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Engineering, Predicting, and Understanding Nicotinamide Cofactor Specificity

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Engineering, Predicting, and Understanding Nicotinamide Cofactor Specificity Engineering, Predicting, and Understanding Nicotinamide Cofactor Specificity Thesis by Jackson Kenai Blender Cahn In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CALIFORNIA INSTITUTE OF TECHNOLOGY Pasadena, California 2016 (Defended January 19, 2016) ii © 2016 Jackson Kenai Blender Cahn All rights reserved except where otherwise noted. iii ACKNOWLEDGEMENTS First, I’d like to thank my advisors Frances Arnold and Steve Mayo for their support and guidance over the last four years. In their labs, I’ve had a great deal of freedom to pursue this work down the various twists and turns it has taken, but Frances and Steve have channeled my natural urge to dabble down the most productive avenues. A particular thank-you to Frances for her guidance in taking my ideas and data and turning them into the narratives that follow. Sabine Brinkmann-Chen brought me into the Arnold Lab, introduced me to cofactor- switching, and taught me nearly everything I know about experimental biochemistry. She has been my main collaborator on everything in this thesis, and none of this would have been possible without her constant assistance and valuable suggestions. I have also been so lucky to have had a number of excellent students who have assisted me. Ruchi Jahagirdar, Lisa Mears, Armin Baumschlager, Nelson Chou, and Caroline Werlang: thank you. Without your observations, your pipetting prowess, and your knowledge of HTML I would still be far from graduating. There are too many members of the Arnold and Mayo Labs for me to adequately thank all of them for their assistance. Devin Trudeau and Martin Engqvist deserve particular gratitude for their experimental wisdom; Bernardo Sosa Padilla Araujo, Emzo de los Santos, and Alex Nisthal gave me valuable python pointers; Sheel Dodani, Andy Buller, and Seth Leiblich have been incredibly valuable for helping me work through ideas. Dozens of others, in my labs and beyond, have supported me with their friendship over these years, celebrating my successes and commiserating my setbacks. Thank you to all of you. You have made my time here a joy. Beyond the lab, I owe gratitude to Pavle Nikolovski and Jens Kaiser of the Molecular observatory for their able assistance in all things X-ray, and to Neil Fromer and Heidi Rusina of the Resnick Sustainability Institute, not only for their assistance in obtaining a graduate fellowship but also for their guidance in presenting my research to a broader audience. Finally, I would like to thank my family. My grandfather John, who served as a model of scientific success and helped me secure my first research opportunity, my father Andy, who taught me a love of science and of puzzles, my mother June, who taught me to work hard, to write clearly, and the prefix ‘sesqui-’ (see Chapter 5), and my little brothers Devin and Torey, who taught me to keep fighting when things get tough. Love to you all. iv ABSTRACT Oxidoreductases, the enzymes that catalyze the transfer of electrons between molecules, represent the largest group of enzymes in metabolism, and the vast majority of these enzymes use the functionally-equivalent cofactors nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) for the storage and transport of the electrons. Understanding the interactions of these proteins with their cofactors is therefore crucial to the engineering of biological pathways and systems that involve these enzymes. In particular, because cells tightly regulate the levels of oxidized and reduced NAD and NADP, it is often valuable to engineer the specificity of enzymes to better integrate them into particular metabolic contexts. The first section of this thesis focuses on this specificity, taking as a model system the ketol-acid reductoisomerase (KARI) enzyme family. Prior to the work described here, all known members of the KARI enzyme family displayed a strict specificity for NADP over NAD. However, the use of these enzymes in a constructed pathway for the production of medium-chain alcohols created a clear need for NAD-specific KARIs to improve yields. Chapter 1 briefly summarizes the prior state of the art in nicotinamide cofactor specificity engineering before describing how a previous switching of the KARI from E. coli was extended to create a simple recipe for the specificity reversal of any KARI enzyme. Chapter 2 then uses the insights into cofactor specificity in KARIs afforded by the engineering in Chapter 1 to search databases of KARI sequences and predict naturally NAD-specific KARIs, resulting in the discovery of extremophilic NAD-utilizing KARIs with properties that outstrip those of the best engineered enzymes. Chapter 3 extends