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GGDEF Enzymes by Zachary Francis Hallberg

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GGDEF Enzymes by Zachary Francis Hallberg Broken Symmetry: Discovery, Characterization, and Structure of Hybrid Promiscuous (HyPr) GGDEF Enzymes By Zachary Francis Hallberg A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Ming Chen Hammond, Chair Professor Matthew Francis Professor Russell Vance Fall 2017 ABSTRACT Broken Symmetry: Discovery, Mechanistic Analysis, and Structure of Hybrid Promiscuous (HyPr) GGDEF Enzymes by Zachary Francis Hallberg Doctor of Philosophy in Chemistry University of California, Berkeley Professor Ming C. Hammond, Chair This past decade has witnessed an expanded diversity in the cast of nucleotide-based second messengers. The small lineup of cyclic AMP, cyclic di-GMP and ppGpp have been joined by new co-stars, including cyclic di-AMP, cyclic oligoA, and two flavors of cyclic AMP-GMP - with one possessing a unique 2’-5’ phosphodiester linkage specific to eukaryotes. As each new signaling pathway has been uncovered, new enzymes classes have been attached to each specific second messenger, suggesting rigid evolutionary pathways in the development of new signal transduction pathways. We have begun to unravel this paradigm with the discovery of a subclass of GGDEF enzyme - previously associated only with cyclic di-GMP synthesis - responsible for syn- thesis of a different cyclic dinucleotide, cyclic AMP-GMP. Intriguingly, this enzyme be- haves nothing like the other two. Our discovery of this new activity from an ancient en- zyme class pinpoints a set of deltaproteobacteria that have this “hybrid promiscuous” GGDEF activity, which preliminary work suggests controls transient surface attachment in diverse bacterial lifestyles. Furthermore, we have begun to unravel how Nature has allowed this newfound activity to act separately from other GGDEF-related pathways to allow precise control over electron acceptor choice in Geobacter species. Our work suggests that there is further underappreciated potential for individual components of nucleotide second messenger signaling enzymes, and establishes a footing for engi- neering new bioorthogonal signaling functions into this venerable enzyme class. !1 For Mary Dolan !i TABLE OF CONTENTS Chapter One: Specificity and Activation of Dinucleotide Cyclases…………..………1 Abstract…………………………………………………………………………….…….2 Intro to CDNs…………………………………………………………………………….3 Discovery and Phylogeny of Cyclic Dinucleotide Synthases……………………….4 Structural Aspects of Nucleotide-based Second-Messenger Enzymes…….……..7 Regulation of Dinucleotide Cyclases………………………………………….……..10 Outlook………………………………………………………………………………….16 Figure………………………………………………………………………………..….18 Bibliography…………………………………………………………………………….21 Chapter Two: Hybrid Promiscuous GGDEF Enzymes Produce Cyclic AMP-GMP..30 Abstract…………………………………………………………………………………31 Results/Discussion…………………………………………………………………….32 Materials and Methods………………………………………………………………..39 Figures………………………………………………………………………………….45 Bibliography………………………………………………………………….…………83 Chapter Three: Kinetic Analysis of Hybrid Promiscuous GGDEFs…………………87 Introduction……………………………………………………………………………. 88 Results…………………………………………………………………………….……88 Discussion……………………………………………………………………….……..90 Materials and Methods………………………………………………………..………92 Figures………………………………………………………………………….………97 Bibliography……………………………………………………………………..……103 Chapter Four: Structural Insights into HyPr GGDEF Function………………….…105 Introduction……………………………………………………………………………106 Results……………………………………………………………………..………….106 Discussion……………………………………………………………………………..112 Materials and Methods……………………………………………………………….114 Figures…………………………………………………………………………………117 Bibliography……………………………………………………………………..……137 Appendix Chapter: Engineering and in vivo Applications of Riboswitches….….140 Abstract………………………………………………………………………………..141 Intro…………………………………………………………………………………….142 Riboswitch Engineering……………………………………………………..….……142 In vivo Applications of Riboswitches…………………………………………….…154 Conclusions……..………………………………………………………………….…158 Figures………………………………………………………………………..….……160 Bibliography…..