Aminoalkylphosphonate N-acetyltransferase with a 6*histidine-tag, which can degradate AMPA. Use BBa_K823004 to construct a new part that can catalyze the acyl transfer reaction between aminomethylphosphoric acid and acetyl-CoA, his tag is used for protein purification.
Under natural conditions, many microorganisms can use the glyphosate oxidoreductase to oxidize glyphosate to aminomethylphosphoric acid (AMPA).
Phn system is a gene cluster for organophosphorus transport and degradation in many microorganisms. The phnO gene encodes the acyltransferase which catalyzes the transfer of acyl to AMPA by acetyl-coa. Through this one-step acyl transfer reaction, AMPA can be converted into glyphosate analogue, which can then be degraded by C-P lyase.
In order to easily purify PhnO, we added a his-tag (6*His) at the C-terminal. we ligased the strong promoter、RBS、Terminator(BBa_J23100、BBa_B0034、BBa_B0015) and the parts (phnO)on the expression vector pSB1C3 by standard assembly. Then the ligation mixture was transformed into E. coli DH5α & E. coli BL21 (DE3), and the correct recombinant one was confirmed by chloramphenicol, enzyme-cut identification and sequencing.
1. Agarose Gel Electrophoresis
When we were building this circuit, enzyme-cut identification was used to certify the plasmid was correct. We used the EcoR I and Pst I to cut the plasmid, then we got the target separate fragment-686bp.
The constructed plasmid was transformed into E. coli BL21 (DE3). Owing to the his-tag, the PhnO could be easily purified through AKTA with Ni-NTA. Compared with the lysate, the PhnO in the eluant can be found at around 16 kDa. Both of them were electrophoresed on a sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining.
3. Determination of enzyme kinetic constants
Velocities for the reaction of PhnO were determined using 4,4′-dithiodipyridine (DTDP) to continuously detect the formation of the product CoA at 324 nm (thiopyridone: ε= 19800 M-1 ·cm-1 ) at 25 °C. (1) Enzyme activity was determined through enzyme-labeled instrument.
Km and kcat of different substrates were determined with a concentration gradient.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BamHI site found at 159
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC
- 1. J. C. Errey, J. S. Blanchard, Functional Annotation and Kinetic Characterization of PhnO from Salmonella enterica. Biochemistry 45, 3033-3039 (2006).