this prediction approach into another enzyme family, xylose reductases, and discusses its strengths and limitations across diverse enzyme folds and families. The next section of the thesis diverges from the topic of cofactor specificity engineering to briefly explore three structural questions which arise from the study of KARIs. Chapter 4 covers an analysis of several new KARI crystal structures, including those obtained during the efforts described in Chapters 1 and 2. From a comprehensive comparison of these structures, two topics are addressed: (1) the effect of insertions and deletions in the cofactor specificity loop on the binding geometry of NAD and NADP, and (2) the conformational motions involved in the binding of cofactor, substrate, and metal ions. Based on a pair of structures from Chapter 4, Chapter 5 experimentally explores the structural evolution of the KARI enzyme family’s two distinct v structural classes, replicating in a class I KARI the structural duplication that produced the class II KARI fold, and demonstrating a remarkable retention of enzymatic activity. Chapter 6 explores a curious sensitivity to mutations observed around the adenine moiety of several KARIs, and extends this observation to a range of other NADP- and NAD-dependent enzymes, with implications both for engineering these proteins and for understanding protein evolution more generally. The third and final section discusses the development of a general method for the cofactor specificity reversal of any NAD(P)-utilizing enzyme. Chapter 7 explains the approach used and how it represents a new paradigm in protein engineering, as well as covering the thorough experimental validation to which the method was subjected. This method was developed into a web applet (CSR-SALAD) for public use, and Chapter 8 discusses the development and use of this tool. This third section represents the culmination of the preceeding work, drawing from an understanding of the sequence and structural determinants of cofactor binding as explored in the preceeding six chapters to create the heuristic picture of specificity which informed the comprehensive engineering approach. vi PUBLISHED CONTENT (* indicates equal contributions by authors) Brinkmann-Chen S.*, Flock T.*, Cahn J. K. B., Snow C. D., Brustad E. M. McIntosh J. A., Meinhold P., Zhang L., Arnold F. H. (2013). General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH, Proceedings of the National Academy of Sciences 110(27), 10946-10951. doi:0.1073/pnas.1306073110 J.K.B.C. participated in the design and performance of research, analysis of data, and writing of the paper. Brinkmann-Chen S.*, Cahn J. K. B.*, and Arnold F. H. (2014). Uncovering rare NADH- preferring ketol-acid reductoisomerases, Metabolic Engineering 26, 17-22. doi:10.1016/j.ymben.2014.08.003 J.K.B.C. participated in the design and performance of research, analysis of data, and writing of the paper. Cahn J. K. B.*, Brinkmann-Chen S.*, Spatzal T., Wiig J. A., Buller A. R., Einsle O., Hu Y., Ribbe M. W., Arnold F. H. (2015). Cofactor specificity motifs and the induced fit mechanism in class I ketol-acid reductoisomerases, Biochemical Journal 468(3), 475-484. doi:10.1042/BJ20150183 J.K.B.C participated in the conception of the project, solved and analyzed the crystal structures, prepared the data, and participated in the writing of the manuscript. Cahn J. K. B.*, Brinkmann-Chen S.*, Buller A. R., Arnold F. H. (2016). Artificial domain duplication replicates evolutionary history of ketol-acid reductoisomerases, Protein Science Epub ahead of print. Doi:10.1002/pro.2852 J.K.B.C. participated in the conception of the project, designed and cloned the constructs, participated in the solving of the crystal structure, analyzed the crystal structure, and participated in the writing of the manuscript. vii Cahn J. K. B.*, Baumschlager A.*, Brinkmann-Chen S., Arnold F. H. (2016). Artificial domain duplication replicates evolutionary history of ketol-acid reductoisomerases, Protein Engineering, Design and Selection 29(1), 31-38. doi:10.1093/protein/gzv057 J.K.B.C participated in the design and performance of research, analysis of data, and writing of the paper. viii TABLE OF CONTENTS Acknowledgements ........................................................................................................ iii Abstract .......................................................................................................................... iv Published Content .......................................................................................................... vi Table of Contents ......................................................................................................... viii List of Illustrations and/or Tables ................................................................................
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