Quantitative 1H-NMR analysis reveals steric and electronic effects on the substrate specificity of benzoate dioxygenase in Ralstonia eutropha B9

The cis-dihydroxylation of arenes by Rieske dearomatizing dioxygenases (RDDs) represents a powerful tool for the production of chiral precursors in organic synthesis. Here, the substrate specificity of the RDD benzoate dioxygenase (BZDO) in Ralstonia eutropha B9 whole cells was explored using quantitative H-1 nuclear magnetic resonance spectroscopy (q(1)H-NMR). The specific activity, specific carbon uptake, and regioselectivity of the dihydroxylation reaction were evaluated in resting cell cultures for a panel of 17 monosubstituted benzoates. Two new substrates of this dioxygenase system were identified (2-methyl- and 3-methoxybenzoic acid) and the corresponding cis-diol metabolites were characterized. Higher activities were observed for benzoates with smaller substituents, predominantly at the 3-position. Elevated activities were also observed in substrates bearing greater partial charge at the C-2 position of the benzoate ring. The regioselectivity of the reaction was directly measured using q(1)H-NMR and found to have positive correlation with increasing substituent size. These results widen the pool of cis-diol metabolites available for synthetic applications and offer a window into the substrate traits that govern specificity for BZDO.

Automated summary

The substrate specificity of RDD benzoate dioxygenase (BZDO) in Ralstonia eutropha B9 cells was investigated using H-1 nuclear magnetic resonance spectroscopy. The enzyme showed higher activity towards benzoates with smaller substituents, particularly at the 3-position. Substrates with larger partial charge at the C-2 position also exhibited elevated activity. The regioselectivity of the reaction correlated positively with increasing substituent size. These findings expand the pool of cis-diol metabolites available for synthesis and provide insight into the substrate features that govern specificity for BZDO.