Evolution tunes functional sub-state interconversion to boost enzyme function
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Abstract
Enzymes do not operate as static structures, but continuously fluctuate between different conformations. Enzymes therefore dynamically sample conformations with varying catalytic activity. However, it remains largely unexplored whether evolution can exploit the conformational dynamics between sub-states to improve activity. Here, we dissect the evolutionary trajectory of the β-lactamase OXA-48 toward improved ceftazidime hydrolysis. Evolution relieved conformational bottlenecks by promoting alternate functional sub-states, gradually shifting the rate-limiting step from substrate binding to sub-state interconversion, and finally to the chemical step. Reorganization of the conformational landscape enhanced OXA-48's ability to hydrolyze ceftazidime and introduced a trade-off in its native activity against meropenem. This trade-off stemmed from catalytic incompatibility between the native and the evolved sub-state populations. Our findings highlight the transitions between functional sub-states as a mechanism of natural selection, shaping functional divergence and offering new strategies for enzyme and antibiotic engineering.
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Evolution tunes functional sub-state interconversion to boost enzyme function
This work offers a compelling view of the emergence of ceftazidime resistance in OXA-48 through conformational dynamics, framing adaptation as selection acting on rare, pre-existing catalytic behavior rather than the invention of new chemistry. A key observation is that adaptive mutations amplify an early, stochastic phase of ceftazidime turnover without changing steady-state kcat, supporting a phenotype-first view of enzyme evolution in which weak, probabilistic activity precedes genetic stabilization. The kinetic trade-offs observed across substrates are consistent with specialization within a constrained conformational landscape, and the ensemble-based interpretation provides a clear framework for linking dynamics, adaptation, and constraint.
A central …
Evolution tunes functional sub-state interconversion to boost enzyme function
This work offers a compelling view of the emergence of ceftazidime resistance in OXA-48 through conformational dynamics, framing adaptation as selection acting on rare, pre-existing catalytic behavior rather than the invention of new chemistry. A key observation is that adaptive mutations amplify an early, stochastic phase of ceftazidime turnover without changing steady-state kcat, supporting a phenotype-first view of enzyme evolution in which weak, probabilistic activity precedes genetic stabilization. The kinetic trade-offs observed across substrates are consistent with specialization within a constrained conformational landscape, and the ensemble-based interpretation provides a clear framework for linking dynamics, adaptation, and constraint.
A central implication of this framing is that productive sub-states must exist before selection acts, with adaptation increasing access to rare activity already present in the ensemble. Do you view the conformational ensemble that gives rise to these weak activities as fixed primarily by protein sequence, with the cellular environment acting mainly as a selector? Or could cellular conditions associated with antibiotic stress (such as altered proteostasis, macromolecular crowding, or chaperone engagement) transiently shift sub-state populations in vivo, changing both which weak activities are expressed and how strongly they translate into fitness early in adaptation? In this view, environmental context would shape not just the visibility of rare catalytic events, but their selective weight. Further, could such stress-associated conditions also influence which mutations are sampled or retained, for example by coupling altered protein dynamics to stress-induced mutagenesis or to differential tolerance of destabilizing variants?
Finally, you frame adaptation as tuning the rates of interconversion between pre-existing functional sub-states. In that view, do conformational dynamics simply provide a permissive background on which selection acts, or do they meaningfully bias which adaptive solutions are most accessible in the first place? More concretely, how would you distinguish a system where many evolutionary outcomes are equally reachable from one where the structure of the conformational ensemble makes certain trajectories more likely under specific substrates or stresses?
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