Skip to main content
SpringerLink
Log in

A Redox-Regulated, Heterodimeric NADH:cinnamate Reductase in Vibrio ruber

  • Published:
Biochemistry (Moscow) Aims and scope Submit manuscript

Abstract

Genes of putative reductases of α,β-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. 6.
Fig. 7.
Fig. 8.

Abbreviations

aCrd:

non-flavinylated Crd protein containing only non-covalently bound flavins

fCrd:

flavinylated Crd protein

*FMN:

covalently bound FMN residue

MS:

mass spectrometry

MV:

methyl viologen

m/z :

mass-to-charge ratio

MD:

molecular dynamics

ROS:

reactive oxygen species

References

  1. Tischer, W., Bader, J., and Simon, H. (1979) Purification and some properties of a hitherto-unknown enzyme reducing the carbon-carbon double bond of α,β-unsaturated carboxylate anions, Eur. J. Biochem., 97, 103-112, https://doi.org/10.1111/j.1432-1033.

    Article  CAS  PubMed  Google Scholar 

  2. Besteiro, S., Biran, M., Biteau, N., Coustou, V., Baltz, T., Canioni, P., and Bringaud, F. (2002) Succinate secreted by Trypanosoma brucei is produced by a novel and unique glycosomal enzyme, NADH-dependent fumarate reductase, J. Biol. Chem., 277, 38001-38012, https://doi.org/10.1074/jbc.M201759200.

    Article  CAS  PubMed  Google Scholar 

  3. Bertsova, Y. V., Oleynikov, I. P., and Bogachev, A. V. (2020) A new water-soluble bacterial NADH: fumarate oxidoreductase, FEMS Microbiol. Lett., 367, fnaa175, https://doi.org/10.1093/femsle/fnaa175.

    Article  CAS  PubMed  Google Scholar 

  4. Bertsova, Y. V., Serebryakova, M. V., Baykov, A. A., and Bogachev, A. V. (2022) A novel, NADH-dependent acrylate reductase in Vibrio harveyi, Appl. Environ. Microbiol., 88, e0051922, https://doi.org/10.1128/aem.00519-22.

    Article  CAS  PubMed  Google Scholar 

  5. Bertsova, Y. V., Kostyrko, V. A., Baykov, A. A., and Bogachev, A. V. (2014) Localization-controlled specificity of FAD:threonine flavin transferases in Klebsiella pneumoniae and its implications for the mechanism of Na+-translocating NADH:quinone oxidoreductase, Biochim. Biophys. Acta, 1837, 1122-1129, https://doi.org/10.1016/j.bbabio.2013.12.006.

    Article  CAS  PubMed  Google Scholar 

  6. Serebryakova, M. V., Bertsova, Y. V., Sokolov, S. S., Kolesnikov, A. A., Baykov, A. A., and Bogachev, A. V. (2018) Catalytically important flavin linked through a phosphoester bond in a eukaryotic fumarate reductase, Biochimie, 149, 34-40, https://doi.org/10.1016/j.biochi.2018.03.013.

    Article  CAS  PubMed  Google Scholar 

  7. Rohdich, F., Wiese, A., Feicht, R., Simon, H., and Bacher, A. (2001) Enoate reductases of Clostridia. Cloning, sequencing, and expression, J. Biol. Chem., 276, 5779-5787, https://doi.org/10.1074/jbc.M008656200.

    Article  CAS  PubMed  Google Scholar 

  8. Kuno, S., Bacher, A., and Simon, H. (1985) Structure of enoate reductase from a Clostridium tyrobutyricum (C. spec. La1), Biol. Chem. Hoppe Seyler, 366, 463-472, https://doi.org/10.1515/bchm3.1985.366.1.463.

    Article  CAS  PubMed  Google Scholar 

  9. Caldeira, J., Feicht, R., White, H., Teixeira, M., Moura, J. J., Simon, H., and Moura, I. (1996) EPR and Mössbauer spectroscopic studies on enoate reductase, J. Biol. Chem., 271, 18743-18748, https://doi.org/10.1074/jbc.271.31.18743.

    Article  CAS  PubMed  Google Scholar 

  10. Giesel, H., and Simon, H. (1983) On the occurrence of enoate reductase and 2-oxo-carboxylate reductase in clostridia and some observations on the amino acid fermentation by Peptostreptococcus anaerobius, Arch. Microbiol., 135, 51-57, https://doi.org/10.1007/BF00419482.

