Coding

Part:BBa_K1819003

Designed by: Lais Ribovski   Group: iGEM15_Brasil-USP   (2015-09-16)
Revision as of 01:09, 21 October 2021 by Vanssy (Talk | contribs)

Rubber oxygenase A - rubber degrading enzyme


Part BBa_K1819003 corresponds to the coding sequence of rubber oxygenase A (Rox A) from Xanthomonas sp. strain 35Y (Gene bank accession code: AGT20506.1.) This 73 kDa protein is an extracellular enzyme that catalyzes oxidative C-C cleavage of poly(cis-1,4-isoprene) to a major product, 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD; C15 tri-isoprenoid), with specific activity measured as 0.3 mol min-1 per mg. Found not only in Xanthomonas sp. but also in other Gram-negative clearing zone formers, RoxA is structurally related to cytochrome c peroxidases and its function is associated to two covalently bound heme groups. Although recently characterized by Birke et al 1, the exact enzymatic mechanism of rubber degradation by RoxA has not yet been elucidated.

This protein was codon optimized and a courtesy of Professor Ph.D. Dieter Jendrossek from the University Stuttgart.


Figure 1 - Schematic representation of BBa_K1819003 insert


Such non-usual restriction sites may serve for cloning purposes that leads to expression of recombinant protein with no additional amino acid residue(s) between its coding sequence and the fused N-terminal tag.

Figure 2 - 3D structure of RoxA generated using PyMOL software (PDB code 4B2N).


Figure 3 - Restriction gel analysis of site-directed mutagenesis of RoxA to eliminate EcoRI restriction site. First column shows propagation plasmid control pUC9 (pUC9 contains a HindII restriction site) with roxA (plasmid backbone was a courtesy of Professor Ph.D. Dieter Jendrossek). Second column shows pUC9 Professor Ph.D. Dieter Jendrossek cleaveaged with EcoRI and HindII restriction enzymes. Third column is relative to transformation reaction of mutated roxA not digested. Fourth column shows mutated roxA digested with HindII and EcoRI


Figure 4 - Restriction gel analysis of RoxA in pSB1C3.



Please note this part was submitted in pSB1C3, the Registry's standard shipping backbone, according to submission requirements. However, pSB1C3 contains a SacI site and if you want to use this part we recommend to move the part into another plasmid backbone what can be easily done.


References

1. Birke J, Jendrossek D. Rubber oxygenase and latex clearing protein cleave rubber to different products and use different cleavage mechanisms. Applied and Environmental Microbiology. 2014;80(16):5012-5020.


Team: SMS_Shenzhen 2021

Rubber oxygenase A - rubber degrading enzyme

RoxA is an extracellular dioxygenase secreted by Xanthomonas sp. strain 35Y during growth on poly(cis-1,4-isoprene) and cleaves rubber to one major product, 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). 

     <img src="T--SMS_Shenzhen--contri1.PNG"  width="500px">

Figure 1 |A.Structure of RoxA, colored from blue at the N terminus to red at the C terminus. The two heme groups and functionally relevant residues are rendered as sticks. B. The RoxA cleaves linear poly-cis-1,4-isoprene into trimeric units of ODTD.[1]

Mature RoxA is a 73-kDa protein with two heme centers that are covalently attached to RoxA via two heme-binding motifs. Electron paramagnetic resonance (EPR) measurements and analysis of the three-dimensional structure revealed that the N-terminal heme represents the active site and has a stably bound dioxygen molecule as an axial ligand. 

     <img src="T--SMS_Shenzhen--contri2.PNG" width="500px">

Figure 2 |A UV(210-nm) absorbance spectrum of RoxA products (black line) and of Lcp products (gray line).[2]

RoxA oxidatively attack the double bonds of rubber molecules to give cleavage products with aldehyde (OCH2OCHO) and keto (CH3OCOOCH2O) end groups and a certain number of isoprene units in between. Compared to LCP, RoxA produces one major polyisoprene cleavage product (ODTD).



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 91
    Illegal BamHI site found at 433
    Illegal BamHI site found at 1171
    Illegal BamHI site found at 1189
    Illegal XhoI site found at 782
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 736
    Illegal NgoMIV site found at 826
    Illegal NgoMIV site found at 1003
    Illegal NgoMIV site found at 1111
    Illegal NgoMIV site found at 1115
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 362
    Illegal SapI.rc site found at 1448


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