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Cited by 38Enzyme Catalysis and ImmobilizationEnzyme Production and CharacterizationAsymmetric Hydrogenation and Catalysis

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Functional characterization and stability improvement of a ‘thermophilic-like’ ene-reductase from Rhodococcus opacus 1CP

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Riedel, Anika, Mehnert, Marika, Paul, Caroline E., Westphal, Adrie H., van Berkel, Willem J. H., Tischler, Dirk·2015·Frontiers in Microbiology
DOI 10.3389/fmicb.2015.01073PMC PMC4589676
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Abstract

Ene-reductases (ERs) are widely applied for the asymmetric synthesis of relevant industrial chemicals. A novel ER OYERo2 was found within a set of 14 putative old yellow enzymes (OYEs) obtained by genome mining of the actinobacterium Rhodococcus opacus 1CP. Multiple sequence alignment suggested that the enzyme belongs to the group of 'thermophilic-like' OYEs. OYERo2 was produced in Escherichia coli and biochemically characterized. The enzyme is strongly NADPH dependent and uses non-covalently bound FMNH2 for the reduction of activated α,β-unsaturated alkenes. In the active form OYERo2 is a dimer. Optimal catalysis occurs at pH 7.3 and 37°C. OYERo2 showed highest specific activities (45-50 U mg(-1)) on maleimides, which are efficiently converted to the corresponding succinimides. The OYERo2-mediated reduction of prochiral alkenes afforded the (R)-products with excellent optical purity (ee > 99%). OYERo2 is not as thermo-resistant as related OYEs. Introduction of a characteristic intermolecular salt bridge by site-specific mutagenesis raised the half-life of enzyme inactivation at 32°C from 28 to 87 min and improved the tolerance toward organic co-solvents. The suitability of OYERo2 for application in industrial biocatalysis is discussed.

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FIGURE 1 Dendrogram of amino acid sequences of old yellow enzymes (OYEs) from Rhodococcus opacus 1CP and characterized OYE enzymes. The maximum likelihood distance tree was solved by usage of Mega 6.06-mac with Clustal W alignment method. Test of phylogeny was performed with 500 bootstrap replications, and resulting numbers are indicated. ‘Thermophilic-like’ OYEs are indicated by a light gray box. Melting temperatures of ‘thermophilic-like’ OYEs are indicated by symbols colored with respect to the thermogram. NCBI accession numbers are in parentheses. OYE1: Saccharomyces pastorianus (carlsbergensis, CAA37666), OYE3: S. cerevisiae (CAA97878), KYE1: Kluyveromyces lactis (AAA98815), EBP1: Candida albicans (AAA18013), OPR1: Solanum lycopersicum (CAB43506), OPR3: S. lycopersicum (CAC21424), NerA: Agrobacterium tumefaciens (CAA74280), MR: Pseudomonas putida (AAC43569), OYERo1 and OYERo3: R. opacus 1CP, NemA: Escherichia coli (BAA13186), PETNR: Enterobacter cloacae (AAB38638), SYE2: Shewanella oneidensis (AAN55487), Achr-OYE3: Achromobacter sp. JA81 (AFK73187), OYERo2 and OYERo4: R. opacus 1CP, RmER: Ralstonia (Cupriavidus) metallidurans CH34 (ABF11721), DrER: Deinococcus radiodurans R1 (AAF11740), TsER: Thermus scotoductus (CAP16804), XenA: P. putida (AAF02538), TOYE: Thermoanaerobacter pseudethanolicus (ABY93685), Chr-OYE3: Chryseobacterium sp. CA49 (AHV90721), YqjM: Bacillus subtilis (BAA12619), GkOYE: Geobacillus kaustophilus (BAD76617) and GeoER: Geobacillus sp. #30 (BAO37313).
