Most biochemical reactions take from hundreds to billions of years to occur spontaneously. However, life depends on highly organized networks of catalyzed chemical reactions that proceed not only rapidly, but specifically and with high fidelity. Biological catalysts are enzymes complicated molecular nanomachines that massively accelerate reactions by positioning specific substrate molecules with such precision that they are compelled to react. The molecular mechanism by which an enzyme executes this remarkable feat involves an exquisitely orchestrated sequence of steps. The structures, mechanisms, and functions of enzymes are all products of millions of years of evolution. Yet despite their fundamental biological importance, we have only a rudimentary understanding of the atomistic basis of the evolutionary changes that create novel enzymes. In this project, we will fully elucidate, at an atomistic level of description, the biophysical principles that underlie the evolutionary changes in structure, dynamics, and mechanism producing novel enzymatic functions. We will resurrect entire evolutionary lineages of ancestral enzymes, solve their structures, characterize their dynamics, and determine their kinetic mechanisms, all correlated with the functional changes observed along these evolutionary trajectories. While we are aggressively pursuing multiple systems to maximize success, our main model system is the malate and lactate dehydrogenase (M/LDH) superfamily. Both enzymes are found in the core metabolism of nearly every organism on the planet. M/LDHs are homologous enzymes that share a fold and catalytic mechanism yet can possess extraordinarily strict specificity for their substrates. The evolution of this family is marked by many important functional innovations, including (1) sharp alterations in substrate specificity, (2) changes in catalytic rate, (3) gain of allosteric control by small effector molecues, (4) acquisition of thermophilic, cryophilic, halophilic, and alkalophilic stability, and (5) the evolution of multimerization via new protein-protein interfaces. Many of these novelties are convergent, having evolved several times independently.
We aim to: 1) reveal the changes in enzyme structure and dynamics responsible for functional convergence; 2) characterize the evolution of the mechanisms of substrate specificity; and 3) resolve the correlated, epistatic mutations that determine enzyme function and specificity. How do substitutions far from the active site affect activity? What is the molecular basis of epistasis? Does specificity increase during evolution? Were the ancestors of M/LDHs promiscuous? By answering these questions, we will provide the first fine-grained description of how enzyme structures and kinetic mechanisms constrain and channel genetic evolutionary processes. The M/LDH superfamily is a classic, well-characterized system, with a common kinetic mechanism and cofactor. Hence, the resulting evolutionary insights will apply broadly to other enzymes and may transform our understanding of how enzymes can be rationally engineered.
This proposal explores the evolutionary mechanisms by which proteins have gained novel functions in a medically important class of enzymes, the malate and lactate dehydrogenases (MDHs and LDHs). Due to their significant differences from the human MDH enzymes, many of the bacterial MDH enzymes are important potential drugs targets, including human pathogens such as malaria, anthrax, Bacteroides, and Pseudomonas. The results of this research will aid in answering several long-standing evolutionary questions and will aid our understanding of how enzymatic functions can be engineered.
|Wirth, Jacob D; Boucher, Jeffrey I; Jacobowitz, Joseph R et al. (2018) Functional and Structural Resilience of the Active Site Loop in the Evolution of Plasmodium Lactate Dehydrogenase. Biochemistry 57:6434-6442|
|Trieu, Melissa M; Devine, Erin L; Lamarche, Lindsey B et al. (2017) Expression, purification, and spectral tuning of RhoGC, a retinylidene/guanylyl cyclase fusion protein and optogenetics tool from the aquatic fungus Blastocladiella emersonii. J Biol Chem 292:10379-10389|
|Nguyen, Vy; Wilson, Christopher; Hoemberger, Marc et al. (2017) Evolutionary drivers of thermoadaptation in enzyme catalysis. Science 355:289-294|
|Lamarche, Lindsey B; Kumar, Ramasamy P; Trieu, Melissa M et al. (2017) Purification and Characterization of RhoPDE, a Retinylidene/Phosphodiesterase Fusion Protein and Potential Optogenetic Tool from the Choanoflagellate Salpingoeca rosetta. Biochemistry 56:5812-5822|
|Devine, Erin L; Theobald, Douglas L; Oprian, Daniel D (2016) Relocating the Active-Site Lysine in Rhodopsin: 2. Evolutionary Intermediates. Biochemistry 55:4864-70|
|Theobald, Douglas L (2016) Presenilin adopts the ClC channel fold. Protein Sci 25:1363-5|
|Steindel, Phillip A; Chen, Emily H; Wirth, Jacob D et al. (2016) Gradual neofunctionalization in the convergent evolution of trichomonad lactate and malate dehydrogenases. Protein Sci 25:1319-31|
|Wilson, C; Agafonov, R V; Hoemberger, M et al. (2015) Kinase dynamics. Using ancient protein kinases to unravel a modern cancer drug's mechanism. Science 347:882-6|
|Mackin, Kristine A; Roy, Richard A; Theobald, Douglas L (2014) An empirical test of convergent evolution in rhodopsins. Mol Biol Evol 31:85-95|
|Boucher, Jeffrey I; Jacobowitz, Joseph R; Beckett, Brian C et al. (2014) An atomic-resolution view of neofunctionalization in the evolution of apicomplexan lactate dehydrogenases. Elife 3:|
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