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 alpha,beta-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.
Svedendahl
Maria
M
Department of Biochemistry, Royal Institute of Technology KTH, AlbaNova University Center, Stockholm, Sweden.
Carlqvist
Peter
P
Branneby
Cecilia
C
Allnér
Olof
O
Frise
Anton
A
Hult
Karl
K
Berglund
Per
P
Brinck
Tore
T
D055162:Biocatalysis; D002175:Candida / Q000201:enzymology*; D003198:Computer Simulation; D004852:Epoxy Compounds / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins; D007700:Kinetics; D008049:Lipase / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D010084:Oxidation-Reduction
D004852:Epoxy Compounds; D005656:Fungal Proteins; D008049:Lipase; C431349:lipase B, Candida antarctica