FIGURE 1 Dendrogram of amino acid sequences of old yellow enzymes (OYEs) from Rhodococcus opacus 1CP and characterized OYE enzymes. The maximum likelihood distance tree was solved by usage of Mega 6.06-mac with Clustal W alignment method. Test of phylogeny was performed with 500 bootstrap replications, and resulting numbers are indicated. ‘Thermophilic-like’ OYEs are indicated by a light gray box. Melting temperatures of ‘thermophilic-like’ OYEs are indicated by symbols colored with respect to the thermogram. NCBI accession numbers are in parentheses. OYE1: Saccharomyces pastorianus (carlsbergensis, CAA37666), OYE3: S. cerevisiae (CAA97878), KYE1: Kluyveromyces lactis (AAA98815), EBP1: Candida albicans (AAA18013), OPR1: Solanum lycopersicum (CAB43506), OPR3: S. lycopersicum (CAC21424), NerA: Agrobacterium tumefaciens (CAA74280), MR: Pseudomonas putida (AAC43569), OYERo1 and OYERo3: R. opacus 1CP, NemA: Escherichia coli (BAA13186), PETNR: Enterobacter cloacae (AAB38638), SYE2: Shewanella oneidensis (AAN55487), Achr-OYE3: Achromobacter sp. JA81 (AFK73187), OYERo2 and OYERo4: R. opacus 1CP, RmER: Ralstonia (Cupriavidus) metallidurans CH34 (ABF11721), DrER: Deinococcus radiodurans R1 (AAF11740), TsER: Thermus scotoductus (CAP16804), XenA: P. putida (AAF02538), TOYE: Thermoanaerobacter pseudethanolicus (ABY93685), Chr-OYE3: Chryseobacterium sp. CA49 (AHV90721), YqjM: Bacillus subtilis (BAA12619), GkOYE: Geobacillus kaustophilus (BAD76617) and GeoER: Geobacillus sp. #30 (BAO37313).CC BY · PubMed Central
FIGURE 2 Multiple sequence alignment of OYERo2 and OYERo2a with eight ‘thermophilic-like’ OYE homologs. Highly conserved residues are shaded in gray, conserved residues involved in the active site are shaded in blue and green. The salt bridges of TsER and TOYE are shaded in yellow. Amino acids that were replaced by site-directed mutagenesis of OYERo2 are also shaded in yellow in the wildtype and in the protein variant OYERo2a. Amino acid numbering of conserved residues is given according to the TOYE sequence (bold font). Other amino acid numberings are given according to each protein sequence. NCBI accession numbers and organism sources: RmER: R. (Cupriavidus) metallidurans CH34 (ABF11721), DrER: D. radiodurans R1 (AAF11740), TsER: T. scotoductus (CAP16804), XenA: P. putida (AAF02538), TOYE: T. pseudethanolicus (ABY93685), Chr-OYE3: Chryseobacterium sp. CA49 (AHV90721), YqjM: B. subtilis (BAA12619) and GkOYE: G. kaustophilus (BAD76617).
FIGURE 2 Multiple sequence alignment of OYERo2 and OYERo2a with eight ‘thermophilic-like’ OYE homologs. Highly conserved residues are shaded in gray, conserved residues involved in the active site are shaded in blue and green. The salt bridges of TsER and TOYE are shaded in yellow. Amino acids that were replaced by site-directed mutagenesis of OYERo2 are also shaded in yellow in the wildtype and in the protein variant OYERo2a. Amino acid numbering of conserved residues is given according to the TOYE sequence (bold font). Other amino acid numberings are given according to each protein sequence. NCBI accession numbers and organism sources: RmER: R. (Cupriavidus) metallidurans CH34 (ABF11721), DrER: D. radiodurans R1 (AAF11740), TsER: T. scotoductus (CAP16804), XenA: P. putida (AAF02538), TOYE: T. pseudethanolicus (ABY93685), Chr-OYE3: Chryseobacterium sp. CA49 (AHV90721), YqjM: B. subtilis (BAA12619) and GkOYE: G. kaustophilus (BAD76617).CC BY · PubMed Central
FIGURE 3 (A) SDS-PAGE analysis of samples of OYERo2 and OYERo2a. Lane 1: marker proteins, lane 2: purified OYERo2, lane 3: purified OYERo2a, lane 4: marker proteins. (B) Analytical gel filtration of purified OYERo2 samples eluted from a Superdex 200 HR 10/30 column. Black line: 119 μM (high concentration), blue line: 59 μM (medium concentration), gray line: 30 μM (low concentration), red line: 30 μM enzyme in presence of 200 μM NADPH. The inset shows the calibration curve obtained from elution of standard proteins. Due to the calibration the following retention volumes are stressed by dotted lines: dimer: 13.8 mL, tetramer: 12.5 ml, octamer: 11.1 mL, hexadecamer: 9.7 mL. ND: no significant activity/conversion detected under steady-state conditions.
