Difference between revisions of "Part:BBa K4378999"

 
 
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<partinfo>BBa_K4378999 short</partinfo>
 
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TphB or 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase (DCDDH) is an enzyme involved in the degradation of terephtalate (TPA). This enzyme plays therefore a key role in the degradation of polyethylenterephthalate (PET) into protocatachuic acid (PCA). The precise reaction performed is the decarboxylation of 1,6-dihydroxycyclohexa-2,4-diene-dicarboxylate (DCD) into PCA. This enzyme is known to assemble as a homodimer whith each unit adapting to an alpha-beta fold with an mixed beta sheet active site.
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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). Samples were then exposed to various phthalates and their respective microbial populations and metabolites characterized thereafter. C. testosteroni emerged from a sample with a microbiota capable of growing on two different phthalate isomers. These were terephthalate (Tph), isophthalate, and p-hydroxybenzoate.
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The designation of the gene TphB is used interchangeably with the name of its product’s function, decarboxylating cis-dihydrodiol dehydrogenase (DCDDH). TphB is unique in its capacity to decarboxylate the reaction product from the TphA1, A2, and A3 actions.
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Bains et al. structurally characterized the 3-dimensional structure of DCDDH at 1.85Å (Bains, 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).
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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 TphB. A comprehensive report on kinetics of phthalate ester metabolism is available from Kluwe et al. (Kluwe, 1982). Apart from the publication from Bains, no experiment has to date provided insight into how TphB accomplishes its reaction.
  
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The coding sequence was optimized for the expression in E. coli K12 cells. To allow further in vitro characterization of this enzyme a His-Tag was added, to allow purification with affinity based chromatography.
 
  
 
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<partinfo>BBa_K4378999 parameters</partinfo>
 
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The old part <partinfo>BBa_K808010</partinfo> was optimized to allow efficient work with TphB in ''Escherichia coli'' (''E. coli'') our team optimized the codons and added a c-terminal histidine tag to allow purification through nickel based affinity chromatography. Addtionally mutated all illegal restriction sites to make this part compatible with the BioBrick standard.
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==SDS-PAGE==
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The results show a very strong overexpression of TphB which is expected to have a size of 33 kDa. Due to very high amounts of expressed protein the band spans over a large size range.
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[[Image:BBa K4378999 TphB overex.PNG|500px|thumb|center| Figure 2. Overexpression at two different temperature (24 °C and 37 °C) and IPTG concentrations (0,1 mM and 1 mM) of TphB using pET24a expression vectors. IF3 was used as control for overexpression. The TphB band is very strong and spans over huge area the expected size is 33 kDa which corresponses to the lower end of the band. The IF3 band is expected to be around 23 kDa.]]
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===References===
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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.
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Wang YZ, Zhou Y, Zylstra GJ. 1995. Molecular analysis of isophthalate and terephthalate degradation by Comamonas testosteroni YZW-D. Environmental Health Perspectives 103: 9–12.

Latest revision as of 09:15, 6 October 2022


TphB with His-Tag

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). Samples were then exposed to various phthalates and their respective microbial populations and metabolites characterized thereafter. C. testosteroni emerged from a sample with a microbiota capable of growing on two different phthalate isomers. These were terephthalate (Tph), isophthalate, and p-hydroxybenzoate.

The designation of the gene TphB is used interchangeably with the name of its product’s function, decarboxylating cis-dihydrodiol dehydrogenase (DCDDH). TphB is unique in its capacity to decarboxylate the reaction product from the TphA1, A2, and A3 actions.

Bains et al. structurally characterized the 3-dimensional structure of DCDDH at 1.85Å (Bains, 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).

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 TphB. A comprehensive report on kinetics of phthalate ester metabolism is available from Kluwe et al. (Kluwe, 1982). Apart from the publication from Bains, no experiment has to date provided insight into how TphB accomplishes its reaction.


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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 219
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 340


The old part BBa_K808010 was optimized to allow efficient work with TphB in Escherichia coli (E. coli) our team optimized the codons and added a c-terminal histidine tag to allow purification through nickel based affinity chromatography. Addtionally mutated all illegal restriction sites to make this part compatible with the BioBrick standard.

SDS-PAGE

The results show a very strong overexpression of TphB which is expected to have a size of 33 kDa. Due to very high amounts of expressed protein the band spans over a large size range.

Figure 2. Overexpression at two different temperature (24 °C and 37 °C) and IPTG concentrations (0,1 mM and 1 mM) of TphB using pET24a expression vectors. IF3 was used as control for overexpression. The TphB band is very strong and spans over huge area the expected size is 33 kDa which corresponses to the lower end of the band. The IF3 band is expected to be around 23 kDa.

References

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.

Wang YZ, Zhou Y, Zylstra GJ. 1995. Molecular analysis of isophthalate and terephthalate degradation by Comamonas testosteroni YZW-D. Environmental Health Perspectives 103: 9–12.