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 Michaelis–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 enzymatic reaction products were identified and characterized, for the first time, through in situ biotransformation 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. Moreover, the transcriptional levels of pcaA and pcaR (gene encoding a LysR-type regulator of the pathway) were also determined, showing an induction when cells were grown on PCA and phenanthrene. Studying key enzymes in biodegradation pathways is significant for bioremediation and for efficient biocatalysts development.

Halotolerant Terephthalic Acid-Degrading Bacteria of the Genus Glutamicibacter

“…The PCA utilization by actinobacteria is known to proceed predominantly through the ortho-pathway [32]. At the same time, there are data on the possibility of PCA degradation by strains of the Micrococcaciae family (genera Arthrobacter and Pseudarthrobacter) via the ortho-and/or meta-pathway [18,33]. Studies of the metabolic pathway of degradation of TPA and PCA by the strains Glutamicibacter spp.…”

Characterization of Gentisate 1,2-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and Its Stabilization by Immobilization on Nickel-Functionalized Magnetic Nanoparticles

“…Crude Sphe3 cell-free extracts were prepared as described previously [52]. The protein concentration was determined spectrophotometrically, according to the Bradford method [53], using the Bio-Rad reagent (Bio-Rad Laboratories, Hercules, CA, USA) and bovine serum albumin (BSA) (Amresco Inc., Solon, OH, USA) as the standard.…”

“…The abovementioned E. coli BL21(DE3)-pET29c (+) host-vector overexpression system was used for the overexpression of the GDO polypeptide, as described elsewhere [51], with the slight modification of harvesting the cells by centrifugation 3 h after the addition of IPTG. For the enzyme purification, cell-free supernatant was applied to a 20 mL HisPrep FF 16/10 column (GE Healthcare, Chicago, IL, USA) using an AKTA FPLC system (GE Healthcare, Chicago, IL, USA), as described before [52].…”

“…All kinetic values are the means of three separate measurements. The effect of pH in the activity of recombinant GDO was investigated in the pH range of 4 to 12 at room temperature, as described elsewhere [52]. To determine the optimal temperature for the GDO, the above assays were performed in a temperature range of 5 • C to 80 • C in the optimal pH.…”

“…and NiCl 2 •6H 2 O, as described before [52]. The influence of different chelating agents on the GDO activity was tested by incubating samples of the purified enzyme (1 µM) with concentrations of EDTA up to 20 µM for 120 min at 0 • C and o-phenanthroline up to 30 µM for 180 min at 0 • C in 50 mM Tris-HCl buffer pH 8.0.…”