Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding

Chen, Qi; Luan, Zheng Jiao; Yu, Hui; Cheng, Xiaolin; Xu, Jian

doi:10.1016/j.jmgm.2015.10.015

Cited by 8 publications

(4 citation statements)

References 38 publications

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“…Detailed analysis of the distance between the carbonyl group of the substrate and the catalytic Ser134 is shown in Figure S1, and the result demonstrates that the substrate binding position is distant from the catalytic residues. Furthermore, we found a strong π–π stacking interaction between the benzene ring of the Phe92 residue and the benzene ring of the catalytic Tyr175 residue, which forms a spatial barrier that blocks substrate binding with the catalytic residues, as has been observed previously for esterases . We speculated that F92 is a key residue affecting the activity of Cg KR1; hence, it was chosen for mutagenesis.…”

Section: Results and Discussionsupporting

confidence: 59%

Preparation of Structurally Diverse Chiral Alcohols by Engineering Ketoreductase CgKR1

et al. 2017

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Ketoreductases are tools for the synthesis of chiral alcohols in industry. However, the low activity of natural enzymes often restricts their use in industrial applications. On the basis of computational analysis and previous reports, two residues (F92 and F94) probably affecting the activity of ketoreductase CgKR1 were identified. By tuning these two residues, the CgKR1-F92C/F94W variant was obtained that exhibited higher activity toward all 28 structurally diverse substrates examined than the wild-type enzyme. Among them, 13 substrates have a specific activity over 50 U mg −1 (54−775 U mg −1 ). Using CgKR1-F92C/F94W as a catalyst, five substrates at high loading (>100 g −1 L −1 ) were reduced completely in gramscale preparative reactions. This approach provides accesses to pharmaceutically relevant chiral alcohols with high enantioselectivity (up to 99.0% ee) and high space-time yield (up to 583 g −1 L −1 day −1 ). Molecular dynamics simulations highlighted the crucial role of residues 92 and 94 in activity improvement. Our findings provide useful guidance for engineering other ketoreductases, especially those possessing a similar active pocket to that in CgKR1.

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Section: Results and Discussionsupporting

confidence: 59%

Preparation of Structurally Diverse Chiral Alcohols by Engineering Ketoreductase CgKR1

et al. 2017

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“…4b ). During hypoxia, mitochondria increased generation of reactive oxygen species (ROS) [ 25 ], which activated mitogen-activated protein kinase (MAPK) [ 26 ]. Hypoxic conditions also lead to cell cycle arrest [ 27 , 28 ] and apoptosis by a number of HIF-1-mediated and independent pathways [ 29 , 30 ].…”

Section: Resultsmentioning

confidence: 99%

Ribosome profiling reveals translational regulation of mammalian cells in response to hypoxic stress

et al. 2017

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BackgroundRetinal pigment epithelium (RPE) cells transfer oxygen and nutrients from choroid to the neural retina. Reduced oxygen to RPE perturbs development and functions of blood vessels in retina. Previous efforts of genome-wide studies have been largely focused on transcriptional changes of cells in response to hypoxia. Recently developed ribosome profiling provides an opportunity to study genome-wide translational changes. To gain systemic insights into the transcriptional and translational regulation of cellular in response to hypoxic stress, we used simultaneous RNA sequencing and ribosome profiling on an RPE cells line, ARPE-19, under hypoxia condition.ResultsBoth HIF-1α and EPAS1 (HIF-2α) proteins were stabilized in ARPE-19 under hypoxic stress treatment at 1 h, 2 h and 4 h. Analysis of simultaneous RNA sequencing and ribosome profiling data showed genome-wide gene expression changes at both transcriptional and translational levels. Comparative analysis of ribosome profiling and RNA-seq data revealed that hypoxia induced changes of more genes at the translational than the transcriptional levels. Ribosomes densities at 5′ untranslated region (UTR) significantly increased under hypoxic stress. Interestingly, the increase in ribosome densities at 5′ UTR is positively correlated with the presence of upstream open reading frames (uORFs) in the 5′ UTR of mRNAs.ConclusionOur results characterized translational profiles of mRNAs for a RPE cell line in response to hypoxia. In particular, uORFs play important roles in the regulation of translation efficiency by affecting ribosomes loading onto mRNAs. This study provides the first attempt to understand translational response of mammalian cells under hypoxic condition.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3996-8) contains supplementary material, which is available to authorized users.

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“…The melting temperature ( T m ) of Rh Est1 M2 was 1.7 °C higher than that of its parent Rh Est1 M1 and nearly 11 °C higher than that of the wild type (Table ). All-atom MD simulations indicated that α-helices and loops in the cap domain exhibit high fluctuations, showing relatively high B-factors . Mutations of these sites might greatly improve the enzyme thermostability and simultaneously decrease the thermal motion and positional disorder of these sites .…”

Section: Resultsmentioning

confidence: 99%

“…All-atom MD simulations indicated that α-helices and loops in the cap domain exhibit high fluctuations, showing relatively high Bfactors. 21 Mutations of these sites might greatly improve the enzyme thermostability and simultaneously decrease the thermal motion and positional disorder of these sites. 22 The improvement of thermostability for this enzyme has provided a significant clue that indicates the relationship between cap domain engineering and enzyme thermostability.…”

Section: Industrial and Engineering Chemistry Researchmentioning

confidence: 99%

Dramatically Improved Performance of an Esterase for Cilastatin Synthesis by Cap Domain Engineering

Luan

et al. 2016

Ind. Eng. Chem. Res.

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Whole-protein random mutation and substrate tunnel evolution have recently been applied to the pharmaceutically relevant esterase RhEst1 for the synthesis of a cilastatin precursor. The mutant RhEst1 M1 (=RhEst1 A147I/V148F/G254A ) was identified from a large library consisting of 1.5 × 10 4 variants. Though the activity of this mutant was improved 5-fold, the enantioselectivity for biohydrolysis decreased at the same time. Herein a smart library (3.0 × 10 3 ) focused on the cap domain of RhEst1 was constructed to improve its catalytic performance comprehensively. As a result, a variant designated as RhEst1 M2 (=RhEst1 M1-A143T ), showed a 6-fold increase in specific activity compared with the wild type. Meanwhile, the decreased enantioselectivity for enzymatic resolution was recovered to the native enzyme level. The melting temperature of RhEst1 M2 was nearly 11 °C higher than that of the wild type. This work provides detailed insight into the vital role of α/β hydrolase cap domains in influencing all aspects of enzyme characteristics. Furthermore, the commercial resin ESR-1 with free amino groups was used for enzyme immobilization to enhance the operational performance of RhEst1 M2 . No obvious activity loss was observed when the immobilized enzyme was incubated at 30 °C for 200 h. The immobilized enzyme could be repeatedly used for up to 20 batches, and the total turnover number (TTN) reached up to 8.0 × 10 5 .

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Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding

Cited by 8 publications

References 38 publications

Preparation of Structurally Diverse Chiral Alcohols by Engineering Ketoreductase CgKR1

Preparation of Structurally Diverse Chiral Alcohols by Engineering Ketoreductase CgKR1

Ribosome profiling reveals translational regulation of mammalian cells in response to hypoxic stress

Dramatically Improved Performance of an Esterase for Cilastatin Synthesis by Cap Domain Engineering

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