Manipulating Conformational Dynamics To Repurpose Ancient Proteins for Modern Catalytic Functions

Gardner, Jasmine M.; Biler, Michal; Risso, Valeria A.; Sánchez‐Ruiz, José M.; Kamerlin, Shina Caroline Lynn

doi:10.1021/acscatal.0c00722

Cited by 46 publications

(56 citation statements)

References 76 publications

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“…Protein dynamics may thus promote non-additive interaction of mutations (Miton and Tokuriki, 2016). Increasing our understanding of the biophysical features that facilitate protein evolution finds practical applications in better directing protein engineering (Johansson and Lindorff-Larsen, 2018;Maria-Solano et al, 2018;Trudeau and Tawfik, 2019;Gardner et al, 2020;Yang et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

“…Protein dynamics are selected for during natural evolution of enzymes, consistent with dynamics being a determinant of function (Henzler-Wildman and Kern, 2007;Liu and Bahar, 2012;Petrovic et al, 2018). Tracking protein dynamics during the course of experimental evolution has equally revealed correlations between protein dynamics, function and evolution (Johansson and Lindorff-Larsen, 2018;Gardner et al, 2020). Ancestral reconstruction of ß-lactamases revealed that protein motions at fast timescales decreased in conjunction with evolution of specificity (Zou et al, 2015).…”

Section: Introductionmentioning

confidence: 94%

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Known Evolutionary Paths Are Accessible to Engineered ß-Lactamases Having Altered Protein Motions at the Timescale of Catalytic Turnover

Alejaldre

Lemay-St-Denis

Perez‐Lopez

et al. 2020

Front. Mol. Biosci.

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The evolution of new protein functions is dependent upon inherent biophysical features of proteins. Whereas, it has been shown that changes in protein dynamics can occur in the course of directed molecular evolution trajectories and contribute to new function, it is not known whether varying protein dynamics modify the course of evolution. We investigate this question using three related ß-lactamases displaying dynamics that differ broadly at the slow timescale that corresponds to catalytic turnover yet have similar fast dynamics, thermal stability, catalytic, and substrate recognition profiles. Introduction of substitutions E104K and G238S, that are known to have a synergistic effect on function in the parent ß-lactamase, showed similar increases in catalytic efficiency toward cefotaxime in the related ß-lactamases. Molecular simulations using Protein Energy Landscape Exploration reveal that this results from stabilizing the catalytically-productive conformations, demonstrating the dominance of the synergistic effect of the E014K and G238S substitutions in vitro in contexts that vary in terms of sequence and dynamics. Furthermore, three rounds of directed molecular evolution demonstrated that known cefotaximase-enhancing mutations were accessible regardless of the differences in dynamics. Interestingly, specific sequence differences between the related ß-lactamases were shown to have a higher effect in evolutionary outcomes than did differences in dynamics. Overall, these ß-lactamase models show tolerance to protein dynamics at the timescale of catalytic turnover in the evolution of a new function.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 94%

Known Evolutionary Paths Are Accessible to Engineered ß-Lactamases Having Altered Protein Motions at the Timescale of Catalytic Turnover

Alejaldre

Lemay-St-Denis

Perez‐Lopez

et al. 2020

Front. Mol. Biosci.

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“…Therefore, the ancestral TIM-barrel described here holds promise as a scaffold for the generation of de novo catalysts, an important and largely unsolved problem in enzyme engineering. We have recently shown 45 that completely new enzyme functions can be generated through a single mutation that generates both a cavity and a catalytic residue, provided that conformational flexibility around the mutation site allows for substrate and transition-state binding 10 , 43 , 44 . The combination of a rigid core that provides stability with high flexibility in specific regions makes the ancestral protein studied here an excellent scaffold to develop this minimalist approach to de novo catalysis (work in progress).…”

Section: Discussionmentioning

confidence: 99%

“…For instance, high stability and substrate/catalytic promiscuity have been described in a number of ancestral resurrection studies 5 , 7 . These two features are known contributors to protein evolvability 8 , 9 , which points to the potential of resurrected ancestral proteins as scaffolds for the engineering of new functionalities 4 , 10 .…”

Section: Introductionmentioning

confidence: 99%

Heme-binding enables allosteric modulation in an ancient TIM-barrel glycosidase

et al. 2021

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Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. Here, we present the exceptional properties of a putative ancestor of bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the ∼5500 reported crystallographic structures of ∼1400 modern glycosidases show a bound porphyrin. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein reconstructions to reveal valuable but unexpected biomolecular features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.

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“…[44] In particular, the flexibility of the protein being used in the design of a new Kemp eliminase has been studied. [45,46]…”

Section: Implications In the De Novo Design Of A Kemp Eliminasementioning

confidence: 99%

Catalytic Effect of Electric Fields on the Kemp Elimination Reactions with Neutral Bases

et al. 2020

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The effect of solvent reaction fields and oriented electric fields on the Kemp elimination reaction between methylamine or imidazole and 5-nitrobenzisoxazole has been theoretically studied. The Kemp reaction is the most widely used for the design of new enzymes. Our results, using the SMD continuous model for solvents, are in quite good agreement with the experimental fact that the rate of the analogous reaction with butylamine is one order of magnitude smaller in water than in acetonitrile. In the case of external electric fields, our results show that they can increase or decrease the energy barrier depending on the magnitude and orientation of the field. A duly oriented electric field may have a notable catalytic effect on the reaction. So, external electric fields and reaction fields due to the medium can contribute to the design of new enzymes. Several factors that must be taken into account to increase the catalytic effect are discussed.

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Manipulating Conformational Dynamics To Repurpose Ancient Proteins for Modern Catalytic Functions

Cited by 46 publications

References 76 publications

Known Evolutionary Paths Are Accessible to Engineered ß-Lactamases Having Altered Protein Motions at the Timescale of Catalytic Turnover

Known Evolutionary Paths Are Accessible to Engineered ß-Lactamases Having Altered Protein Motions at the Timescale of Catalytic Turnover

Heme-binding enables allosteric modulation in an ancient TIM-barrel glycosidase

Catalytic Effect of Electric Fields on the Kemp Elimination Reactions with Neutral Bases

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