Revealing 3-hydroxybenzoate-4-hydroxylase from Pseudarthrobacter phenanthrenivorans Sphe3 via In-Cell NMR analysis and molecular cloning

This study aims at the functional and kinetic characterization of 3-Hydroxybenzoate-4-Hydroxylase (3HB4H) from Pseudarthrobacter phenanthrenivorans Sphe3. Bioinformatic sequence and structural analyses suggested that Asphe3_36590 gene encodes a putative 3HB4H, a finding supported by transcriptional data of a ~ 2000-fold induction when Sphe3 cells were grown on 3-Hydroxybenzoate (3-HBA) compared to glucose. The recombinant 3HB4H is highly specific for 3-HBA and follows Michaelis–Menten kinetics, with Km and Vmax values of 72.6 µM and 42.5 µM × min− 1, respectively, at pH 8.5 and 25 ◦C. It accepts both NADH and NADPH as cofactors and primarily hydroxylates 3-HBA to protocatechuate (PCA), while also converting 4-Hydroxybenzoate and gentisic acid. Combining heterologous expression with in-cell NMR using living Escherichia coli (E. coli) cells overexpressing 3HB4H, demonstrated that 3HB4H encoded by Asphe3_36590 gene catalyzes the hydroxylation of 3-HBA. Real-time NMR revealed rapid intracellular conversion of 3-HBA to PCA, providing direct functional validation of the enzyme in a living cellular environment. Sphe3 3HB4H represents the first biochemically characterized single-subunit FAD-depended hydroxylase (monooxygenase) in Actinobacteria. Furthermore, these findings highlight the exceptional power of in-cell NMR to capture enzymatic activity non-invasively, allowing direct observation of substrate consumption and PCA formation without the need for derivatization or chromatographic separation. This approach offers a powerful platform for investigating enzymatic activity and metabolic transformations in living cells. Graphical Abstract