FIGURE 3 (A) SDS-PAGE analysis of samples of OYERo2 and OYERo2a. Lane 1: marker proteins, lane 2: purified OYERo2, lane 3: purified OYERo2a, lane 4: marker proteins. (B) Analytical gel filtration of purified OYERo2 samples eluted from a Superdex 200 HR 10/30 column. Black line: 119 μM (high concentration), blue line: 59 μM (medium concentration), gray line: 30 μM (low concentration), red line: 30 μM enzyme in presence of 200 μM NADPH. The inset shows the calibration curve obtained from elution of standard proteins. Due to the calibration the following retention volumes are stressed by dotted lines: dimer: 13.8 mL, tetramer: 12.5 ml, octamer: 11.1 mL, hexadecamer: 9.7 mL. ND: no significant activity/conversion detected under steady-state conditions.CC BY · PubMed Central
FIGURE 4 Binding of flavin cofactor to OYERo2 and OYERo2a. (A) UV-VIS absorption spectra of 100 μM purified OYERo2 and of 100 μM purified OYERo2a (orange lines, overlapping) and a similar amount of free FMN (black line). (B) RP-HPLC on flavin samples extracted from purified OYERo2 and OYERo2a (black lines, overlapping) and on flavin standards FAD, FMN and riboflavin (orange line, with offset). (C) Spectral changes in OYERo2 upon titration with p-chlorophenol (0–4.2 mM) at 15°C. The enzyme concentration was 13.8 μM in 50 mM phosphate buffer (pH 7.1).
FIGURE 4 Binding of flavin cofactor to OYERo2 and OYERo2a. (A) UV-VIS absorption spectra of 100 μM purified OYERo2 and of 100 μM purified OYERo2a (orange lines, overlapping) and a similar amount of free FMN (black line). (B) RP-HPLC on flavin samples extracted from purified OYERo2 and OYERo2a (black lines, overlapping) and on flavin standards FAD, FMN and riboflavin (orange line, with offset). (C) Spectral changes in OYERo2 upon titration with p-chlorophenol (0–4.2 mM) at 15°C. The enzyme concentration was 13.8 μM in 50 mM phosphate buffer (pH 7.1).CC BY · PubMed Central
FIGURE 5 Temperature and pH optima of purified OYERo2 () and purified OYERo2a (). (A) Normalized activity-temperature plot: 140 μM NADPH and 1 mM maleimide in 50 mM phosphate buffer (pH 7.1) were incubated at various temperatures (10-50°C) for 10 min. The reaction was started through the addition of 30 nM enzyme (not tempered). (B) Normalized activity-pH plot: 140 μM NADPH, 1 mM maleimide in 50 mM phosphate buffers (pH 5.5 to 8.8) and 30 nM enzyme, measured at 25°C.
FIGURE 5 Temperature and pH optima of purified OYERo2 () and purified OYERo2a (). (A) Normalized activity-temperature plot: 140 μM NADPH and 1 mM maleimide in 50 mM phosphate buffer (pH 7.1) were incubated at various temperatures (10-50°C) for 10 min. The reaction was started through the addition of 30 nM enzyme (not tempered). (B) Normalized activity-pH plot: 140 μM NADPH, 1 mM maleimide in 50 mM phosphate buffers (pH 5.5 to 8.8) and 30 nM enzyme, measured at 25°C.CC BY · PubMed Central
FIGURE 6 Thermal stability of purified OYERo2 () and OYERo2a (). (A,B) Thermal stability was measured applying the standard assay. Protein was incubated in 50 mM phosphate buffer (pH 7.1) at various temperatures (1–50°C) and added to the assay in a final concentration of 30 nM. (C) Inactivation at 32°C with constant high protein concentrations of 7 μM (hc) and varying NADPH concentrations: 1 mM (red; continuous lines) and 0 mM (ox; dashed lines). (D) Inactivation at 32°C with various protein concentrations under reducing conditions: 7 μM protein and 1 mM NADPH (hc; continuous lines) and 30 nM protein and 0.14 mM NADPH (lc, dashed lines).
