Computational Studies of Candida Antarctica Lipase B to Test Its Capability as a Starting Point To Redesign New Diels–Alderases

Abstract

The design of new biocatalysts is a target that is receiving increasing attention. One of the most popular reactions in this regard is the Diels-Alder cycloaddition due to its applications in organic synthesis and the absence of efficient natural enzymes that catalyze it. In this paper, the possibilities of using the highly promiscuous Candida Antarctica lipase B (CALB) as a protein scaffold to re-design a Diels-Alderase has been explored by means of theoretical quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations. Free energy surfaces have been computed for two reactions in the wild-type and in several mutants with hybrid AM1/MM potentials with corrections at M06-2X/MM level. The study of the counterpart reactions in solution has allowed performing comparative analysis that render interesting conclusion. Since the dienophile anchors very well in the oxyanion hole of all tested protein variants, the slight electronic changes from reactant complex to the transition state suggest that mutations should be focused in favoring the formation of reactive conformations of reactant complex that, in turn, would reduce the energy barrier.

Exploring the Origin of Amidase Substrate Promiscuity in CALB by a Computational Approach

“…Kinetic isotope effects (KIEs) were calculated, at M06-2X/MM and AM1/MM levels, from isotopic substitution of the key atoms in the reactants and the different transition state structures, as described in previous papers. ,, Details and further discussions are provided in the Supporting Information.…”

Computational Studies Suggest Promiscuous Candida antarctica Lipase B as an Environmentally Friendly Alternative for the Production of Epoxides

“…In this first step, a negative charge is developed in the carbonyl oxygen atom of the substrate, which is stabilized by the oxyanion hole formed by Thr40 and Gln106. The role of this oxyanion hole is well known and has been stated in other reactions catalyzed by CALB. − , The formation of the epoxide ring by the bond forming between C 2 and O 1 takes place in the second step. This second step is assisted by the acid/base character of the His224 that permits the transfer of the proton, H 1 , to oxygen, O 2 , thus forming a water molecule.…”

“…The role of this oxyanion hole is well known and has been stated in other reactions catalyzed by CALB. [22][23][24]65 The formation of the epoxide ring by the bond forming between C 2 and O 1 takes place in the second step. This second step is assisted by the acid/base character of the His224 that permits the transfer of the proton, H 1 , to oxygen, O 2 , thus forming a water molecule.…”

“…One of the best-characterized enzymes in this field is Candida antarctica lipase B (CALB). CALB is widely used in industries and has served as a model to study enzyme promiscuity, either by experimental or theoretical approaches. ,− CALB has been demonstrated to be able to perform a wide variety of promiscuous reactions, such as synthesis of esters and amides, transacylation of alcohols, epoxidation reactions, aldol additions, and Michael-type additions, , among many others. , It has been proposed that this serine hydrolase performs its activity due to the presence of a catalytic triad Ser/His/Asp in the active site, as well as an oxyanion hole formed by Thr and Gln, which stabilizes the negative charge accumulating on the carbonyl oxygen atom of the substrate in different states along the reaction. The hydrolytic character of CALB is achieved by the catalytic Ser105.…”