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The evolution of enzyme functions in the metallo-β-lactamase superfamily

University of British Columbia

Enzyme superfamilies have expanded over billions of years from the descendants of a potentially single common ancestral function. Understanding the evolution of their functional diversity is central to biochemistry, molecular and evolutionary biology. The overarching question of my thesis is how enzyme promiscuity, the serendipitous ability to catalyze non-native reactions and reactions, connects enzyme functions and facilitates molecular evolution by providing evolutionary starting points towards new functions. In particular, I primarily focus on proteins across the metallo-β-lactamase (MBL) superfamily by comparing evolutionary and functional connectivity based on the functional profiling of 24 enzymes against 10 distinct hydrolytic MBL reactions. This analysis revealed that MBL enzymes are generally promiscuous, as each enzyme catalyzes on average 1.5 reactions in addition to its native one, which leads to high functional connectivity. Furthermore, the ability to promiscuously bind different metal ions, enzymatic co- factors of MBL enzymes, provide additional mechanisms whereby the function profile of some MBL enzymes can be broadened, and thus further extends the connectivity between functions. In addition, I expand and compare the analyses of function connectivity through promiscuity to three previously published superfamily-wide function profiling studies, which revealed common trends that are discussed in the context of enzyme superfamily evolution. Finally, I assess the evolvability of promiscuous enzymes to determine their potential as evolutionary starting points towards a novel function by performing a comparative laboratory evolution experiment of two related β-lactamases, NDM1 and VIM2, towards a shared promiscuous phosphonate monoester hydrolase activity. Both trajectories accumulate 13 mutations over ten rounds of directed evolution, however the mutational solutions and evolvability is strikingly different for the two enzymes. NDM1 improves catalytic efficiency by over 20,000-fold and loses much of its solubility, i.e. the amount of functional enzyme in the cell. Contrarily, VIM2 improves catalytic efficiency only by 60-fold, but improves solubility. Detailed structural analysis, combined with molecular dynamics simulations, reveals a molecular understanding for the observed differences in evolvability between NDM1 and VIM2. Overall, my research contributes to our understanding of enzyme evolution and will help to advance functional annotation and engineering of enzyme.

Text

2017-04-18

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/61252

Genome Science and Technology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/