BBa_K2198000:E1_1 The naphthalene 1,2-dioxygenase part 1 (E1_1) from Pseudomonas putida Ref. [1.A]
Forms a complex with naphthalene 1,2-dioxygenase part 2 (E1_2) to form an active enzyme to oxidize PAH that can be used as substrate. This oxidation with dioxygen form a vicinal diol and add two hydrogens on the ring. This step need energy to break the aromaticity. This complex enzyme need NADH,H+.
BBa_K2198001:E1_2 The naphthalene 1,2-dioxygenase part 1 (E1_2) from Pseudomonas putida Ref. [1.B]
Gets complex with naphthalene 1,2-dioxygenase part 1 (E1_1) to form an active enzyme to oxidize PAH that can be used as substrate. This oxidation with dioxygen form a vicinal diol and add two hydrogens on the ring. This step need energy to break the aromaticity. This complex enzyme need NADH,H+.
BBa_K2198002:E2 The dihydrodiol dehydrogenase from Ralstonia sp. Ref. [2]
This step allows the restoration of the aromatic with a dehydrogenation of the vicinal diol. Thanks to this, E3 will be able to open the ring with an oxidative cleavage.
BBa_K2198003:E3 The catechol 2,3-dioxygenase from Pseudomonas putida Ref. [3]
In our degradation pathway the key step is to remove ring from PAH to reduce their toxicity. The E3 is able to do an extradiol ring cleavage. Thanks to this step, the PAH are now less toxic and available for an alkyl removal by E4. If this step is the last one of the process all the products can be handle by chemistry thanks to this alkyl chain. Furthermore E3 can begin an iterative degradation with E4 if the E4 main product enter in the range of substrate specificity.
BBa_K2198004:E4 The trans-2-carboxybenzalpyruvate hydratase-aldolase from Nocardioides sp Ref. [4]
The last step of our degradation pathway allow us to remove a ring from the PAH by producing a py-ruvic acid. This product is a common harmless cell intermediate molecule. Moreover, this step pro-duce a smaller PAH if there is more than 2 ring at the beginning. This second generation PAH can reintegrate the pathway at the previous step thanks to a low substrate specificity E3
BBa_K2198005:S1 The Major ampullate Spidroin 1 (MaSp1) from Euprosthenops australis Ref. [5]
In order to carry out the reaction on a solid support, we designed a scaffold made of spider silk that can polymerize.
Figure 1: Biochemical degradation pathway for Anthracene
The catalytic degradation of PAH is ensured by four enzymes. (E1_1, E1_2) homodimer catalyzes the di-hydroxylation, E2 the dehydrogenation of the vicinal diol, E3 oxidative cleavage of the aromatic cycle and E4 cleavage of a linear unsaturated α-β enone to release a pyruvate molecule.
Open reading frames for the four enzymes were designed using Geneious 11.0.2 Ref. [5] software with Uniprot database sequences. Each sequence was designed with Biobrick prefix and suffix Registry in the 5’ and 3’ regions respectively. In addition, His(6) tag, spacer and TeV cleavage sites were included at the N-terminus for purification and release of the His(6) -tag. BamHI site were inserted in the construct after the prefix and before the suffix respectively for insertion into pET43.1a(+) or pET32a(+) (Merck-Millipore) expression plasmids. Genes were ordered from Eurofins Genomics after sequence optimization for Biobrick and cloning into pET vector compatibility. Sequences were further optimized for expression in Escherichia coli and gene synthesis.
References
1. A.: J Bacteriol. 2000 Mar;182(6):1641-9. Substrate specificity of naphthalene dioxygenase: effect of specific amino acids at the active site of the enzyme. Parales RE1, Lee K, Resnick SM, Jiang H, Lessner DJ, Gibson DT
1. B.: J Bacteriol. 1983 Aug;155(2):505-11. Naphthalene dioxygenase: purification and properties of a terminal oxygenase component. Ensley BD, Gibson DT
2. : J Bacteriol. 1998 May; 180(9): 2522–2530. A Gene Cluster Encoding Steps in Conversion of Naphthalene to Gentisate in Pseudomonas sp. Strain U2 Sergio L. Fuenmayor,1 Mark Wild,2 Alastair L. Boyes,2 and Peter A. Williams1
3 : Int J Biol Macromol. 2017 Aug 25. pii: S0141-8130(17)32824-6. doi: 10.1016/j.ijbiomac.2017.08.113. [Epub ahead of print] Isolation and characterization of three novel catechol 2,3-dioxygenase from three novel haloalkaliphilic BTEX-degrading Pseudomonas strains. Hassan HA1, Aly AA2.
4 : J Bacteriol. 1998 Feb; 180(4): 945–949. PMCID: PMC106976 - Biochemical and Genetic Characterization of trans-2′-Carboxybenzalpyruvate Hydratase-Aldolase from a Phenanthrene-Degrading Nocardioides Strain - Tokuro Iwabuchi† and Shigeaki Harayama
5 : “Geneious version (the version you are using) (https://www.geneious.com, Kearse et al., 2012)” Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S., Cooper, A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Mentjies, P., Drummond, A. (2012). Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data.Bioinformatics, 28(12), 1647-1649.
6 : Biomacromolecules 2007, 8, 1695-1701 // Macroscopic Fibers Self-Assembled from Recombinant Miniature Spider Silk Proteins; Margareta Stark, Stefan Grip, Anna Rising, My Hedhammar, Wilhelm Engström, Goran Hjälm, and Jan Johansson*, Department of Anatomy, Physiology, and Biochemistry, The Biomedical Centre, Swedish University of Agricultural Sciences, SE-751 23 Uppsala, Sweden, and Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden Received January 16, 2007; Revised Manuscript Received February 20, 2007
