Characterization of protocatechuate 4,5-dioxygenase from pseudarthrobacter phenanthrenivorans sphe3 and in situ reaction monitoring in the NMR tube

Abstract

The current study aims at the functional and kinetic characterization of protocatechuate (PCA) 4,5-dioxygenase (PcaA) from Pseudarthrobacter phenanthrenivorans Sphe3. This is the first single subunit Type II dioxygenase characterized in Actinobacteria. RT-PCR analysis demonstrated that pcaA and the adjacent putative genes implicated in the PCA meta-cleavage pathway comprise a single transcriptional unit. The recombinant PcaA is highly specific for PCA and exhibits Michae-lis–Menten kinetics with Km and Vmax values of 21 ± 1.6 μM and 44.8 ± 4.0 U × mg−1, respectively, in pH 9.5 and at 20 °C. PcaA also converted gallate from a broad range of substrates tested. The enzy-matic reaction products were identified and characterized, for the first time, through in situ bio-transformation monitoring inside an NMR tube. The PCA reaction product demonstrated a keto-enol tautomerization, whereas the gallate reaction product was present only in the keto form. More-over, the transcriptional levels of pcaA and pcaR (gene encoding a LysR-type regulator of the path-way) were also determined, showing an induction when cells were grown on PCA and phenan-threne. Studying key enzymes in biodegradation pathways is significant for bioremediation and for efficient biocatalysts development.