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Glucagon stands out as a pivotal peptide hormone, instrumental in controlling blood glucose levels and lipid metabolism. While the formation of glucagon amyloid fibrils has been documented, their biological functions remain enigmatic. Recently, we demonstrated experimentally that glucagon amyloid fibrils can act as catalysts in several biological reactions including esterolysis, lipid hydrolysis, and dephosphorylation. Herein, we present a multiscale quantum mechanics/molecular mechanics (QM/MM) simulation of the acylation step in the esterolysis of para-nitrophenyl acetate (p-NPA), catalyzed by native glucagon amyloid fibrils, serving as a model system to elucidate their catalytic function. This step entails a concerted mechanism, involving proton transfer from serine to histidine, followed by the nucleophilic attack of the serine oxy anion on the carbonyl carbon of p-NPA. We computed the binding energy and free-energy profiles of this reaction using the protein-dipole Langevin-dipole (PDLD) within the linear response approximation (LRA) framework (PDLD/S-LRA-2000) and the empirical valence bond (EVB) methods. This included simulations of the reaction in an aqueous environment and in the fibril, enabling us to estimate the catalytic effect of the fibril. Our EVB calculations obtained a barrier of 23.4 kcal mol-1 for the enzyme-catalyzed reaction compared to the experimental value of 21.9 kcal mol-1 (and a calculated catalytic effect of 3.2 kcal mol-1 compared to the observed effect of 4.7 kcal mol-1). This close agreement together with the barrier reduction when transitioning from the reference solution reaction to the amyloid fibril provides supporting evidence to the catalytic role of glucagon amyloid fibrils. Moreover, employing the PDLD/S-LRA-2000 approach further reinforced exclusively the enzyme's catalytic role. The results presented in this study contribute significantly to our understanding of the catalytic role of glucagon amyloid fibrils, marking, to the best of our knowledge, the first-principles mechanistic investigation of fibrils using QM/MM methods. Therefore, our findings offer fruitful insights for future research into the mechanisms of related amyloid catalysis.
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Aliphatic Polyester Recognition and Reactivity at the Active Cleft of a Fungal Cutinase
Protein engineering of cutinases is a promising strategy for the biocatalytic degradation of non-natural polyesters. We report a mechanistic study addressing the hydrolysis of the aliphatic polyester poly(butylene succinate, or PBS) by the fungal Apergillus oryzae cutinase enzyme. Through atomistic molecular dynamics simulations and advanced alchemical transformations, we reveal how three units of a model PBS substrate fit the active site cleft of the enzyme, interacting with hydrophobic side chains. The substrate ester moiety approaches the Asp–His–Ser catalytic triad, displaying catalytically competent conformations. Acylation and deacylation hydrolytic reactions were modeled according to a canonical esterase mechanism using umbrella sampling simulations at the quantum mechanical/molecular mechanical DFT(B3LYP)/6–31G**/AMBERff level. The free energy profiles of both steps show a high-energy tetrahedral intermediate resulting from the nucleophilic attack on the ester’s carboxylic carbon. The free energy barrier of the acylation step is higher (20.2 ± 0.6 kcal mol–1) than that of the deacylation step (13.6 ± 0.6 kcal mol–1). This is likely due to the interaction of the ester’s carboxylic oxygen with the oxyanion hole in the reactive conformation of the deacylation step. In contrast, these interactions form as the reaction proceeds during the acylation step. The formation of an additional hydrogen bond interaction with the side chain of Ser48 is crucial to stabilizing the developing charge at the carboxylic oxygen, thus lowering the activation free energy barrier. These mechanistic insights will inform the design of enzyme variants with improved activity for plastic degradation.
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- PAR ID:
- 10591133
- Publisher / Repository:
- American Chemical Society
- Date Published:
- Journal Name:
- Journal of Chemical Information and Modeling
- Volume:
- 65
- Issue:
- 9
- ISSN:
- 1549-9596
- Page Range / eLocation ID:
- 4662-4673
- Subject(s) / Keyword(s):
- cutinase enzyme polyester reactivity qm/mm quantum mechanics molecular mechanics molecular dynamics amber quick
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
-
Accepted Manuscript
- Journal Article:
- https://doi.org/10.1021/acs.jcim.5c00739
