrid • Stable up to 75oC • pI ~ 6.0 • Spectrum similar to CYP119 • Shift to 450 with CO shows an active P450 (B2 refers to the hybrid).

10. Substrate binding analysis via UV-vis spectroscopy • Substrates tested: • Styrene • Lauric acid • Sterol • Capric acid • Palmitic acid • Camphor • Naphthalene • Pyrene • Benzene • Toluene • Ethylbenzene • Stilbene

11. Change in spectra due to lauric acid binding Hybrid with no substrate Hybrid with substrate bound Negligible change in spectra with other substrates.

12. Conclusions • A thermostable hybrid P450 produced by combining CYP101 and CYP119 • Protein purified to homogeneity by two-step purification protocol • Substrate binding using UV-vis technique shows active site similar to CYP119 • Further testing using more sensitive techniques such as NMR needed to better understand the substrate pocket.

13. Acknowledgements • Funding was provided by DOE, NPTO • Dr. Paul Ortiz deMontellano for cyp101 gene, and Dr. Stephen Sligar for cyp119 gene. • In-kind support provided by industrial partners: • ChevronTexaco