Substrate promiscuity and active site differences in gentisate 1,2-dioxygenases: electron paramagnetic resonance study

  • Aleksey Aleshintsev
  • Erik Eppinger
  • Janosch A. D. Gröning
  • Andreas Stolz
  • Rupal GuptaEmail author
Original Paper


Gentisate 1,2-dioxygenases (GDOs) are non-heme iron enzymes that catalyze the oxidation of dihydroxylated aromatic substrate, gentisate (2,5-dihydroxybenzoate). Salicylate 1,2-dioxygenase (SDO), a member of the GDO family, performs the ring scission of monohydroxylated substrates such as salicylate, thereby oxidizing a broader range of substrates compared to GDOs. Although the two types of enzymes share a high degree of sequence similarity, the origin of substrate specificity between SDO and GDOs is not understood. We present electron paramagnetic resonance (EPR) investigation of ferrous-nitrosyl complexes of SDO and a GDO from the bacterium Corynebacterium glutamicum (GDOCg). The EPR spectra of these complexes, which mimic the Fe-substrate-O2 intermediates in the catalytic cycle, show unexpected differences in the substrate binding mode and the coordination geometry of the metal cofactor in the two enzymes. Binding of substrate to the ferrous center increases the symmetry of the Fe(II)–NO complex in SDO, while a reverse trend is observed in GDOCg where substrate ligation reduces the symmetry of the nitrosyl complex. Identical EPR spectra were obtained for the NO derivatives of a variant of GDOCg(A112G), which can oxidize salicylate, and wild-type GDOCg revealing that the A112G mutation does not alter the nature of the Fe-substrate-O2 ternary complex.


Non-heme dioxygenases Gentisate dioxygenase Salicylate dioxygenase EPR Iron-nitrosyl complex 



Salicylate 1,2 dioxygenase


Gentisate 1,2 dioxygenase


Electron paramagnetic resonance



This work was supported by the City University of New York and Research Foundation startup funds to RG.

Supplementary material

775_2019_1646_MOESM1_ESM.pdf (322 kb)
Supplementary material 1 (PDF 322 kb)