………………………………………………………………………164 !ii “It’s not about you.” - (now-Dr.) Cindy Wang Per the usual, Cindy was right: I couldn't do this alone. I have so many fabulous colleagues who helped keep me sane through this process. They were especially instrumental the last few weeks, when I was on the verge of mental collapse due to lack of sleep and needed coherent people to make sure that what I was writing (on no sleep) (a) made sense and (b) were true statements. And I have a boss who is willing to put in near-superhuman hours to make sure that the things we write are of high quality. More than any other professor in chemistry that I've seen, my adviser is not ashamed to be right next to her students in the arena. That's a topic for later, though. But that's the point: I can't do this alone. Any of this. None of us can. I’m going to get religious at this point, so if that’s not your cup of tea, then please move on to page two. Audrey Assad (one of my favorite Christian artists) has an album titled "Fortunate Fall." The content is all based around the idea of the "Felix Culpa" (Happy Fault) as espoused by St. Augustine - his solution to the problem of evil is that "God judged it better to bring good out of evil than not to permit any evil to exist," and thus, the original sin of Adam was what led to the Incarnation of God in human form. The album itself is gorgeous (seriously, listen), in both lyrical and musical content. If you want a stronger recommendation - I've spent the past week alternating between this and her album "Heart." Anyways - different artists have different messages that they want to communicate with the world - being a semi-Platonist, I'd argue that the "sparks" that lead us to different forms of expression and creativity stem from us seeing different fundamental aspects of God. Think of it this way - God is multidimensional in His aspects, and by multidimensional I'd argue we're talking thousands if not millions of dimensions (most likely infinite. Word's still out on whether it's countably infinite or uncountable). While we are, ourselves, multidimensional in our personalities, we can only experience a finite amount of the dimensions of God at a time (the same way we can only perceive 3 dimensions spatially at a time). And it's those dimensions that we experience that we are most comfortable communicating as a message to others. I suppose the message that comes through my experience of God I sum up as "Not By, Through." It's the fundamental idea that everything that I've ever accomplished has happened only because I've had the chance to stand on the shoulders of giants - friends, mentors, colleagues, historical figures, grad students from 50 years ago, or, well, God. There is nothing that I have done that can't be traced back to someone else. I think that's an important exercise for everyone to do. Find something that you like about where you are in life, trace it back through, and figure out how many people helped make that into a reality. I know, that's a super simple - almost stupidly simple - exercise that we all probably did in kindergarten. But do it. Give yourself the gift of remembering just how loved you are. What follows is my meditation upon this, and my thanks to each and every one of those people. I may miss some people - understand that it’s not personal, that I’m running on not-so-much sleep, and that there is a finite deadline of less than 8 hours before I have to submit this thing as a whole. Also as a quick note - if the name Dr. is in parentheses, it is because they became a PhD between when I met them and now. If the name Dr. is not in parentheses, it is because they were already a PhD when I had met them. !iii Although my PhD was earned studying the biochemistry of an anaerobe, my science has never existed in a vacuum. From the earliest days that I thought about becoming a scientist, to the present, I have been blessed beyond measure with communities of diverse and passionate people who have supported my endeavors. It is a daily reminder to me that often environment has a stronger effect on personal outcomes than genetics. So, without further adieu - these are the people through whom I found the strength, courage, energy, and passion to keep doing science (almost) every day for five and a half years. To my immediate family - I know that I may put you to sleep talking about science. I would say that I’m sorry, but part of telling a young person that they should pursue their dreams and cultivate their love of science suggests to me that I shouldn’t be that sorry. To my parents, who were the first to nurture that love of science - thank you for all that you have done. I see now how lucky I am to have had parents who made it a priority that I graduate from college debt free. I see now how lucky I am to have had parents who made building a stable home life a priority over having the most auspicious career or the most money. I see now how lucky I am to have been given a strong moral compass grounded in love, grace, and integrity. I see now how lucky I am to have been given gifts outside of science - primarily music - to cultivate when my spirit needs respite. Let’s face it - the past two years were scary for our family. If there is one thing that I regret not saying often enough - it is that I love you both so much. To my siblings, I am sorry that you had to go through school living in the midst of my shadow.
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