The final portion of the PETerminator enzymatic pathway is accomplished by the enzyme decarboxylating cis-dihydrodiol dehydrogenase (DCDDH).
This remarkable enzyme was first discovered from studies in the late 1990s.
The first documented degradation of phthalates in soil bacteria comes from a paper published 1995.
Researchers sampled sediment from the riverbed of the Passaic River in New Jersey (Wang, 1995).
Researchers sampled sediment from the riverbed of the Passaic River in New Jersey (Wang<em>et al</em>. 1995).
Samples were then exposed to various phthalates and their respective microbial populations and metabolites characterized thereafter. <em>C. testosteroni</em> emerged from a sample with a microbiota capable of growing on two different phthalate isomers.
These were terephthalate (TPA), isophthalate, and p-hydroxybenzoate (see Figure 1).
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<imgstyle="width: 60%;"src='https://static.igem.wiki/teams/4378/wiki/img/lab-enzymes/dcddh/dcddh-gene-architecture.png'alt="Gene architecture of tph genes">
<p><em>Figure 1: Architecture of the terephthalate-degrading gene isolated from C. testosteroni.
Based on illustration by Wang et al (1995)</em></p>
Based on illustration by Wang <em>et al</em> (1995)</em></p>
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Bains et al. structurally characterized the 3-dimensional structure of DCDDH at 1.85å (Bains <em>et al</em>, 2012).
Bains <em>et al</em>. structurally characterized the 3-dimensional structure of DCDDH at 1.85å (Bains <em>et al</em> 2012).
The method used for structural characterization was iodide single wavelength anomalous dispersion.
Computational modeling yielded information about how DCDDH acts on its substrate (Bains, 2012).
Computational modeling yielded information about how DCDDH acts on its substrate (Bains<em>et al</em> 2012).
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No genetic engineering has been carried out to improve catalytic activity of DCDDH.
All published efforts outline characterizations of either mechanistic or structural interest.
Similarly, little information exists as to the kinetics of the above catalysis accomplished by DCDDH.
A comprehensive report on kinetics of phthalate ester metabolism is available from Kluwe et al. (Kluwe, 1982).
A comprehensive report on kinetics of phthalate ester metabolism is available from Kluwe <em>et al</em>. (Kluwe 1982).
Apart from the publication from Bains, no experiment has to date provided insight into how DCDDH accomplishes its reaction.
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Protein overexpression was induced according to the following conditions (see Table 1).
The best overexpression conditions were those of row two in Table 1 (see below).
We used a plasmid containing the gene for IF3 as a control in the case of overexpression with the pET24a vector a positive control.
We chose IF3 as a control because of confirmed clear overexpression bands at 24 kDa (Hershey et al., 1977) (see Figure 5).
We chose IF3 as a control because of confirmed clear overexpression bands at 24 kDa (Forster <em>et al</em>. 1977) (see Figure 5).
Wang Y Z , Zhou Y, Zylstra GJ. 1995. Molecular Analysis of Isophthalate and terephthalate degradation by Comamonas testosteroni YZW-D. Environmental Health Perspective. June 1995, 103: 9-12.
<br><br>Bains J, Wulff JE, Boulanger MJ. 2012. Investigating terephthalate biodegradation: structural characterization of a putative decarboxylating cis-dihydrodiol dehydrogenase. Journal of Molecular Biology 423: 284-293.
<br><br>Kluwe, W M. 1982. Overview of phthalate ester pharmacokinetics in mammalian species. Environmental Health Persp. 45: 3-10.
Bains J, Wulff JE, Boulanger MJ. 2012. Investigating terephthalate biodegradation: structural characterization of a putative decarboxylating cis-dihydrodiol dehydrogenase. Journal of Molecular Biology 423: 284-293.
<br><br>Bao L, Menon PNK, Liljeruhm J, Forster AC. 2020. Overcoming chromoprotein limitations by engineering a red fluorescent protein. Analytical Biochemistry 611: 113936.
<br><br>Forster AC, Weissbach H, Blacklow SC. 2001. A simplified reconstitution of mRNA-directed epsilon enhancer and an unnatural amino acid. Anal. Biochem.. 297(1): 60-70.
<br><br>Bao L, Menon PNK, Liljeruhm J, Forster AC. 2020. Overcoming chromoprotein limitations by engineering a red fluorescent protein. Analytical Biochemistry 611: 113936.
<br><br>Kluwe, W M. 1982. Overview of phthalate ester pharmacokinetics in mammalian species. Environmental Health Persp. 45: 3-10.
<br><br>Wang Y Z , Zhou Y, Zylstra GJ. 1995. Molecular Analysis of Isophthalate and terephthalate degradation by Comamonas testosteroni YZW-D. Environmental Health Perspective. June 1995, 103: 9-12.
