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LABORATORY EVOLUTION of CYTOCHROME P450 PEROXYGENASE ACTIVITY Thesis by Patrick C. Cirino in Partial Fulfillment of the Requirem

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LABORATORY EVOLUTION of CYTOCHROME P450 PEROXYGENASE ACTIVITY Thesis by Patrick C. Cirino in Partial Fulfillment of the Requirem LABORATORY EVOLUTION OF CYTOCHROME P450 PEROXYGENASE ACTIVITY Thesis by Patrick C. Cirino In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy California Institute of Technology Pasadena, California 2004 (Defended June 2, 2003) ii © 2004 Patrick C. Cirino All Rights Reserved iii ACKNOWLEDGEMENTS My tenure at Caltech has been a wonderful and rewarding experience. I leave this chapter of my life feeling enlightened because of what I have learned, and charmed because of the people that I have had the fortune to spend time with. I am forever indebted to these people for their contributions to my scientific and personal growth over the years. I wish to thank my advisor Frances Arnold. I thank her for having confidence in me and for giving me the opportunity to work in her lab. I thank her for her constant encouragement, support, and guidance. She has been incredibly patient and has given me the time to grow professionally and mature personally. My respect and admiration for Frances continues to grow, and she has become a source of inspiration as I begin my own academic career. I could not have had a better advisor and I will never be able to thank her enough for all that she has given me. I wish to acknowledge the entire Division of Chemistry and Chemical Engineering for their resources, their courses, and for providing such a supportive and inspiring work environment. In particular, the following faculty members have been extremely helpful: I thank Dave Tirrell for all his generosity. As Division Chair, Dave has provided invaluable services which I have been able to take advantage of. I thank Dave for his supportive letters of recommendation, for giving me the opportunity to collaborate with his group, and for serving on my thesis committee. I thank Harry Gray for serving on my candidacy and thesis committees, for supplying advice and perspective throughout the years, and for amusing and stimulating conversation. I thank Mark Davis for serving on my thesis committee, for putting in a good word for me, and for good advice. I also acknowledge George Gavalas and Jack Richards for serving on my candidacy committee. I thank Geethani Bandara, not just for keeping the lab running smoothly and helping out wherever needed, but also for helping me to keep my life in proper perspective. I want to acknowledge Donna Johnson and Kathy Bubash for all of their assistance and administrative services, and Suresh Guptha for being so helpful and for many pleasant conversations. iv Zhanglin Lin was an excellent mentor who taught me most of what I know about experimental molecular biology. Zhanglin made laboratory work fun. Uli Schwaneberg and Carlos Martinez played important roles in my scientific development and particularly in my knowledge of P450s. Other Arnold lab members who have been exceptionally helpful include Volker Sieber, Holger Berk, Edgardo Farinas and Daisuke Umeno. I consider all of these people to be great friends and terrific scientists, and I look forward to future interactions with everyone. Edgardo Farinas has become one of my closest friends over the years. Once again, I must thank Frances for putting Edgardo and I together. We were an instant team and together we explored cytochrome P450 research and established most of the lab’s P450 protocols. Outside the lab, our desire to match hard work with hard play led us into memorable mischief. I thank Yi Tang for his friendship and all his wisdom, and for helping to make our first year together tolerable. Chris Voigt has also been a good friend and a good person to talk to. Yi and Chris set the upper limit on what can be expected from our generation of scientists and I will continue to learn from both of them. I would like to acknowledge all members of the Arnold lab and Tirrell lab for their help, advice, generosity and friendship. More importantly, I thank them for putting up with me. It’s no secret that I have a loud personality and a loud stereo to go with it. I only hope that my influence has rubbed off and Springsteen will continue to echo through those halls. Isaac Carrico and Pin Wang in the Tirrell group have served as reliable sources of information and insight… these guys know everything. Denis Shcherbakov was a hard working SURF student who endured the trials of assay development with me. Anders Olson helped with screening and became a great friend. Oriana Salazar was always uplifting and a delight to work with. Mona Shahgholi provided helpful assistance with MALDI-TOF mass spectral analyses, and Nathan Dalleska and Peter Green assisted with GC procedures. I thank my friends and family for their continued support and encouragement. My parents and sisters are the most important people in my life and they have been with me every v step of the way. There is no “significant