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.

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