4.5 Article

Gentisate 1,2-dioxygenase from the gram-positive bacteria Rhodococcus opacus 1CP: Identical active sites vs. different substrate selectivities

Journal

BIOCHIMIE
Volume 180, Issue -, Pages 90-103

Publisher

ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER
DOI: 10.1016/j.biochi.2020.10.016

Keywords

Rhodococcus; 3-Hydroxybenzoate; Gentisate 1; 2-dioxygenase; Cupin; Structure and function relationship; Substrate specificity

Funding

  1. Russian Foundation for Basic Research (RFBR) [13-04-00880]

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The research investigated the gentisate 1,2-dioxygenase from Rhodococcus opacus 1CP, revealing its narrow substrate specificity and potential application in oxidizing gentisate. Analysis of the rho-GDO structure and function unveiled a new mechanism of substrate specificity for this family of enzymes.
Gentisate 1,2-dioxygenases belong to the class III ring-cleaving dioxygenases catalyzing key reactions of aromatic compounds degradation by aerobic microorganisms. In the present work, the results of complete molecular, structural, and functional investigations of the gentisate 1,2-dioxygenase (rho-GDO) from a gram-positive bacterium Rhodococcus opacus 1CP growing on 3-hydroxybenzoate as a sole source of carbon and energy are presented. The purified enzyme showed a narrow substrate specificity. Among fourteen investigated substrate analogues only gentisate was oxidized by the enzyme, what can be potentially applied in biosensor technologies. The rho-GDO encoding gene was identified in the genomic DNA of the R. opacus 1CP. According to phylogenetic analysis, the rho-GDO belongs to the group of apparently most recently acquired activities in bacterial genera Rhodococcus, Arthrobacter, Corynebacterium, Nocardia, Amycolatopsis, Comamonas, and Streptomyces. Homology modeling the rho-GDO 3D-structure demonstrates the composition identity of the first-sphere residues of the active site of rho-GDO and salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans (RCSB PDB: 2PHD), despite of their different substrate specificities. The phenomenon described for the first time for this family of enzymes supposes a more complicated mechanism of substrate specificity than previously imagined, and makes the rho-GDO a convenient model for a novel direction of structure-function relationship studies. (C) 2020 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

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