    Article  CAS  PubMed  Google Scholar 

  11. Mordaka, P. M., Hall, S. J., Minton, N., and Stephens, G. (2018) Recombinant expression and characterisation of the oxygen-sensitive 2-enoate reductase from Clostridium sporogenes, Microbiology (Reading), 164, 122-132, https://doi.org/10.1099/mic.0.000568.

    Article  CAS  PubMed  Google Scholar 

  12. Shieh, W. Y., Chen, Y. W., Chaw, S. M., and Chiu, H. H. (2003) Vibrio ruber sp. nov., a red, facultatively anaerobic, marine bacterium isolated from sea water, Int. J. Syst. Evol. Microbiol., 53, 479-484, https://doi.org/10.1099/ijs.0.02307-0.

    Article  PubMed  Google Scholar 

  13. Bogachev, A. V., Bertsova, Y. V., Bloch, D. A., and Verkhovsky, M. I. (2012) Urocanate reductase: Identification of a novel anaerobic respiratory pathway in Shewanella oneidensis MR-1, Mol. Microbiol., 86, 1452-1463, https://doi.org/10.1111/mmi.12067.

    Article  CAS  PubMed  Google Scholar 

  14. Light, S. H., Méheust, R., Ferrell, J. L., Cho, J., Deng, D., Agostoni, M., Iavarone, A. T., Banfield, J. F., D’Orazio, S. E. F., and Portnoy, D. A. (2019) Extracellular electron transfer powers flavinylated extracellular reductases in Gram-positive bacteria, Proc. Natl. Acad. Sci. USA, 116, 26892-26899, https://doi.org/10.1073/pnas.1915678116.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bogachev, A. V., Baykov, A. A., and Bertsova, Y. V. (2018) Flavin transferase: the maturation factor of flavin-containing oxidoreductases, Biochem. Soc. Trans., 46, 1161-1169, https://doi.org/10.1042/BST20180524.

    Article  CAS  PubMed  Google Scholar 

  16. Koike, H., Sasaki, H., Kobori, T., Zenno, S., Saigo, K., Murphy, M. E., Adman, E. T., and Tanokura, M. (1998) 1.8 Å crystal structure of the major NAD(P)H:FMN oxidoreductase of a bioluminescent bacterium, Vibrio fischeri: overall structure, cofactor and substrate-analog binding, and comparison with related flavoproteins, J. Mol. Biol., 280, 259-273, https://doi.org/10.1006/jmbi.1998.1871.

    Article  CAS  PubMed  Google Scholar 

  17. Bertsova, Y. V., Kulik, L. V., Mamedov, M. D., Baykov, A. A., and Bogachev, A. V. (2019) Flavodoxin with an air-stable flavin semiquinone in a green sulfur bacterium, Photosynth. Res., 142, 127-136, https://doi.org/10.1007/s11120-019-00658-1.

    Article  CAS  PubMed  Google Scholar 

  18. Bertsova, Y. V., Fadeeva, M. S., Kostyrko, V. A., Serebryakova, M. V., Baykov, A. A., and Bogachev, A. V. (2013) Alternative pyrimidine biosynthesis protein ApbE is a flavin transferase catalyzing covalent attachment of FMN to a threonine residue in bacterial flavoproteins, J. Biol. Chem., 288, 14276-14286, https://doi.org/10.1074/jbc.M113.455402.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227, 680-685, https://doi.org/10.1038/227680a0.

    Article  CAS  PubMed  Google Scholar 

  20. Ells, A. H. (1959) A colorimetric method for the assay of soluble succinic dehydogenase and pyridinenucleotide-linked dehydrogenases, Arch. Biochem. Biophys., 85, 561-562, https://doi.org/10.1016/0003-9861(59)90527-2.

    Article  CAS  PubMed  Google Scholar 

  21. Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Žídek, A., Potapenko, A., Bridgland, A., Meyer, C., Kohl, S. A. A., Ballard, A. J., Cowie, A., Romera-Paredes, B., Nikolov, S., Jain, R., Adler, J., Back, T., Petersen, S., Reiman, D., Clancy, E., Zielinski, M., Steinegger, M., Pacholska, M., Berghammer, T., Bodenstein, S., Silver, D., Vinyals, O., Senior, A. W., Kavukcuoglu, K., Kohli, P., and Hassabis, D. (2021) Highly accurate protein structure prediction with AlphaFold, Nature, 596, 583-589, https://doi.org/10.1038/s41586-021-03819-2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Mirdita, M., Schütze, K., Moriwaki, Y., Heo, L., Ovchinnikov, S., and Steinegger, M. (2022) ColabFold: making protein folding accessible to all, Nat. Meth., 19, 679-682, https://doi.org/10.1038/s41592-022-01488-1.

