Learning from Directed Evolution: Further Lessons from Theoretical Investigations into Cooperative Mutations in Lipase Enantioselectivity

Reetz, Manfred T.; Puls, Michael; Carballeira, José Daniel; Vogel, Andreas; Jaeger, Karl‐Erich; Eggert, Thorsten; Thiel, Walter; Bocola, Marco; Otte, Nikolaj

doi:10.1002/cbic.200600359

Cited by 111 publications

(80 citation statements)

References 55 publications

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“…Theoretical methodology in asymmetric biocatalysis has relied mainly on substrate docking or molecular dynamics simulations of the enzyme-substrate (ES) complexes in the ground state. [7,8] To some extent, this has been successful in qualitatively guiding the experimental work as to which parts of the active site to manipulate and also to provide some rationalization for observed trends. These approaches are, however, inherently deficient since they do not consider the transition states and can thus not be fully quantitative.…”

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confidence: 99%

Quantum Chemistry as a Tool in Asymmetric Biocatalysis: Limonene Epoxide Hydrolase Test Case

Lind

Himo

2013

Angewandte Chemie

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Quantum chemical models of enzyme active sites have in recent years proven to be a very powerful tool in the elucidation of enzymatic reaction mechanisms. [1] In the socalled cluster approach, a limited part of the enzyme around the active site is cut out and treated using relatively accurate electronic structure methods, typically hybrid density functional theory (DFT). The missing enzyme surrounding is approximated by a homogeneous polarizable continuum model with some assumed dielectric constant. [2] A large variety of enzymatic systems have been investigated quite successfully using this approach and a wealth of mechanistic insight has been gained. [3] A few years ago, active site models consisted typically of less than 100 atoms. However, with today s computers and using the same computational protocol, it is possible to treat more than 250 atoms quite routinely. These developments have paved the way for wider applications of the cluster approach, beyond the pure mechanistic investigations.To investigate and explain sources of various kinds of selectivities, in particular enantioselectivity, one has typically to reproduce relative transition-state energies on the order of 1 kcal mol À1 . The accuracy of modern DFT methods has been proven to be sufficiently high to achieve this. In particular, these methods have in recent years been applied very successfully to a multitude of problems in the field of asymmetric homogenous catalysis. [4] Modeling enzymatic enantioselectivity with the cluster approach has remained somewhat out of reach because larger active-site models are in general required to create the chiral environment provided by the enzyme. Herein, we will demonstrate that this kind of modeling approach is indeed able to reproduce and rationalize enantioselectivity in enzymes and has thus the potential to become a valuable tool also in the field of asymmetric biocatalysis.Today enzymes are increasingly used in synthetic chemistry for the production of base and fine chemicals. [5] Indeed, biocatalytic processes are starting to replace transition-metalcatalyzed reactions for large-scale production of drug compounds. [6] One of the most attractive features of enzymes in this respect is their high levels of selectivity. Various engineering techniques have been developed to manipulate the properties of enzymes to achieve the desired function. These techniques range from pure combinatorial approaches to semi-rational and fully rational designs, which are based on the detailed knowledge of the structure and mechanism of the enzymes. Theoretical methodology in asymmetric biocatalysis has relied mainly on substrate docking or molecular dynamics simulations of the enzyme-substrate (ES) complexes in the ground state. [7,8] To some extent, this has been successful in qualitatively guiding the experimental work as to which parts of the active site to manipulate and also to provide some rationalization for observed trends. These approaches are, however, inherently deficient since they do not consider the transition ...

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confidence: 99%

Quantum Chemistry as a Tool in Asymmetric Biocatalysis: Limonene Epoxide Hydrolase Test Case

Lind

Himo

2013

Angewandte Chemie

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“…With the aid of the wild-type LipA crystal structure (72), molecular dynamics simulations were carried out to predict the roles of the six amino acids, and the main source of enantioselectivity was traced to the cooperative influence of two of the six mutations, namely, S53P and L162G (12). Based on this analysis, several single and multiple mutants were generated by site-directed mutagenesis, and it was shown that the double S53P L162G mutant was even more enantioselective than its parent with six mutations (89). The folding of active LipA involves the participation of the lipase-specific foldase Lif, which is encoded upstream of lipA.…”

