Part:BBa_K5043009

phtAc from M. vanbaalenii Pyr-1

PhtAc codes for phthalate dioxygenase ferredoxin subunit. It is a subunit of ring-hydroxylating oxygenase enzyme complexes which participate in biodegradation of polycyclic aromatic hydrocarbons. [1] This part encodes for the same protein as BBa_J73042. It only differs in synonym codon usage.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Introduction

In this project, we aimed to establish a pyrene degradation pathway in Pseudomonas vancouverensis and to integrate it with the organism's native phenanthrene degradation pathway [2]. It is established that PhtAcAd, as electron transfer components, along with a ring-hydroxylating dioxygenase system formed by pdoA2 and pdoB2, aggregate to create a complex exhibiting dioxygenase activity [3, 4, 5]. This complex is capable of converting phenanthrene-4-carboxylate, an intermediate product of pyrene degradation, into cis-3,4-dihydroxyphenanthrene an intermediate in the native phenanthrene degradation pathway of P. vancouverensis, thereby facilitating the conjunction of the two pathways [4, 5]. Given that this enzyme complex serves as a critical junction within the pathways, we opted to conduct a more detailed characterization. The components of the electron transport chain, PhtAc a ferredoxin reductase and ferredoxin PhtAd, were produced and purified to elucidate their kinetic parameters. Subsequently, we aimed to analyze the complex formation with PdoA2B2 through HPLC analysis.

Enzyme production and purification

Enzymes coding sequences were cloned in pQE bacterial expression plasmid with a cleavable N-terminal, 6x-His tag. Proteins were expressed in E. coli BL21 (DE3). Main cultures were incubated at 37°C until an OD600 of 0.5 was reached. Subsequently, cultures were induced with a final concentration of 0.5 mM IPTG and incubated overnight at 30°C. Purification was performed using Immobilized Metal Affinity Chromatography (IMAC). Both production and purification samples were analyzed via SDS-PAGE and Coomassie Staining (data not shown). Enzyme concentration was determined using the Bradford Assay and for most of the enzymes we were able to get a high yield.

Characterization of ferredoxin reductase PhtAd and ferredoxin PhtAc

The characterization of PhtAd (insert part) activity was performed by evaluating its ability to reduce 2,6-dichlorophenolindophenol (DCPIP), which serves as an electron acceptor [6]. In its oxidized form, DCPIP displays a blue color, which transitions to colorless upon reduction, facilitating the evaluation of PhtAd activity [6]. The decrease in absorbance was monitored at 600 nm [6].

The activity of PhtAc was assessed using a coupled assay. The increase in absorbance resulting from an electron transfer to Cytochrome c signifies an interaction between ferredoxin PhtAc and ferredoxin reductase PhtAd [6]. The increase in absorbance was monitored at 5500 nm [6]. Both reactions needed NADH and FADH as cofactors [6].

References

[1] S.-J. Kim, O. Kweon, R. C. Jones, J. P. Freeman, R. D. Edmondson, and C. E. Cerniglia, "Complete and integrated pyrene degradation pathway in Mycobacterium vanbaalenii PYR-1 based on systems biology," Journal of bacteriology, vol. 189, no. 2, pp. 464–472, 2007, doi: 10.1128/JB.01310-06. [2] Y. Yang, R. F. Chen, and M. P. Shiaris, "Metabolism of naphthalene, fluorene, and phenanthrene: preliminary characterization of a cloned gene cluster from Pseudomonas putida NCIB 9816," Journal of bacteriology, vol. 176, no. 8, pp. 2158–2164, 1994, doi 10.1128/jb.176.8.2158-2164.1994. [3] Pagnout, C., Frache, G., Poupin, P., Maunit, B., Muller, J., & Férard, J. (2007). Isolation and characterization of a gene cluster involved in PAH degradation in Mycobacterium sp. strain SNP11: Expression in Mycobacterium smegmatis mc2155. Research in Microbiology, 158(2), 175–186. https://doi.org/10.1016/j.resmic.2006.11.002 [4] Yuan, K., Xie, X., Wang, X., Lin, L., Yang, L., Luan, T., & Chen, B. (2018). Transcriptional response of Mycobacterium sp. strain A1-PYR to multiple polycyclic aromatic hydrocarbon contaminations. Environmental Pollution, 243, 824–832. https://doi.org/10.1016/j.envpol.2018.09.001 [5] Krivobok, S., Kuony, S., Meyer, C., Louwagie, M., Willison, J. C., & Jouanneau, Y. (2003). Identification of Pyrene-Induced Proteins in Mycobacterium sp. Strain 6PY1: Evidence for Two Ring-Hydroxylating Dioxygenases. Journal of Bacteriology, 185(13), 3828–3841. https://doi.org/10.1128/jb.185.13.3828-3841.2003 [6] Wu, Y., Xu, Y., & Zhou, N. (2019). A newly defined dioxygenase system from Mycobacterium vanbaalenii PYR-1 endowed with an enhanced activity of dihydroxylation of high-molecular-weight polyaromatic hydrocarbons. Frontiers of Environmental Science & Engineering, 14(1). https://doi.org/10.1007/s11783-019-1193-5