    Article  CAS  Google Scholar 

  23. Trott, O., and Olson, A. J. (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading, J. Comput. Chem., 31, 455-461, https://doi.org/10.1002/jcc.21334.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Case, D.A., Aktulga, H. M., Belfon, K., Ben-Shalom, I. Y., Berryman, J. T., Brozell, S. R., et al. (2022) Amber 2022, University of California, San Francisco.

  25. Bolanos-Garcia, V. M., and Davies, O. R. (2006) Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography, Biochim. Biophys. Acta, 1760, 1304-1313, https://doi.org/10.1016/j.bbagen.2006.03.027.

    Article  CAS  PubMed  Google Scholar 

  26. Bertsova, Y. V., Serebryakova, M. V., Baykov, A. A., and Bogachev, A. V. (2021) The flavin transferase ApbE flavinylates the ferredoxin:NAD+-oxidoreductase Rnf required for N2 fixation in Azotobacter vinelandii, FEMS Microbiol. Lett., 368, fnab130, https://doi.org/10.1093/femsle/fnab130.

    Article  CAS  PubMed  Google Scholar 

  27. Hertzberger, R., Arents, J., Dekker, H. L., Pridmore, R. D., Gysler, C., Kleerebezem, M., and de Mattos, M. J. (2014) H2O2 production in species of the Lactobacillus acidophilus group: a central role for a novel NADH-dependent flavin reductase, Appl. Environ. Microbiol., 80, 2229-2239, https://doi.org/10.1128/AEM.04272213.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Kim, S., Kim, C. M., Son, Y. J., Choi, J. Y., Siegenthaler, R. K., Lee, Y., Jang, T. H., Song, J., Kang, H., Kaiser, C. A., and Park, H. H. (2018) Molecular basis of maintaining an oxidizing environment under anaerobiosis by soluble fumarate reductase, Nat. Commun., 9, 4867, https://doi.org/10.1038/s41467-018-07285-9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Agarwal, R., Bonanno, J. B., Burley, S. K., and Swaminathan, S. (2006) Structure determination of an FMN reductase from Pseudomonas aeruginosa PA01 using sulfur anomalous signal, Acta Crystallogr. D Biol. Crystallogr., 62, 383-391, https://doi.org/10.1107/S0907444906001600.

    Article  CAS  PubMed  Google Scholar 

  30. Borshchevskiy, V., Round, E., Bertsova, Y., Polovinkin, V., Gushchin, I., Ishchenko, A., Kovalev, K., Mishin, A., Kachalova, G., Popov, A., Bogachev, A., and Gordeliy, V. (2015) Structural and functional investigation of flavin binding center of the NqrC subunit of sodium-translocating NADH:quinone oxidoreductase from Vibrio harveyi, PLoS One, 10, e0118548, https://doi.org/10.1371/journal.pone.0118548.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Venskutonytė, R., Koh, A., Stenström, O., Khan, M. T., Lundqvist, A., Akke, M., Bäckhed, F., and Lindkvist-Petersson, K. (2021) Structural characterization of the microbial enzyme urocanate reductase mediating imidazole propionate production, Nat. Commun., 12, 1347, https://doi.org/10.1038/s41467-021-21548-y.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Coustou, V., Besteiro, S., Rivière, L., Biran, M., Biteau, N., Franconi, J. M., Boshart, M., Baltz, T., and Bringaud, F. (2005) A mitochondrial NADH-dependent fumarate reductase involved in the production of succinate excreted by procyclic Trypanosoma brucei, J. Biol. Chem., 280, 16559-16570, https://doi.org/10.1074/jbc.M500343200.

    Article  CAS  PubMed  Google Scholar 

  33. Wargnies, M., Plazolles, N., Schenk, R., Villafraz, O., Dupuy, J. W., Biran, M., Bachmaier, S., Baudouin, H., Clayton, C., Boshart, M., and Bringaud, F. (2021) Metabolic selection of a homologous recombination-mediated gene loss protects Trypanosoma brucei from ROS production by glycosomal fumarate reductase, J. Biol. Chem., 296, 100548, https://doi.org/10.1016/j.jbc.2021.100548.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Page, C. C., Moser, C. C., Chen, X., and Dutton, P. L. (1999) Natural engineering principles of electron tunnelling in biological oxidation-reduction, Nature, 402, 47-52, https://doi.org/10.1038/46972.

