Computational design of a lipase for catalysis of the Diels-Alder reaction

Abstract

Combined molecular docking, molecular dynamics (MD) and density functional theory (DFT) studies have been employed to study catalysis of the Diels-Alder reaction by a modified lipase. Six variants of the versatile enzyme {\em Candida Antarctica} lipase B (CALB) have been rationally engineered {\em in silico} based on the specific characteristics of the pericyclic addition. A kinetic analysis reveals that hydrogen bond stabilization of the transition state and substrate binding are key components of the catalytic process. In the case of substrate binding, which has the greater potential for optimization, both binding strength and positioning of the substrates are important for catalytic efficiency. The binding strength is determined by hydrophobic interactions and can be tuned by careful selection of solvent and substrates. The MD simulations show that substrate positioning is sensitive to cavity shape and size, and can be controlled by a few rational mutations. The well-documented S105A mutation is essential to enable sufficient space in the vicinity of the oxyanion hole. Moreover, bulky residues on the edge of the active site hinders the formation of a sandwich-like near-attack conformer (NAC), and the I189A mutation is needed to obtain enough space above the face of the $\alpha$,$\beta$-double bond on the dienophile. The double mutant S105A/I189A performs quite well for two of three dienophiles. Based on binding constants and NAC energies obtained from MD simulations combined with activation energies from DFT computations, relative catalytic rates ($v_{cat}/v_{uncat}$) of up to $10^3$ are predicted.Response to Reviewers: We are happy with the favorable comments by the reviewers. We have have corrected the manuscript as suggested by reviewer 1 1. A sentence explaining catalytic promiscuity has been added. 2. A paragraph discussing the relationship between the NAC concept and the Reaction Force has been added.We have shortened the manuscript as suggested by reviewer 2. In particular, we have moved information regarding the structural relaxation of protein structure in MD simulations to the supplementary material. Abstract Combined molecular docking, molecular dynamics (MD) and density functional theory (DFT) studies have been employed to study catalysis of the DielsAlder reaction by a modified lipase. Six variants of the versatile enzyme Candida Antarctica lipase B (CALB) have been rationally engineered in silico based on the specific characteristics of the pericyclic addition. A kinetic analysis reveals that hydrogen bond stabilization of the transition state and substrate binding are key components of the catalytic process. In the case of substrate binding, which has the greater potential for optimization, both binding strength and positioning of the substrates are important for catalytic efficiency. The binding strength is determined by hydrophobic interactions and can be tuned by careful selection of solvent and substrates. The MD simulations show that substrate positioning is sensitive to cavity s...

Designing a New Diels–Alderase: A Combinatorial, Semirational Approach Including Dynamic Optimization.

“…As seen from the corresponding columns in Table 3, the 'absolute' TS stabilization is small and amounts to at most À2.2 kcal/mol (1c+2). This result correlates with previous findings by ourselves 19,20 and others, 15,16 stating that the stabilization of the TS is a relatively small portion of the net catalytic effect. The fact that ΔG NAC ‡ for 1d is larger than the corresponding uncatalyzed energy is a consequence of interactions in the active site that hinders the TS formation.…”

“…We have been able to reach a high degree of shape complementarity, leading to extremely high formation of 'near-attack conformers' compared to previous designs. 9,19 Understanding and designing enzyme catalysis is a complex problem which requires a range of methods, and with this work, we have shown that a combination of several standard, well-established techniques gives detailed information and control of the design process. We have improved the chance of success by not designing to a specific pair of reagents, instead selecting reagent pairs most likely to be optimum for a given set of mutations.…”

Algorithm‐based coevolution network identification reveals key functional residues of the α/β hydrolase subfamilies

“…The predicted functional covariant residues are shown in Figure A whose function includes the activity, thermostability, and enantioselectivity. And the residues in red are reported functional sites . For example, E188 has a strong correlation with W104, D134, and M129 (Figure B).…”

Computational Enzyme Design: Advances, Hurdles and Possible Ways Forward

“…We have been interested in re-designing enzymes containing an 'oxyanion hole’ moiety[68] to catalyze the intermolecular Diels-Alder reaction, which is virtually unknown in nature[69–71] and therefore highly interesting for de novo design. [2] Our work was initially guided by a rational design framework (Figure 1a),[72] which can be said to rely on the concept of 'catalytic promiscuity’. [73] Catalytic promiscuity is in turn a manifest of evolutionary heritage,[23, 74, 75] where a wide array of reactions can be catalyzed by structurally and functionally similar enzymes.…”

“…Another key feature of our approach is that we rely on molecular dynamics (MD) simulations for evaluation, selection and quantification of different variants,[72, 76] a practice that has recently begun appearing in several other studies (Figure 1b). [21, 67, 79] We refer to this as ‘dynamic design’.…”