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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
  • 55 Downloads

Abstract

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.

Keywords

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

Abbreviations

SDO

Salicylate 1,2 dioxygenase

GDO

Gentisate 1,2 dioxygenase

EPR

Electron paramagnetic resonance

Notes

Acknowledgements

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)

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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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