ABSTRACT
Engineering artificial enzymes with high activity and catalytic mechanism different from naturally occurring enzymes is a challenge in protein design. For example, many attempts have been made to obtain active hydrolases by introducing a Ser â Cys exchange at the respective catalytic triads, but this generally induced a breakdown of activity. We now report that this long-standing dogma no longer pertains, provided additional mutations are introduced by directed evolution. By employing Candida antarctica lipase B (CALB) as the model enzyme with the Ser-His-Asp catalytic triad, a highly active cysteine-lipase having a Cys-His-Asp catalytic triad and additional mutations W104V/A281Y/A282Y/V149G can be evolved, showing a 40-fold higher catalytic efficiency than wild-type CALB in the hydrolysis of 4-nitrophenyl benzoate, and tolerating bulky substrates. Crystal structures, kinetics, MD simulations and QM/MM calculations reveal dynamic features and explain all results, including the preference of a two-step mechanism involving the zwitterionic pair Cys105-/His224+ rather than a concerted process.
Subject(s)
Cysteine/chemistry , Lipase/chemistry , Binding Sites , Candida/enzymology , Catalysis , Catalytic Domain , Crystallography, X-Ray , Enzyme Activation , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Hydrolysis , Kinetics , Lipase/genetics , Lipase/metabolism , Models, Molecular , Mutation , Protein Conformation , Protein Engineering/methods , Substrate SpecificityABSTRACT
Chiral α-functionalized carboxylic acids are valuable precursors for a variety of medicines and natural products. Herein, we described an engineered fatty acid photodecarboxylase (CvFAP)-catalyzed kinetic resolution of α-amino acids and α-hydroxy acids, which provides the unreacted R-configured substrates with high yields and excellent stereoselectivity (ee up to 99 %). This efficient light-driven process requires neither NADPH recycling nor prior preparation of esters, which were required in previous biocatalytic approaches. The structure-guided engineering strategy is based on the scanning of large amino acids at hotspots to narrow the substrate binding tunnel. To the best of our knowledge, this is the first example of asymmetric catalysis by an engineered CvFAP.
