Computationally designed libraries for rapid enzyme stabilization

Wijma, Hein J.; Floor, R.J.; Jekel, Peter A.; Baker, David; Marrink, Siewert J.; Janssen, Dick B.

doi:10.1093/protein/gzt061

Cited by 217 publications

(268 citation statements)

References 69 publications

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“…The thermal shift analysis of the CH55-LEH defines clearly an apparent T M of 79.7°C, which to the best of our knowledge is the highest T M value estimated to date for a natural EH and significantly higher than that estimated for the Re-LEH (50°C) [14]. Instead, although the apparent T M of the Tomsk-LEH is 74.5°C (Fig.…”

Section: Recombinant Expression In Escherichia Coli Of the Leh Homolomentioning

confidence: 56%

“…For example, Zheng and Reetz have significantly improved [up to > 99% enantiomeric excess (ee)] and even inverted the stereoselectivity of Re-LEH towards the model substrate cyclopentene oxide by using iterative saturation mutagenesis [13]. In another recent study, the Re-LEH thermostability has been significantly improved by screening computationally designed libraries, resulting in an increase of the melting temperature from 50 to 85°C [14].…”

Section: Introductionmentioning

confidence: 99%

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Discovery and characterization of thermophilic limonene‐1,2‐epoxide hydrolases from hot spring metagenomic libraries

et al. 2015

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The epoxide hydrolases (EHs) represent an attractive option for the synthesis of chiral epoxides and 1,2-diols which are valuable building blocks for the synthesis of several pharmaceutical compounds. A metagenomic approach has been used to identify two new members of the atypical EH limonene-1,2-epoxide hydrolase (LEH) family of enzymes. These two LEHs (Tomsk-LEH and CH55-LEH) show EH activities towards different epoxide substrates, differing in most cases from those previously identified for Rhodococcus erythropolis (Re-LEH) in terms of stereoselectivity. Tomsk-LEH and CH55-LEH, both from thermophilic sources, have higher optimal temperatures and apparent melting temperatures than Re-LEH. The new LEH enzymes have been crystallized and their structures solved to high resolution in the native form and in complex with the inhibitor valpromide for Tomsk-LEH and poly(ethylene glycol) for CH55-LEH. The structural analysis has provided insights into the LEH mechanism, substrate specificity and stereoselectivity of these new LEH enzymes, which has been supported by mutagenesis studies. DatabaseThe atomic coordinates and structure factors of the crystal structures have been deposited in the Protein Data Bank with the codes 5AIF (Tomsk-LEH native structure), 5AIG (Tomsk-LEH valpromide complex), 5AIH (CH55-LEH native structure) and 5AII (CH55-LEH PEG complex). Nucleotide sequence data are available in the GenBank databases under the accession numbers KP765711 (Tomsk-LEH) and KP765710 (CH55-LEH).Abbreviations CH55-LEH, limonene-1,2-epoxide hydrolase from the metagenomic DNA from the Chinese sample; de, diastereomeric excess; ee, enantiomeric excess; EH, epoxide hydrolase; LEH, limonene-1,2-epoxide hydrolase; Mt-LEH, Mycobacterium tuberculosis limonene-1,2-epoxide hydrolase; PEG, poly(ethylene glycol); Re-LEH, Rhodococcus erythropolis limonene-1,2-epoxide hydrolase; Tomsk-LEH, limonene-1,2-epoxide hydrolase from the metagenomic DNA from the Tomsk sample.

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Section: Recombinant Expression In Escherichia Coli Of the Leh Homolomentioning

confidence: 56%

Section: Introductionmentioning

confidence: 99%

Discovery and characterization of thermophilic limonene‐1,2‐epoxide hydrolases from hot spring metagenomic libraries

et al. 2015

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“…We find a promising example in the work by Wijma et al, where a hierarchical protocol is used to increase thermostabilty [139]. In this line, we expect the development of additional techniques combining quick bioinformatics (or knowledge-based) screening of a large sequence space, with a molecular modeling refinement of selected mutants.…”

Section: Catalytic Rate Constant Enhancementmentioning

confidence: 94%

“…Recently, Wijma and coworkers developed, and applied with success, a mixed approach which aims to obtain highly thermostable protein variants in a short time with minimum experimental screening [139]. In their computational workflow, potentially stabilizing mutations were firstly produced and scored with Rosetta [119] and FoldX [27].…”