    Article  CAS  PubMed  Google Scholar 

  35. Sogin, E. M., Michellod, D., Gruber-Vodicka, H. R., Bourceau, P., Geier, B., Meier, D. V., Seidel, M., Ahmerkamp, S., Schorn, S., D’Angelo, G., Procaccini, G., Dubilier, N., and Liebeke, M. (2022) Sugars dominate the seagrass rhizosphere, Nat. Ecol. Evol., 6, 866-877, https://doi.org/10.1038/s41559-022-01740-z.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Bertsch, J., Parthasarathy, A., Buckel, W., and Müller, V. (2013) An electron-bifurcating caffeyl-CoA reductase, J. Biol. Chem., 288, 11304-11311, https://doi.org/10.1074/jbc.M112.444919.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Martone, P. T., Estevez, J. M., Lu, F., Ruel, K., Denny, M. W., Somerville, C., and Ralph, J. (2009) Discovery of lignin in seaweed reveals convergent evolution of cell-wall architecture, Curr. Biol., 19, 169-175, https://doi.org/10.1016/j.cub.2008.12.031.

    Article  CAS  PubMed  Google Scholar 

  38. Fernando, I. P., Kim, M., Son, K. T., Jeong, Y., and Jeon, Y. J. (2016) Antioxidant activity of marine algal polyphenolic compounds: a mechanistic approach, J. Med. Food, 19, 615-628, https://doi.org/10.1089/jmf.2016.3706.

    Article  PubMed  Google Scholar 

  39. Rameshkumar, N., and Nair, S. (2009) Isolation and molecular characterization of genetically diverse antagonistic, diazotrophic red-pigmented vibrios from different mangrove rhizospheres, FEMS Microbiol. Ecol., 67, 455-467, https://doi.org/10.1111/j.1574-6941.2008.00638.x.

    Article  CAS  PubMed  Google Scholar 

  40. Smith, E. A., and Macfarlane, G. T. (1996) Enumeration of human colonic bacteria producing phenolic and indolic compounds: effects of pH, carbohydrate availability and retention time on dissimilatory aromatic amino acid metabolism, J. Appl. Bacteriol., 81, 288-302, https://doi.org/10.1111/j.1365-2672.1996.tb04331.x.

    Article  CAS  PubMed  Google Scholar 

  41. Knaus, T., Toogood, H. S., and Scrutton, N. S. (2016) Ene-Reductases and Their Applications. Green Biocatalysis, John Wiley and Sons, Inc., pp. 473-488, https://doi.org/10.1002/9781118828083.ch18.

  42. Sievers, F., and Higgins, D. G. (2018) Clustal Omega for making accurate alignments of many protein sequences, Protein Sci., 27, 135-145, https://doi.org/10.1002/pro.3290.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

MALDI MS and laser scanner facilities became available to us in the framework of the Moscow State University Development Program PNG 5.13. This article is devoted to cherished memory of Vladimir P. Skulachev.

Funding

This work was supported by the Russian Science Foundation (project no. 22-24-00133).

Author information

Authors and Affiliations

  1. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119234, Moscow, Russia

    Yulia V. Bertsova, Marina V. Serebryakova, Victor A. Anashkin, Alexander A. Baykov & Alexander V. Bogachev

Authors
  1. Yulia V. Bertsova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marina V. Serebryakova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Victor A. Anashkin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alexander A. Baykov
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Alexander V. Bogachev
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

AVB the conception of the study; YVB, MVS, VAA, AAB, and AVB the acquisition and analysis of the data; AAB and AVB writing of the manuscript.

Corresponding author

Correspondence to Alexander V. Bogachev.

Ethics declarations

The authors declare no conflicts of interest. This article does not contain description of studies with the involvement of humans or animal subjects.

Additional information

Publisher’s Note. Pleiades Publishing remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bertsova, Y.V., Serebryakova, M.V., Anashkin, V.A. et al. A Redox-Regulated, Heterodimeric NADH:cinnamate Reductase in Vibrio ruber. Biochemistry Moscow 89, 241–256 (2024). https://doi.org/10.1134/S0006297924020056

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0006297924020056

Keywords

Search

Navigation