Team:SMS Shenzhen/Contribution
Here we document two existing parts, Rubber oxygenase A (BBa_K1819003) and Latex Clearing Protein (BBa_K1819001).
BBa_K1819003
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).
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.
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).
BBa_K1819001
Latex Clearing Protein (Lcp) - rubber degradation
Lcp is widespread in Gram-positive rubber degraders, and is responsible for the primary attack on polyisoprene by actinomycetes.
Lcp has a molecular mass of about 42 kDa. The amino acid sequences of Lcps have no heme binding motifs that could serve as covalent attachment sites for heme groups.
Figure 3 |Two conformers of Lcp-K30.[3]
LCP is identified as a b-type cytochromes. Lcp was classed as a globin and the structural importance of several residues contributing to the stability of the protein, especially Arg195 and Arg202 was stated. The conserved residues (Arg164, Thr168 and His198) of the recently biochemically characterised Lcp-specific domain of unknown function 2236 (DUF2236) are located close to the haem cofactor and were identified as crucial active site residues.
Figure 4 |Tegradation products of oxidative cleavage of poly(cis-1,4-isoprene). [4]
Lcp cleaves rubber to multiple products ranging from C20 tetra-isoprenoid to at least C35 hepta-isoprenoid. The Lcp was assume that is more surface exposed and should be located close to the substrate binding site. Depending on the binding of the polymer to the protein, the substrate can be cleaved at different relative positions with respect to the end of the polymer molecule, resulting in cleavage products that differ largely in the number of isoprene units.
References
[1] Seidel, J., Schmitt, G., Hoffmann, M., Jendrossek, D., & Einsle, O. (2013). Structure of the processive rubber oxygenase RoxA from Xanthomonas sp. Proceedings of the National Academy of Sciences of the United States of America, 110(34), 13833–13838. https://doi.org/10.1073/pnas.1305560110
[2] Birke, J., & Jendrossek, D. (2014). Rubber oxygenase and latex clearing protein cleave rubber to different products and use different cleavage mechanisms. Applied and environmental microbiology, 80(16), 5012–5020. https://doi.org/10.1128/AEM.01271-14
[3]Ilcu, L., Röther, W., Birke, J., Brausemann, A., Einsle, O., & Jendrossek, D. (2017). Structural and Functional Analysis of Latex Clearing Protein (Lcp) Provides Insight into the Enzymatic Cleavage of Rubber. Scientific reports, 7(1), 6179. https://doi.org/10.1038/s41598-017-05268-2
[4]Birke, J., Röther, W., & Jendrossek, D. (2015). Latex Clearing Protein (Lcp) of Streptomyces sp. Strain K30 Is a b-Type Cytochrome and Differs from Rubber Oxygenase A (RoxA) in Its Biophysical Properties. Applied and environmental microbiology, 81(11), 3793–3799. https://doi.org/10.1128/AEM.00275-15