Section: Protein Stability Improvementmentioning

confidence: 99%

Molecular Modeling in Enzyme Design, Toward In Silico Guided Directed Evolution

Monza

Acebes

Lucas

et al. 2017

Directed Enzyme Evolution: Advances and Applications

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Directed evolution creates diversity in subsequent rounds of mutagenesis in the quest of increased protein stability, substrate binding and catalysis. Although this technique does not require any structural/mechanistic knowledge of the system, the frequency of improved mutations is usually low. For this reason, computational tools are increasingly used to focus the search in sequence space, enhancing the efficiency of laboratory evolution. In particular, molecular modeling methods provide a unique tool to grasp the sequence/structure/function relationship The final publication is available at Springer via https://link.springer.com/chapter/10.1007/978-3-319-50413-1_10/fulltext.html of the protein to evolve, with the only condition that a structural model is provided. With this book chapter, we tried to guide the reader through the state of art of molecular modeling, discussing their strengths, limitations and directions. In addition, we suggest a possible future template for in silico directed evolution where we underline two main points: a hierarchical computational protocol combining several different techniques, and a synergic effort between simulations and experimental validation. IntroductionBiotechnology needs catalysts that can work under harsh conditions, catalyze a broad range of substrates, generate maximum amount of product, and tolerate changes in the environment. Enzymes, which are biodegradable and reusable catalysts [1], in addition to remarkable reaction rates, can work in environmentally friendly pH and temperature ranges, and display control over stereochemistry and regioselectivity which makes them ideal for many applications [2,3]. When thinking about enzymes, people normally associate them to expressions such as "perfect catalysts" or "outstanding reaction rate". In fact, there are examples of enzymes that catalyze reactions at extremely high rates such as triose phosphate isomerase, superoxide dismutase or carbonic anhydrase [4]. These are often limited only by the rate of ligand diffusion into the active site (diffusion-controlled rate). Nevertheless, an extensive analysis by Bar-Even et al., of nearly 2000 enzymes, showed that the median maximal turnover rate value over all measured enzymes is about 10 s −1 nowhere close to the values of 10 5 or 10 6 normally associated with catalysts [5,6].So, it would appear that natural enzymes are "just good enough" for the function they must perform in a given organism [7]. One might conclude that if they had evolved to their optimum performance then trying to improve them (from a kinetic point of view) would be attempting the impossible. On the contrary, as seen by the distribution of reaction rates, k cat , most enzymes function at a lower rate than the diffusion-limit and thus, there is space to further increase their kinetic properties to meet industrial needs. Additionally, we need enzymes capable of catalyzing reactions for which no known enzymes exist, to work with different substrates and for particular conditions that are industrially...

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“…The relative change in folding free energy (ΔΔG) based on point mutation was predicted using the FoldX software. The predicted ΔΔG equals the ΔG for the protein carrying the point mutation minus the ΔG for the wild-type protein (38). The potentially stabilizing mutations were selected based on the predicted ΔΔG and verified experimentally.…”

Section: Methodsmentioning

confidence: 99%

Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis

Zhang

Kong

et al. 2017

Appl Environ Microbiol

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Streptomyces coelicolor CR1 (ScCR1) has been shown to be a promising biocatalyst for the synthesis of an atorvastatin precursor, ethyl-(S)-4-chloro-3-hydroxybutyrate [(S)-CHBE]. However, limitations of ScCR1 observed for practical application include low activity and poor stability. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. First, the crystal structure of ScCR1 complexed with NADH and cosubstrate 2-propanol was solved, and the specific activity of ScCR1 was increased from 38.8 U/mg to 168 U/mg (ScCR1 I158V/P168S ) by structure-guided engineering. Second, directed evolution was performed to improve the stability using ScCR1 I158V/P168S as a template, affording a triple mutant, ScCR1 A60T/I158V/P168S , whose thermostability (T 50 15 , defined as the temperature at which 50% of initial enzyme activity is lost following a heat treatment for 15 min) and substrate tolerance (C 50 15 , defined as the concentration at which 50% of initial enzyme activity is lost following incubation for 15 min) were 6.2°C and 4.7-fold higher than those of the wild-type enzyme. Interestingly, the specific activity of the triple mutant was further increased to 260 U/mg. Protein modeling and docking analysis shed light on the origin of the improved activity and stability. In the asymmetric reduction of ethyl-4-chloro-3-oxobutyrate (COBE) on a 300-ml scale, 100 g/liter COBE could be completely converted by only 2 g/liter of lyophilized ScCR1 A60T/I158V/P168S within 9 h, affording an excellent enantiomeric excess (ee) of Ͼ99% and a space-time yield of 255 g liter Ϫ1 day Ϫ1 . These results suggest high efficiency of the protein engineering strategy and good potential of the resulting variant for efficient synthesis of the atorvastatin precursor.IMPORTANCE Application of the carbonyl reductase ScCR1 in asymmetrically synthesizing (S)-CHBE, a key precursor for the blockbuster drug Lipitor, from COBE has been hindered by its low catalytic activity and poor thermostability and substrate tolerance. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. The catalytic efficiency, thermostability, and substrate tolerance of ScCR1 were significantly improved by structure-guided engineering and directed evolution. The engineered ScCR1 may serve as a promising biocatalyst for the biosynthesis of (S)-CHBE, and the protein engineering strategy adopted in this work would serve as a useful approach for future engineering of other reductases toward potential application in organic synthesis.

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Computationally designed libraries for rapid enzyme stabilization

Cited by 217 publications

References 69 publications

Discovery and characterization of thermophilic limonene‐1,2‐epoxide hydrolases from hot spring metagenomic libraries

Discovery and characterization of thermophilic limonene‐1,2‐epoxide hydrolases from hot spring metagenomic libraries

Molecular Modeling in Enzyme Design, Toward In Silico Guided Directed Evolution

Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis

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