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

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.

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Declaration of competing interest The authors declare that they have not known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. All authors have not and will have not any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations.