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Cited by 24Enzyme Catalysis and ImmobilizationBiochemical and Structural CharacterizationEnzyme Structure and Function

The DOI matches its Crossref record; 47 of the 47 references it cites were found in a registry or an index.

Fixing Flavins: Hijacking a Flavin Transferase for Equipping Flavoproteins with a Covalent Flavin Cofactor

Open access
Tong, Yapei, Kaya, Saniye G., Russo, Sara, Rozeboom, Henriette J., Wijma, Hein J., Fraaije, Marco W.·2023·Journal of the American Chemical Society
DOI 10.1021/jacs.3c12009PMC PMC10722498

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Abstract

Most flavin-dependent enzymes contain a dissociable flavin cofactor. We present a new approach for installing in vivo a covalent bond between a flavin cofactor and its host protein. By using a flavin transferase and carving a flavinylation motif in target proteins, we demonstrate that "dissociable" flavoproteins can be turned into covalent flavoproteins. Specifically, four different flavin mononucleotide-containing proteins were engineered to undergo covalent flavinylation: a light-oxygen-voltage domain protein, a mini singlet oxygen generator, a nitroreductase, and an old yellow enzyme-type ene reductase. Optimizing the flavinylation motif and expression conditions led to the covalent flavinylation of all four flavoproteins. The engineered covalent flavoproteins retained function and often exhibited improved performance, such as higher thermostability or catalytic performance. The crystal structures of the designed covalent flavoproteins confirmed the designed threonyl-phosphate linkage. The targeted flavoproteins differ in fold and function, indicating that this method of introducing a covalent flavin-protein bond is a powerful new method to create flavoproteins that cannot lose their cofactor, boosting their performance.

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Crossref · 10.1021/jacs.3c12009

Cites

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Figures

Figure 1 FMN binding pocket of PpSB1-LOV and the modeled variants. (a) Overall structure of the wild-type PpSB1-LOV protein (PDB: 5J3W). (b) Close-up view of the FMN binding state of PpSB1-LOV wild-type. (c) Predicted FMN binding state of PpSB1-LOV-F1. (d) Predicted FMN binding state of PpSB1-LOV-F2.
Figure 1 FMN binding pocket of PpSB1-LOV and the modeled variants. (a) Overall structure of the wild-type PpSB1-LOV protein (PDB: 5J3W). (b) Close-up view of the FMN binding state of PpSB1-LOV wild-type. (c) Predicted FMN binding state of PpSB1-LOV-F1. (d) Predicted FMN binding state of PpSB1-LOV-F2.CC BY · PubMed Central
Figure 2 Expression and mass identification of PpSB1-LOV and its variants. (a) Polyacrylamide gel (SDS-PAGE) of purified PpSB1-LOV proteins: wild-type, variant F1, and variant F2. In-gel fluorescence (right) and protein staining (left) of the same gel are shown. (b) Electrospray ionization mass spectral analysis of PpSB1-LOV-F1. (c) Electrospray ionization mass spectral analysis of PpSB1-LOV-F2. Cells were grown at 30 °C for 20 h in 30 mL TB.
Figure 2 Expression and mass identification of PpSB1-LOV and its variants. (a) Polyacrylamide gel (SDS-PAGE) of purified PpSB1-LOV proteins: wild-type, variant F1, and variant F2. In-gel fluorescence (right) and protein staining (left) of the same gel are shown. (b) Electrospray ionization mass spectral analysis of PpSB1-LOV-F1. (c) Electrospray ionization mass spectral analysis of PpSB1-LOV-F2. Cells were grown at 30 °C for 20 h in 30 mL TB.CC BY · PubMed Central
Figure 3 UV/vis spectra for PpSB1-LOV variants. Spectra of (a) PpSB1-LOV-F1, (b) PpSB1-LOV-F2, and (c) wild-type PpSB1-LOV. The dark-adapted (black) and dark-recovered states (green) are shown, while a spectrum during the recovery process is also shown (blue). (d) Recovery traces of wild-type PpSB1-LOV and the two covalent variants (F1 and F2) were recorded as absorbances at 480 nm. Spectra were recorded at 25 °C in the dark after 30 s of blue-light illumination: wild type (red), F1 (green), and F2 (blue).
Figure 3 UV/vis spectra for PpSB1-LOV variants. Spectra of (a) PpSB1-LOV-F1, (b) PpSB1-LOV-F2, and (c) wild-type PpSB1-LOV. The dark-adapted (black) and dark-recovered states (green) are shown, while a spectrum during the recovery process is also shown (blue). (d) Recovery traces of wild-type PpSB1-LOV and the two covalent variants (F1 and F2) were recorded as absorbances at 480 nm. Spectra were recorded at 25 °C in the dark after 30 s of blue-light illumination: wild type (red), F1 (green), and F2 (blue).CC BY · PubMed Central
Figure 4 Crystal structures of the engineered covalent flavoproteins. (a) PpSB1-LOV-F1, (b) miniSOG-F2, and (c) BtNR-F3. The mutated residues, shown in the stick presentation, are colored green, purple, and slate, respectively. FMN atoms, in sticks, are colored orange. 2Fo-Fc electron density maps, in gray, of the mutated residues and FMN are contoured at 1.0 σ. Hydrogen bonds are shown as dashed lines, and large arrows indicate the bond linking the phosphate to the threonine residue.
Figure 4 Crystal structures of the engineered covalent flavoproteins. (a) PpSB1-LOV-F1, (b) miniSOG-F2, and (c) BtNR-F3. The mutated residues, shown in the stick presentation, are colored green, purple, and slate, respectively. FMN atoms, in sticks, are colored orange. 2Fo-Fc electron density maps, in gray, of the mutated residues and FMN are contoured at 1.0 σ. Hydrogen bonds are shown as dashed lines, and large arrows indicate the bond linking the phosphate to the threonine residue.CC BY · PubMed Central

