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.
Highlights
Protocatechuic acid (PCA) is an important ring-cleavage common intermediate formed from the catabolism of low molecular weight polycyclic aromatic hydrocarbons, namely ferulic, vanillic, and p-coumaric acid
Higher activity was recorded in cells grown on PCA (0.025 U × mg−1 ) than on phenanthrene (0.012 U × mg−1 ) whereas no activity was detected in cells grown on glucose
We have formerly demonstrated that P. phenanthrenivorans Sphe3, isolated from a creosote-oil contaminated soil [24], degrades phenanthrene via o-phthalate, leading to the formation of PCA, which in turn could be metabolized via both 3,4- and 4,5-ring cleavage pathways [29]
Summary
Protocatechuic acid (PCA) is an important ring-cleavage common intermediate formed from the catabolism of low molecular weight polycyclic aromatic hydrocarbons, namely ferulic, vanillic, and p-coumaric acid. These compounds are derived from the apopolymerization of lignin, phthalic acid isomers, chlorobenzoic acids, and methylated aromatic hydrocarbons such as toluene and xylene [1]. The ortho-cleavage pathway of PCA is widely distributed amongst Actinobacteria and Proteobacteria [6] and PCA 3,4-dioxygenase is the most commonly characterized enzyme [7]. Homologues of PCA 4,5-dioxygenases (meta-cleavage pathway) were found almost exclusively in Proteobacteria [8,9], whereas Actinobacteria degrade PCA predominantly via the ortho-cleavage pathway [9]. The meta-cleavage enzymes have been studied less relative to the respective ortho-cleavage enzymes, probably due to their sparser distribution among bacteria and/or their relative instability [11]
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