Directed Evolution of Enantioselective Enzymes: Iterative Cycles of CASTing for Probing Protein‐Sequence Space

Reetz, Manfred T.; Wang, Liwen; Bocola, Marco

doi:10.1002/ange.200502746

Cited by 120 publications

(144 citation statements)

References 91 publications

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“…While previous studies of mutationally accessible pathways to high-fitness sequences (29)(30)(31)(32)40) have explored the evolution of novel functions, pathways were not penalized if they simultaneously caused the organism to lose other functionality. In this sense, our experiment provides the first glimpse of the constraint imposed on evolutionary pathways by the adaptive conflict between inhibitor resistance and maintenance of sufficient endogenous activity.…”

Section: Discussionmentioning

confidence: 99%

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Stepwise acquisition of pyrimethamine resistance in the malaria parasite

Lozovsky

Chookajorn

Brown

et al. 2009

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

257

328

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The spread of high-level pyrimethamine resistance in Africa threatens to curtail the therapeutic lifetime of antifolate antimalarials. We studied the possible evolutionary pathways in the evolution of pyrimethamine resistance using an approach in which all possible mutational intermediates were created by site-directed mutagenesis and assayed for their level of drug resistance. The coding sequence for dihydrofolate reductase (DHFR) from the malaria parasite Plasmodium falciparum was mutagenized, and tests were carried out in Escherichia coli under conditions in which the endogenous bacterial enzyme was selectively inhibited. We studied 4 key amino acid replacements implicated in pyrimethamine resistance: N51I, C59R, S108N, and I164L. Using empirical estimates of the mutational spectrum in P. falciparum and probabilities of fixation based on the relative levels of resistance, we found that the predicted favored pathways of drug resistance are consistent with those reported in previous kinetic studies, as well as DHFR polymorphisms observed in natural populations. We found that 3 pathways account for nearly 90% of the simulated realizations of the evolution of pyrimethamine resistance. The most frequent pathway (S108N and then C59R, N51I, and I164L) accounts for more than half of the simulated realizations. Our results also suggest an explanation for why I164L is detected in Southeast Asia and South America, but not at significant frequencies in Africa.adaptive landscape ͉ drug resistance ͉ evolution

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Section: Discussionmentioning

confidence: 99%

“…In previous experiments carried out to examine possible pathways for the evolution of various functions in various proteins (29)(30)(31)(32), epistasis among the mutant sites substantially reduced the number of selectively accessible mutational pathways. Mutational bias and adaptive conflicts were not explicitly addressed, however.…”

mentioning

confidence: 99%

Stepwise acquisition of pyrimethamine resistance in the malaria parasite

Lozovsky

Chookajorn

Brown

et al. 2009

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

257

328

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“…Although substrate acceptance has been made possible by our method, activities are modest. Combining the present approach with such techniques as DNA shuffling (7)(8)(9)(10)(11) or iterative saturation mutagenesis at sites aligning the reshaped binding pocket (11,12) may allow for further improvements.…”

Section: Resultsmentioning

confidence: 99%

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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120

118

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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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“…To this end, we have proposed and implemented iterative saturation mutagenesis (ISM) as a general approach which comes closer to the above goal (Reetz et al 2006b;Reetz et al 2006c;. The concept of ISM can be used to influence very different catalytic properties of an enzyme, such as thermostability in the form of B-FIT (Reetz et al 2006b; or substrate acceptance and enantioselectivity using CASTing (Reetz et al 2006c;Bartsch et al 2008;Liang et al 2007).…”

Section: Introductionmentioning

confidence: 99%

“…The concept of ISM can be used to influence very different catalytic properties of an enzyme, such as thermostability in the form of B-FIT (Reetz et al 2006b; or substrate acceptance and enantioselectivity using CASTing (Reetz et al 2006c;Bartsch et al 2008;Liang et al 2007). Based on structural information of the enzyme and by focusing on positions expected to be crucial for a given catalytic property, several sites, composed of one or more residues, can be selected and randomized.…”

Section: Introductionmentioning

confidence: 99%

Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates

Sanchis

Fernández

Carballeira

et al. 2008

Appl Microbiol Biotechnol

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126

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Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene's QuikChange™ sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.

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Directed Evolution of Enantioselective Enzymes: Iterative Cycles of CASTing for Probing Protein‐Sequence Space

Abstract: Umbaumaßnahmen am Enzym: Der jüngst eingeführte CAST (=combinatorial active‐site saturation test) wurde in Iterationszyklen bei der gerichteten Evolution enantioselektiver Wildtyp‐Epoxidhydrolasen (WT‐EH) angewendet.

Cited by 120 publications

References 91 publications

Stepwise acquisition of pyrimethamine resistance in the malaria parasite

Stepwise acquisition of pyrimethamine resistance in the malaria parasite

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

Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates

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