Project description: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.
Project description:Pseudarthrobacter phenanthrenivorans strain MHSD1 is a bacterial endophyte isolated from sterilized leaves of Pellaea calomelanos, a medicinal plant capable of growing in arid environments. Here, we report the draft genome sequence and annotation of this bacterial endophyte. The draft genome sequence of P. phenanthrenivorans strain MHSD1 has 4 450 468 bp with a G + C content of 65.30%. The National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline identified a total of 4004 protein-coding genes, 56 genes coding for RNAs, and 82 pseudogenes. Biosynthesis pathways for various phytohormones such as auxin, salicylic acid, ethylene, cytokinin, jasmonic acid, abscisic acid, and gibberellins were identified. Putative genes involved in various characteristics of bacterial endophyte lifestyle such as transport, motility, adhesion, membrane proteins, secretion and delivery systems, plant cell wall modification, and detoxification were identified. Phylogenomic analysis showed P. phenanthrenivorans strain MHSD1 to be a subspecies of P. phenanthrenivorans Sphe3.