FIGURE 6 Thermal stability of purified OYERo2 () and OYERo2a (). (A,B) Thermal stability was measured applying the standard assay. Protein was incubated in 50 mM phosphate buffer (pH 7.1) at various temperatures (1–50°C) and added to the assay in a final concentration of 30 nM. (C) Inactivation at 32°C with constant high protein concentrations of 7 μM (hc) and varying NADPH concentrations: 1 mM (red; continuous lines) and 0 mM (ox; dashed lines). (D) Inactivation at 32°C with various protein concentrations under reducing conditions: 7 μM protein and 1 mM NADPH (hc; continuous lines) and 30 nM protein and 0.14 mM NADPH (lc, dashed lines).CC BY · PubMed Central
FIGURE 7 Activity and stability of OYERo2 () and OYERo2a () in the presence of organic co-solvents. (A) Activity of enzymes against 10% (vol/vol) of selected organic co-solvents was measured under assay conditions applying 140 μM NADPH, 25 mM phosphate buffer (pH 7.1) and 30 nM enzyme without additional incubation time. (B) Dependency of enzyme activity on ethanol concentration (0–30%). (C) Residual activity after pre-incubation (0–20 min) of the enzyme with 20% ethanol.
FIGURE 7 Activity and stability of OYERo2 () and OYERo2a () in the presence of organic co-solvents. (A) Activity of enzymes against 10% (vol/vol) of selected organic co-solvents was measured under assay conditions applying 140 μM NADPH, 25 mM phosphate buffer (pH 7.1) and 30 nM enzyme without additional incubation time. (B) Dependency of enzyme activity on ethanol concentration (0–30%). (C) Residual activity after pre-incubation (0–20 min) of the enzyme with 20% ethanol.CC BY · PubMed Central
FIGURE 8 Cartoon diagram of the dimeric model structure of R. opacus 1CP variant OYERo2a (PyMOL Molecular Graphics System, Version 1.5.0.4). OYERo2a is represented in its oxidized form with the FMN cofactor (purple), showing the five residues from the introduced characteristic salt bridge (red) and the Asp35 residue from the adjacent monomer (blue).
FIGURE 8 Cartoon diagram of the dimeric model structure of R. opacus 1CP variant OYERo2a (PyMOL Molecular Graphics System, Version 1.5.0.4). OYERo2a is represented in its oxidized form with the FMN cofactor (purple), showing the five residues from the introduced characteristic salt bridge (red) and the Asp35 residue from the adjacent monomer (blue).CC BY · PubMed Central

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Authors
Riedel, A., Mehnert, M., Paul, C. E., Westphal, A. H., van Berkel, W. J. H., & Tischler, D.
Affiliations
  • TU Bergakademie Freiberg (DE)
  • Delft University of Technology (NL)
  • Wageningen University & Research (NL)
Corresponding author
  • Riedel, Anika
  • Tischler, Dirk
ORCID
Venue
Frontiers in Microbiology
Publisher
Frontiers Media SA
Published
2015-10-01
Volume
6
Pages
1073-1073
ISSN
1664-302X (ISSN-L)
DOI
10.3389/fmicb.2015.01073
PMC
PMC4589676
OpenAlex
W2178030934
Cited by
38
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en
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Funders
  • Technische Universität Bergakademie Freiberg
  • European Social Fund (100101363)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
  • Physical Sciences › Chemistry › Inorganic Chemistry › Asymmetric Hydrogenation and Catalysis
Keywords
ene-reductases, old yellow enzyme, asymmetric synthesis, succinimide synthesis, NADPH-dependent enzyme, enzyme dimerization, enzyme thermostability, salt bridges, site-directed mutagenesis, industrial biocatalysis

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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