Difference between revisions of "Part:BBa K215090"

Latest revision as of 00:46, 11 October 2022

OpdA (phosphotriesterase)

OpdA is a phophotriesterase from Agrobacterium that can detoxify a broad range of organophosphate pesticides and nerve agents.

Usage and Biology

To test BBa _K215090 it was inserted into BBa_K215000 using standard biobrick assembly, resulting in BBa_K215091. OpdA was then produced and purified as described in the [http://2009.igem.org/Team:Washington/Notebook/IMAC_protocol UW 2009 iGEM team Notebook]. The purified protein was then tested for activity against paraoxon, for a detailed description of the assay please see the [http://2009.igem.org/Team:Washington/Project/Target#BioBricking_and_Characterization_of_OpdA 2009 UW iGEM wiki]. The resulting data is shown below.

The substrate vs. velocity curve above plots the rate of paraoxon degradation (Vobs, y-axis) as a function of substrate concentration (x-axis). As observed in the curve above, at high substrate concentration this enzyme suffers from substrate inhibition, in the conditions it was assayed in. At lower concentrations it shows standard Michaelis-Menten kinetics, as depicted in the zoom in plot on the left. When this data was fit to a canonical substrate inhibition curve we obtained the following kinetic parameters:
kcat (s-1): 17.6
Km (mM): 0.011
Ksi (mM): 1.06
kcat/Km (M-1 s-1): 1.6 x 10⁶

More Information (added by CCA_San_Diego 2020)

Author: Ayush Agrawal, Andrew Sun

Summary: Provides specific background information on the OpdA gene as well as connections between different organisms with the gene. We also introduce a common storage method for proteins that can be applied for more effective OpdA storage during experiments.

Documentation:

When a BLAST search of the NCBI GenBank database was conducted on this plasmid sequence, the closest match was synthetic construct parathion hydrolase gene derived from OPH gene of Flavobacterium sp., with an 81% identity. The study using this synthetic construct suggests that removal of the signal peptide encoding DNA increases expression of the rOPH. OpdA has also been shown to be able to hydrolyze dimethyl OPs phosmet and fenthion, a quality not shown by OPH. Both phosmet and fenthion are organophosphates and similarly work by cholinesterase inhibition (Satvik Iyengar et al, 2015).

Opd and OpdA genes have been discovered in organisms from different geographical locations and in 4 distinct organisms. This gives more insight to the development of the OpdA gene as it most likely originated in some common ancestor. OpdA gene is chromosomally located in A. radiobacter P230, however due to inability of A. tumefaciens C58 to hydrolyze organophosphates and no result when using a OpdA hybridization probe, it is believed that OpdA was introduced to A. radiobacter P230 through lateral gene transfer.

Storage (CCA_San_Diego 2020)

A common storage method of proteins is lyophilization. However, lyophilization poses many risks to the enzymes such as change in pH, excipient damage/crystallization, as well as ice crystal formation. Therefore, there exists a need for a method of stabilization. Sugar excipients are an optimal solution to this issue, as the hydrogen bonds formed between the sugars and the proteins remain stable after the water is removed. Maltose, trehalose, and mannose have been shown to be the most effective sugar excipients for stabilization of the enzymes during/after lyophilization (Horne et al, 2002).

References (CCA_San_Diego 2020)

[1] Ali, M., Naqvi, T.A., Kanwal, M. et al. Detection of the organophosphate degrading gene opdA in the newly isolated bacterial strain Bacillus pumilus W1. Ann Microbiol 62, 233–239 (2012). https://doi.org/10.1007/s13213-011-0251-4.

[2] Horne, I., Sutherland, T., Harcourt, R., Russell, R., & Oakeshott, J. (2002, July). Identification of an opd (organophosphate degradation) gene in an Agrobacterium isolate. Retrieved October 22, 2020, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC126808/

[3] Satvik Iyengar AR, Tripathy RK, Bajaj P, Pande AH. Improving storage stability of recombinant organophosphorus hydrolase. Protein Expr Purif. 2015 Jul;111:28-35. doi: 10.1016/j.pep.2015.01.012. Epub 2015 Mar 14. PMID: 25782740.

Improvement

Improvement by SSTi-SZGD 2017

2017 SSTi-SZGD has improved the this part by add a TorA signal peptide to N-terminus of opdA gene and gave it a new name TorA-opdA(BBa_K2244003).

Improvement by IISc-Bangalore 2021

This part was codon-optimized for expression in E. coli. We also attached SpyTag002 and sfGFP modules to the OpdA enzyme to give rise to the OpdA-SpyTag002 family of fusion proteins. See BBa_K3765008, BBa_K3765013, BBa_K3765014 and BBa_K3765015 for more details.

Sequence and Features

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Characterization of the active site

Characterization of the OPH (Organophosphate hydrolase) protein's active site (added by Concordia-Montreal 2022)

Author: Boubia, Maria

Summary: OPH is a phosphodiesterase capable of hydrolyzing organophosphate chemicals. We have shown the active site in the OPH protein, in its crystal dimer form.

Description: OPH has a great potential to be used in industries because its protein can degrade one of the most toxic chemicals, organophosphates. These chemicals are used as pesticides but they are harmful to human health. Therefore, finding a novel way to remediate them back into the environment is essential. And this starts by characterizing their active site !

Red - large group pocket (His254, His257, Leu271, and Met317) [1]
Green - small group pocket (Gly60, Ile106, Thr303, and Ser308) [1]
These two pockets make the catalytic site.
Yellow - metal ligand pocket (His55, His57, Lys169, His201, His230 and Asp301) [1]

References

[1] Katyal, P., Chu, S., & Montclare, J. K. (2020). Enhancing organophosphate hydrolase efficacy via protein engineering and immobilization strategies. Annals of the New York Academy of Sciences, 1480(1), 54–72. https://doi.org/10.1111/nyas.14451