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

Autor:	Andreas Stolz, Erik Eppinger, Rupal Gupta, Janosch A. D. Gröning, Aleksey Aleshintsev
Rok vydání:	2019
Předmět:	010402 general chemistry 01 natural sciences Biochemistry Cofactor Dioxygenases Substrate Specificity law.invention Inorganic Chemistry law Catalytic Domain Electron paramagnetic resonance Ternary complex Bond cleavage Coordination geometry biology 010405 organic chemistry Chemistry Electron Spin Resonance Spectroscopy Active site Substrate (chemistry) 0104 chemical sciences Corynebacterium glutamicum Crystallography Catalytic cycle Biocatalysis biology.protein
Zdroj:	JBIC Journal of Biological Inorganic Chemistry. 24:287-296
ISSN:	1432-1327 0949-8257
Popis:	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.
Databáze:	OpenAIRE
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