Cecilia Branneby

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.

Transaminations with isopropyl amine: equilibrium displacement with yeast alcohol dehydrogenase coupled to in situ cofactor regeneration

Enantiopure chiral amines synthesis using omega-transaminases is hindered by an unfavourable equilibrium, but when using isopropylamine as the amine donor the equilibrium can be completely displaced by using a specific dehydrogenase in situ for removal of formed acetone.

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.

Rational design of Pseudozyma antarctica lipase B yielding a general esterification catalyst.

Pseudozyma antarctica lipase B (CALB) shows activity in the acrylation of hydroxypropylcarbamate, a racemic mixture of enantiomers of primary and secondary alcohols. However, full conversion is hampered by the slowly reacting S enantiomer of the secondary alcohol. The same is true for a wide range of secondary alcohols, for example, octan‐2‐ and ‐3‐ol. In order to get high conversion in these reactions in a short time, the stereospecificity pocket of CALB was redesigned by using predictions from molecular modeling. Positions 278, 104, and 47 were targeted, and a library for two‐site saturation mutagenesis at positions 104 and 278 was constructed. The library was then screened for hydrolysis of acrylated hydroxypropylcarbamates. The best mutants L278A, L278V, L278A/W104F, and L278A/W104F/S47A showed an increased conversion in hydrolysis and transesterification of more than 30 %. While the wild‐type showed only 73 % conversion in the acrylation of hydroxypropylcarbamate after 6 h, 97 % conversion was achieved by L278A in this time. Besides this, L278A/W104F reached >96 % conversion in the acrylation of octan‐2‐ and ‐3‐ol within 48 h and showed a significant decrease in stereoselectivity, while the wild‐type reached only 68 and 59 % conversion, respectively. Thus the new biocatalysts can be used for efficient transformation of racemic alcohols and esters with high activity when the high stereoselectivity of the wild‐type hampers complete conversion of racemic substrates in a short time.

7.18 C-X Bond Formation : Transaminases as Chiral Catalysts: Mechanism, Engineering, and Applications

Enantiomerically pure amines and amino acids are important building blocks in academic research as well as in industrial-scale chemical production. Transaminases are versatile enzymes providing access to such compounds of high enantiomeric excess. This chapter illustrates the available strategies with transaminases such as kinetic resolution or stereoselective synthesis and highlights many successful examples for amino acid and chiral amines synthesis. There are some known challenges linked to the use of transaminases, for example in terms of unfavorable equilibria and inhibition. Several successful examples to overcome these limitations are presented. Also, the classification of transaminases, mechanistic details, and various strategies for optimization are discussed.