Maria Svedendahl

Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions

Michael‐type additions of various thiols and α,β‐unsaturated carbonyl compounds were performed in organic solvent catalyzed by wild‐type and a rationally redesigned mutant of Candida antarctica lipase B. The mutant lacks the nucleophilic serine 105 in the active‐site; this results in a changed catalytic mechanism of the enzyme. The possibility of utilizing this mutant for Michael‐type additions was initially explored by quantum‐chemical calculations on the reaction between acrolein and methanethiol in a model system. The model system was constructed on the basis of docking and molecular‐dynamics simulations and was designed to simulate the catalytic properties of the active site. The catalytic system was explored experimentally with a range of different substrates. The kcat values were found to be in the range of 10−3 to 4 min−1, similar to the values obtained with aldolase antibodies. The enzyme proficiency was 107. Furthermore, the Michael‐type reactions followed saturation kinetics and were confirmed to take place in the enzyme active site.

Direct Epoxidation in Candida antarctica Lipase B Studied by Experiment and Theory

Candida antarctica lipase B (CALB) is a promiscuous serine hydrolase that, besides its native function, catalyzes different side reactions, such as direct epoxidation. A single‐point mutant of CALB demonstrated a direct epoxidation reaction mechanism for the epoxidation of α,β‐unsaturated aldehydes by hydrogen peroxide in aqueous and organic solution. Mutation of the catalytically active Ser105 to alanine made the previously assumed indirect epoxidation reaction mechanism impossible. Gibbs free energies, activation parameters, and substrate selectivities were determined both computationally and experimentally. The energetics and mechanism for the direct epoxidation in CALB Ser105Ala were investigated by density functional theory calculations, and it was demonstrated that the reaction proceeds through a two step‐mechanism with formation of an oxyanionic intermediate. The active‐site residue His224 functions as a general acid‐base catalyst with support from Asp187. Oxyanion stabilization is facilitated by two hydrogen bonds from Thr40.

Reversed Enantiopreference of an ω-Transaminase by a Single-Point Mutation

Altering the characteristics of an active‐site loop in an (S)‐selective ω‐transaminase from Arthrobacter citreus (variant CNB05‐01) influences the enantioselectivity. This active‐site loop belongs to the second subunit of the dimeric enzyme structure that participates in the coordination of pyridoxal‐5′‐phosphate (PLP) in the so called “phosphate group binding cup”. Three amino acid residues (E326, V328, and Y331) in this loop are selected by homology modeling for site‐directed mutagenesis aiming to increase the enzyme enantioselectivity for 4‐fluorophenylacetone. By combining these mutations, five enzyme variants are created. The performance of these variants is explored using a model system consisting of isopropylamine and 4‐fluorophenylacetone or 4‐nitroacetophenone in asymmetric synthesis using a whole‐cell system approach. Three of the five variants show increased enantioselectivity for 4‐fluorophenylacetone compared to CNB05‐01. Variant CNB05‐01/Y331C increases the enantioselectivity from 98 % ee to over 99.5 % ee. A single‐point mutation, V328A, turn the (S)‐selective ω‐transaminase into an (R)‐selective enzyme. This switch in enantioselectivity is substrate dependent, exhibiting (R) selectivity for 4‐fluorophenylacetone and retaining (S) selectivity for 4‐nitroacetophenone. The shift in enantiopreference is further confirmed by molecular docking simulations. Homology modeling is shown to be a powerful tool to target important amino acid residues in this enzyme in order to improve enantioselectivity by rational design.

Suppressed Native Hydrolytic Activity of a Lipase to Reveal Promiscuous Michael Addition Activity in Water

Suppression of the native hydrolytic activity of Pseudozyma antarctica lipase B (PalB) (formerly Candida antarctica lipase B) in water is demonstrated. By replacing the catalytic Ser 105 residue with an alanine unit, promiscuous Michael addition activity is favored. A Michael addition reaction between methyl acrylate and acetylacetone was explored as a model system. For the PalB Ser 105 Ala mutant, the hydrolytic activity was suppressed more than 1000 times and, at the same time, the Michael addition activity was increased by a factor of 100. Docking studies and molecular dynamics simulations revealed an increased ability of the PalB Ser 105 Ala mutant to harbor the substrates close to a catalytically competent conformation.