Section: Pseudomonas Physiological and Metabolic Diversitymentioning

confidence: 99%

Pseudomonas 2007

et al. 2008

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“…Remote effects in the directed evolution of enantioselective enzymes have been noted earlier but these do not involve allostery (7)(8)(9)(10)(11)16). Here, we describe the experimental implementation of induced allostery using a Baeyer-Villiger monooxygenase (BVMO) as the model enzyme (17)(18)(19)(20)(21).…”

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confidence: 91%

“…When applying this technique to enzymes displaying strong allosteric effects or coupled motions along the reaction coordinate (15), it is necessary to consider such phenomena to make reasonable decisions regarding the appropriate randomization sites, that is, to identify those residues that do align the binding pocket when the actual catalytic transformation occurs. A very different approach would be the idea of "inducing" allosteric communication at a remote site that results in altered structure and dynamics at the active site, thereby influencing enzyme activity and enantioselectivity.Remote effects in the directed evolution of enantioselective enzymes have been noted earlier but these do not involve allostery (7)(8)(9)(10)(11)16). Here, we describe the experimental implementation of induced allostery using a Baeyer-Villiger monooxygenase (BVMO) as the model enzyme (17-21).…”

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Induced allostery in the directed evolution of an enantioselective Baeyer–Villiger monooxygenase

Acevedo

Reetz

2010

Proc. Natl. Acad. Sci. U.S.A.

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The molecular basis of allosteric effects, known to be caused by an effector docking to an enzyme at a site distal from the binding pocket, has been studied recently by applying directed evolution. Here, we utilize laboratory evolution in a different way, namely to induce allostery by introducing appropriate distal mutations that cause domain movements with concomitant reshaping of the binding pocket in the absence of an effector. To test this concept, the thermostable Baeyer-Villiger monooxygenase, phenylacetone monooxygenase (PAMO), was chosen as the enzyme to be employed in asymmetric Baeyer-Villiger reactions of substrates that are not accepted by the wild type. By using the known X-ray structure of PAMO, a decision was made regarding an appropriate site at which saturation mutagenesis is most likely to generate mutants capable of inducing allostery without any effector compound being present. After screening only 400 transformants, a double mutant was discovered that catalyzes the asymmetric oxidative kinetic resolution of a set of structurally different 2-substituted cyclohexanone derivatives as well as the desymmetrization of three different 4-substituted cyclohexanones, all with high enantioselectivity. Molecular dynamics (MD) simulations and covariance maps unveiled the origin of increased substrate scope as being due to allostery. Large domain movements occur that expose and reshape the binding pocket. This type of focused library production, aimed at inducing significant allosteric effects, is a viable alternative to traditional approaches to "designed" directed evolution that address the binding site directly.allosteric effects | enzymes | molecular dynamics simulations | protein engineering P rotein allostery has been recognized as a positive or negative cooperative event, leading to a structural change at the binding site as a result of distal docking of a molecule acting as an effector (1-5). Allosteric effects can be influenced by such factors as variation in pH, temperature, ionic strength, and covalent modification as well as mutational changes. A variety of experimental and computational techniques have been utilized to unravel the intricacies of this phenomenon (1-5), but also to achieve useful applications such as the creation of protein switches (6). Among the techniques that have been applied to address these challenges is directed evolution, a method that is generally used to engineer the catalytic profiles of enzymes or the binding properties of proteins (7-11). In the endeavor to make directed evolution of enantioselective enzymes more efficient than in the past, we recently introduced the concept of iterative saturation mutagenesis according to which sites around the binding pocket are randomized iteratively, a given site being composed of one or more amino acid positions in the protein (12-14.) When applying this technique to enzymes displaying strong allosteric effects or coupled motions along the reaction coordinate (15), it is necessary to consider such phenomena to make reasonable...

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Learning from Directed Evolution: Further Lessons from Theoretical Investigations into Cooperative Mutations in Lipase Enantioselectivity

Cited by 111 publications

References 55 publications

Quantum Chemistry as a Tool in Asymmetric Biocatalysis: Limonene Epoxide Hydrolase Test Case

Quantum Chemistry as a Tool in Asymmetric Biocatalysis: Limonene Epoxide Hydrolase Test Case

Pseudomonas 2007

Induced allostery in the directed evolution of an enantioselective Baeyer–Villiger monooxygenase

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