Part:BBa_K2287027
purF K326Q
It is the coding sequence of the K326Q mutant of glutamine phosphoribosylpyrophosphate amidotransferase that catalyzes the transformation from PRPP to PRA in de novo purine biosynthesis. We introduced a mutation K326Q to the enzyme based on "Identification of Sites for Feedback Regulation of Glutamine 5-Phosphoribosylpyrophosphate Amidotransferase by Nucleotides and Relationship to Residues Important for Catalysis"(Zhou G. et al. ,1993) to remit the feedback inhibition by GMP.
Usage and Biology
De novo purine nucleotide synthesis is a necessary procedure in almost every organism to satisfy the basic living and reproducing needs. In the prime step of the pathway, PRPP is transformed from R5P with the catalysis of Prs. An ATP provides a pyrophosphate and turns into AMP in the same procedure.(fig.1)
The formation of PRA is the first committed step of the pathway. PurF catalyzes the attachment of an amino group to C-1 of PRPP.(fig.2)
The resulting PRA is highly volatile. Purine rings are then built up on this structure. The second step is the addition of three atoms from glycine to turn PRA into GRA, helped along by enzyme PurD and energy provided by an ATP.(fig.3).
Then N10-formyltetrahydrofolate formylates the glycine amino group which turns GAR into FGAR, and a nitrogen is contributed by glutamine transforming FGAR to FGAM, before dehydration and ring closure yield the AIR by PurM.(fig.4)
To complete the second ring in the purine structure, a carboxyl group is first added and transforms AIR into CAIR. Then, CAIR is transformed into SACAIR with enzyme PurC and energy provided by an ATP.(fig.5)
After another 3 steps, we have the first intermediate with a complete purine ring is inosinate (IMP). The total pathway of de novo purine biosynthesis is showed below.(fig.6)
Relieve the feedback inhibition
Zhou G.(1993) pointed out that, at concentrations of GMP which completely inhibited the wild type(fig.7), whearas the K326Q enzyme retained full activity. The experiment demonstrated that inactivation of glutamine PRPP amidotransferase was caused by covalent labeling by FSBA. It appears that Lys326 may have been labeled by FSBA in the secondary phase of the reaction which did not contribute to inactivation, so we introduced a mutagenesis from AAA to CAG (979-981) based on E.coli codon bias.
We overexpressed PurF K326Q to achieve the mass synthesis of purine. Consequently, more purines than before participant in the circuits we designed and were used to synthesize uric acid when co-expressed with rhXOR.
References
1.Lehninger, A. L., Nelson, D. L. 1., & Cox, M. M. (2008). Lehninger principles of biochemistry (5th ed.). New York ; New Delhi: W.H. Freeman.
2.Zhou G, Charbonneau H, Colman RF, Zalkin H. Identification of sites for feedback regulation of glutamine 5-phosphoribosylpyrophosphate amidotransferase by nucleotides and relationship to residues important for catalysis. J Biol Chem. 1993 May 15;268(14):10471–10481.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 876
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 94
- 1000COMPATIBLE WITH RFC[1000]
| None |