other” to acknowledge, but my life-long friends Dan, Jeremy, Jay, and G are closer to me than brothers. I keep nothing inside; every worry, emotion and anxiety is poured onto my loved ones. There is nothing more comforting in my life than when someone knows the right thing to say to ease my mind. While this is not a complete list of the friends who have enriched my life and provided encouragement over the years, the following people (who have not already appeared above) have been particularly supportive: Tim, Ilias & Eric, Kathy & Mark, Karen (my Bruce buddy), Eimear, and Allen (Chud!). Finally, I would like to acknowledge the organizations that have helped to fund my research: British Petroleum, the Biotechnology Research and Development Corporation (BRDC) and the NSF. vi ABSTRACT The ability of the cytochrome P450 heme monooxygenases to catalyze difficult oxidation reactions, often with high specificity and selectivity, makes them attractive for numerous bio- technological applications. However they are generally limited by low turnover rates and low stability, and their minimum requirements for catalysis include a cofactor as source of electrons (NAD(P)H), partner proteins for electron transfer, and dioxygen. Some P450s are capable of supporting low levels of peroxygenase activity, in which a peroxide is utilized to drive catalysis via a “shunt” pathway. This mechanism for substrate oxidation, although inefficient and not generally utilized in nature, simplifies P450 catalysis by eliminating the need for NAD(P)H. Our goal was to engineer an efficient P450 peroxygenase which utilizes hydrogen peroxide (H2O2). Directed evolution is a powerful enzyme engineering methodology which mimics nature's algorithm for evolution. Enzyme libraries are generated via DNA mutagenesis or recombination techniques, and variants with improved function are isolated using an appropriate screen or selection. Using this strategy, in combination with site-directed mutagenesis, we have created P450 BM-3 heme domain variants with more than 100-fold improved H2O2-driven hydroxylation activity compared to wild-type, showing both an improved kcat as well as a lower Km for H2O2. Thermostability was also improved by directed evolution. We have engineered a cell-free, biomimetic hydroxylase that requires only H2O2 to exploit the hydroxylating power of P450 BM-3. Peroxide-mediated inactivation as a result of heme destruction remains a major obstacle and presents an important enzyme engineering challenge. This research has broadened the potential applications of P450 biocatalysis by exploiting the versatility of heme-containing proteins. vii THESIS SUMMARY Nature provides an arsenal of biocatalysts whose capabilities we are learning to exploit and perfect through protein engineering. Oxygenases comprise several protein families that introduce one (monooxygenases) or two oxygen atoms (dioxygenases) into their substrates, typically through the activation of dioxygen (O2) using reduction equivalents supplied from the cofactors NADH or NADPH (NAD(P)H) via electron transfer proteins (e.g., reductase). In spite of this rather complex catalytic system, these enzymes and the reactions they catalyze are highly sought-after on all levels of chemical synthesis. Among the array of transformations catalyzed by oxygenases, the ability of the heme- containing “superfamily” of enzymes termed the cytochrome P450 monooxygenases to oxidize unactivated C-H bonds is perhaps the most useful and certainly the most impressive from an applied catalysis perspective. Throughout nature these enzymes play important roles in metabolism and biosynthesis, and they are of primary importance in the design of pharmaceuticals. It is no surprise that the cytochromes P450 are the most studied of the monooxygenases and are marveled for their unmatched biocatalytic potential, diversity, and complexity. It is often advantageous to employ in vitro enzyme catalysis (e.g., to avoid metabolism of desired products, for substrates that cannot permeate cell walls, or where sterile conditions are desired). In vitro P450 biocatalysis would require continuous regeneration of the expensive cofactor NAD(P)H from NAD(P)+, adding complexity and cost to the reaction system. In vitro P450 catalysis is also sometimes desired in applications where it would not be feasible to use the natural cofactor to drive catalysis, viii such as in the use of P450s in laundry detergents for the oxidation of surfactants and clothing stains. Many P450s are capable of using peroxides as a source of oxygen via a peroxide “shunt” pathway: P450 RH + R’OOH Æ ROH + R’OH This mechanism for substrate oxidation offers the opportunity to take advantage of P450 catalysis without the need for a cofactor, and eliminates the rate-limiting electron transfer step carried out by the reductase. However, low efficiency is a major limitation as a result of this pathway not generally being utilized in nature. Our goal was to improve and harness this unnatural P450 reaction. The heme cofactor is ubiquitous in nature and found in many enzyme families.
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