  1. 1.
    Buongiorno D, Straganz GD (2013) Structure and function of atypically coordinated enzymatic mononuclear non-heme-Fe(II) centers. Coord Chem Rev 257(2):541–563. CrossRefGoogle Scholar
  2. 2.
    Crawford RL, Hutton SW, Chapman PJ (1975) Purification and properties of gentisate 1,2-dioxygenase from Moraxella osloensis. J Bacteriol 121(3):794–799Google Scholar
  3. 3.
    Hintner J-P, Lechner C, Riegert U, Kuhm AE, Storm T, Reemtsma T, Stolz A (2001) Direct ring fission of salicylate by a salicylate 1,2-dioxygenase activity from Pseudaminobacter salicylatoxidans. J Bacteriol 183(23):6936–6942. CrossRefGoogle Scholar
  4. 4.
    Harpel MR, Lipscomb JD (1990) Gentisate 1,2-dioxygenase from Pseudomonas acidovorans. Methods Enzymol 188:101–107CrossRefGoogle Scholar
  5. 5.
    Adams MA, Singh VK, Keller BO, Jia Z (2006) Structural and biochemical characterization of gentisate 1,2-dioxygenase from Escherichia coli O157:H7. Mol Microbiol 61(6):1469–1484. CrossRefGoogle Scholar
  6. 6.
    Chen J, Li W, Wang M, Zhu G, Liu D, Sun F, Hao N, Li X, Rao Z, Zhang XC (2008) Crystal structure and mutagenic analysis of GDOsp, a gentisate 1,2-dioxygenase from Silicibacter pomeroyi. Protein Sci 17(8):1362–1373. CrossRefGoogle Scholar
  7. 7.
    Kal S, Que L (2017) Dioxygen activation by nonheme iron enzymes with the 2-His-1-carboxylate facial triad that generate high-valent oxoiron oxidants. J Biol Inorg Chem 22(2):339–365. CrossRefGoogle Scholar
  8. 8.
    Wang Y, Li J, Liu A (2017) Oxygen activation by mononuclear nonheme iron dioxygenases involved in the degradation of aromatics. J Biol Inorg Chem 22(2):395–405. CrossRefGoogle Scholar
  9. 9.
    Harpel MR, Lipscomb JD (1990) Gentisate 1,2-dioxygenase from Pseudomonas Substrate coordination to active site Fe2+ and mechanism of turnover. J Biol Chem 265(36):22187–22196Google Scholar
  10. 10.
    Hintner J-P, Reemtsma T, Stolz A (2004) Biochemical and molecular characterization of a ring fission dioxygenase with the ability to oxidize (substituted) salicylate(s) from Pseudaminobacter salicylatoxidans. J Biol Chem 279(36):37250–37260. CrossRefGoogle Scholar
  11. 11.
    Matera I, Ferraroni M, Bürger S, Scozzafava A, Stolz A, Briganti F (2008) Salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans: crystal structure of a peculiar ring-cleaving dioxygenase. J Mol Biol 380(5):856–868. CrossRefGoogle Scholar
  12. 12.
    Ferraroni M, Matera I, Steimer L, Bürger S, Scozzafava A, Stolz A, Briganti F (2012) Crystal structures of salicylate 1,2-dioxygenase-substrates adducts: a step towards the comprehension of the structural basis for substrate selection in class III ring cleaving dioxygenases. J Struct Biol 177(2):431–438. CrossRefGoogle Scholar
  13. 13.
    Ferraroni M, Steimer L, Matera I, Bürger S, Scozzafava A, Stolz A, Briganti F (2012) The generation of a 1-hydroxy-2-naphthoate 1,2-dioxygenase by single point mutations of salicylate 1,2-dioxygenase–rational design of mutants and the crystal structures of the A85H and W104Y variants. J Struct Biol 180(3):563–571. CrossRefGoogle Scholar
  14. 14.
    Ferraroni M, Matera I, Bürger S, Reichert S, Steimer L, Scozzafava A, Stolz A, Briganti F (2013) The salicylate 1,2-dioxygenase as a model for a conventional gentisate 1,2-dioxygenase: crystal structures of the G106A mutant and its adducts with gentisate and salicylate. FEBS J 280(7):1643–1652. CrossRefGoogle Scholar
  15. 15.
    Eppinger E, Ferraroni M, Bürger S, Steimer L, Peng G, Briganti F, Stolz A (2015) Function of different amino acid residues in the reaction mechanism of gentisate 1,2-dioxygenases deduced from the analysis of mutants of the salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans. Biochim Biophys Acta 11854:425–1437. Google Scholar
  16. 16.
    Eppinger E, Stolz A (2017) Expansion of the substrate range of the gentisate 1,2-dioxygenase from Corynebacterium glutamicum for the conversion of monohydroxylated benzoates. Protein Eng Des Sel 30(1):57–65. Google Scholar
  17. 17.
    Emerson JP, Farquhar ER, Que L (2007) Structural “snapshots” along reaction pathways of non-heme iron enzymes. Angew Chem Int Ed 46(45):8553–8556. CrossRefGoogle Scholar
  18. 18.
    Roy S, Kästner J (2016) Synergistic substrate and oxygen activation in salicylate dioxygenase revealed by QM/MM simulations. Angew Chem Int Ed 55(3):1168–1172. CrossRefGoogle Scholar
  19. 19.
    Roy S, Kästner J (2017) Catalytic mechanism of salicylate dioxygenase: QM/MM simulations reveal the origin of unexpected regioselectivity of the ring cleavage. Chem Eur J 23(37):8949–8962. CrossRefGoogle Scholar
  20. 20.
    Tamanaha E, Zhang B, Guo Y, Chang WC, Barr EW, Xing G, Clair J, Ye S, Neese F, Bollinger JM, Krebs C (2016) Spectroscopic evidence for the two C-H-cleaving intermediates of Aspergillus nidulans isopenicillin N synthase. J Am Chem Soc 138(28):8862–8874. CrossRefGoogle Scholar
  21. 21.
    Mbughuni MM, Chakrabarti M, Hayden JA, Meier KK, Dalluge JJ, Hendrich MP, Munck E, Lipscomb JD (2011) Oxy intermediates of homoprotocatechuate 2,3-dioxygenase: facile electron transfer between substrates. Biochemistry 50(47):10262–10274. CrossRefGoogle Scholar
  22. 22.
    Visser SPD (2011) Experimental and computational studies on the catalytic mechanism of non-heme iron dioxygenases. Iron-containing enzymes: versatile catalysts of hydroxylation reactions in nature. R Soc Chem 5:1–41. Google Scholar
  23. 23.
    Zhang Y, Pavlosky MA, Brown CA, Westre TE, Hedman B, Hodgson KO, Solomon EI (1992) Spectroscopic and theoretical description of the electronic structure of the S = 3/2 nitrosyl complex of non-heme iron enzymes. J Am Chem Soc 114(23):9189–9191. CrossRefGoogle Scholar
  24. 24.
    Brown CA, Pavlosky MA, Westre TE, Zhang Y, Hedman B, Hodgson KO, Solomon EI (1995) Spectroscopic and theoretical description of the electronic structure of S = 3/2 iron-nitrosyl complexes and their relation to O2 activation by non-heme iron enzyme active sites. J Am Chem Soc 117(2):715–732. CrossRefGoogle Scholar
  25. 25.
    Harpel MR, Lipscomb JD (1990) Gentisate 1,2-dioxygenase from Pseudomonas. Purification, characterization, and comparison of the enzymes from Pseudomonas testosteroni and Pseudomonas acidovorans. J Biol Chem 265(11):6301–6311Google Scholar
  26. 26.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1):248–254. CrossRefGoogle Scholar
  27. 27.
    Ford-Smith MH, Sutin N (1961) The kinetics of the reactions of substituted 1,10-phenanthroline, 2,2′-dipyridine and 2,2′,2″-tripyridine complexes of iron(III) with iron(II) ions1. J Am Chem Soc 83(8):1830–1834. CrossRefGoogle Scholar
  28. 28.
    Golombek AP, Hendrich MP (2003) Quantitative analysis of dinuclear manganese(II) EPR spectra. J Magn Reson 165(1):33–48. CrossRefGoogle Scholar
  29. 29.
    Enemark JH, Feltham RD (1974) Principles of structure, bonding, and reactivity for metal nitrosyl complexes. Coord Chem Rev 13(4):339–406. CrossRefGoogle Scholar

Copyright information

© Society for Biological Inorganic Chemistry (SBIC) 2019

Authors and Affiliations

  1. 1.Institut für MikrobiologieUniversität StuttgartStuttgartGermany
  2. 2.Department of Chemistry and Biochemistry, College of Staten IslandCity University of New YorkNew YorkUSA
  3. 3.Program in BiochemistryThe Graduate Center of the City University of New YorkNew YorkUSA
  4. 4.Program in ChemistryThe Graduate Center of the City University of New YorkNew YorkUSA

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