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Metadata

Authors
Tong, Y., Kaya, S. G., Russo, S., Rozeboom, H. J., Wijma, H. J., & Fraaije, M. W.
Affiliations
  • University of Groningen (NL)
  • Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
Corresponding author
Fraaije, Marco W.
ORCID
Venue
Journal of the American Chemical Society
Publisher
American Chemical Society (ACS)
Published
2023-12-04
Volume
145
Issue
49
Pages
27140-27148
ISSN
  • 0002-7863 (ISSN-L)
  • 1520-5126
DOI
10.1021/jacs.3c12009
PMC
PMC10722498
OpenAlex
W4389295058
Cited by
24
References
47
Citations by year
2026: 8 · 2025: 9 · 2024: 5 · 2023: 2
FWCI
2.14
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of the American Chemical Society (publishedVersion, cc-by)
  • ChemRxiv (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • University of Groningen research database (University of Groningen / Centre for Information Technology) (submittedVersion, cc-by)
  • University of Groningen research database (University of Groningen / Centre for Information Technology) (submittedVersion, other-oa)
Funders
China Scholarship Council
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
Keywords
flavoprotein, flavin mononucleotide, protein engineering, light-oxygen-voltage domain, singlet oxygen generator, nitroreductase, old yellow enzyme, ene-reductases, thermal stability
MeSH terms
Dinitrocresols, Flavin-Adenine Dinucleotide, Flavins, Flavoproteins, Protein Binding, Transferases

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CoChat. (2026). Citation check: Fixing Flavins: Hijacking a Flavin Transferase for Equipping Flavoproteins with a Covalent Flavin Cofactor. https://app.cochat.ai/papers/10.1021/jacs.3c12009
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Tong, Y., Kaya, S. G., Russo, S., Rozeboom, H. J., Wijma, H. J., & Fraaije, M. W. (2023). Fixing Flavins: Hijacking a Flavin Transferase for Equipping Flavoproteins with a Covalent Flavin Cofactor. Journal of the American Chemical Society. https://doi.org/10.1021/jacs.3c12009

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