| 1. |
- Jost, Etta, et al.
(författare)
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Variants of the Acyltransferase from Mycobacterium smegmatis Enable Enantioselective Acyl Transfer in Water
- 2020
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Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 10:18, s. 10500-10507
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Tidskriftsartikel (refereegranskat)abstract
- The acyltransferase from Mycobacterium smegmatis (MsAcT) complements the well-established acylation activity of hydrolases in organic solvents with its activity to perform acylation reactions (among other reactions) in an aqueous environment. The enzyme’s potential is however limited, due to its poor regio- and stereoselectivity with enantioselectivities (E-values) below 20 for bulky (aromatic) substrates. By applying computer-guided rational design, a library of single variants was designed that allowed conversion of a set of previously challenging substrates with good activity and E-values up to >200. The computational predictions were found to be in agreement with experimental data, which in turn allowed for the generation of even more active and selective double variants. Overall, the produced set of variants provides a toolbox for the enantioselective acylation of challenging alcohols in water, effectively contributing to an alternative to reactions in organic solvents.
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| 2. |
- Sheng, Xiang, et al.
(författare)
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Computational Study of the Fries Rearrangement Catalyzed by Acyltransferase from Pseudomonas protegens
- 2024
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Ingår i: ChemistryOpen. - 2191-1363.
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Tidskriftsartikel (refereegranskat)abstract
- The acyltransferase from Pseudomonas protegens (PpATase) catalyzes in nature the reversible transformation of monoacetylphloroglucinol to diacetylphloroglucinol and phloroglucinol. Interestingly, this enzyme has been shown to catalyze the promiscuous transformation of 3-hydroxyphenyl acetate to 2′,4′-dihydroxyacetophenone, representing a biological version of the Fries rearrangement. In the present study, we report a mechanistic investigation of this activity of PpATase using quantum chemical calculations. A detailed mechanism is proposed, and the energy profile for the reaction is presented. The calculations show that the acylation of the enzyme is highly exothermic, while the acetyl transfer back to the substrate is only slightly exothermic. The deprotonation of the C6−H of the substrate is rate-limiting, and a remote aspartate residue (Asp137) is proposed to be the general base group in this step. Analysis of the binding energies of various acetyl acceptors shows that PpATase can promote both intramolecular and intermolecular Fries rearrangement towards diverse compounds.
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| 3. |
- Sheng, Xiang, et al.
(författare)
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Mechanism of Biocatalytic Friedel-Crafts Acylation by Acyltransferase from Pseudomonas protegens
- 2020
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Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 10:1, s. 570-577
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Tidskriftsartikel (refereegranskat)abstract
- Acyltransferases isolated from Pseudomonas protegens (PpATase) and Pseudomonas fluorescens (PfATase) have recently been reported to catalyze the Friedel-Crafts acylation, providing a biological version of this classical organic reaction. These enzymes catalyze the cofactor-independent acylation of monoacetylphloroglucinol (MAPG) to diacetylphloroglucinol (DAPG) and phloroglucinol (PG) and have been demonstrated to have a wide substrate scope, making them valuable for potential applications in biocatalysis. Herein, we present a detailed reaction mechanism of PpATase on the basis of quantum chemical calculations, employing a large model of the active site. The proposed mechanism is consistent with available kinetics, mutagenesis, and structural data. The roles of various active site residues are analyzed. Very importantly, the Asp137 residue, located more than 10 angstrom from the substrate, is predicted to be the proton source for the protonation of the substrate in the rate-determining step. This key prediction is corroborated by site-directed mutagenesis experiments. Based on the current calculations, the regioselectivity of PpATase and its specificity toward non-natural substrates can be rationalized.
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