Part:BBa_K3215005

phn Operon Correction Cassette

This part enables it's user to fix and improve the already defective phn Operon in E. coli K-12. Utilizing a recombination technique, both flanking regions will be responsible for the removal of the phnEFG cistrons and insertion of the corrected phnEG cistrons. The removal of the phnF repressor and insertion of the corrected and optimized phnEG cistrons improve the organism's ability to metabolize phosphonates.

Usage and Biology

The phn Operon is one of the nine transcriptional units that are present in the Phosphate Regulon (Pho), a global regulatory mechanism involved in bacterial Pi management. It is responsible for the catabolism of phosphates and it has 14 cistrons (phnCDEFGHIJKLMNOP) that, together, are responsible for controlling the Carbon-Phosphorus (C-P) lyase activity. The C-P lyase pathway is activated upon conditions of phosphate starvation and it is responsible for converting phosphonates into 5-phosphoribosyl-α-1-diphosphate (PRPP), utilizing ATP.

phnCDE

It encodes an ATP-binding cassette, where phnC has the ATP-binding site, phnD is a periplasmic binding protein for phosphonates and phnE is a membrane spanning transport protein.

The complex formed is composed of two proteins derived from phnC, two from phnE and one solute-binding protein from phnD. Altogether, these proteins are responsible for the transport of alkylphosphonates and aminoalkylphosphonates, through the reactions:

phnF

The cistron phnF is responsible for the production of a transcriptional regulator with two domains, an amino-terminal domain that contains a potential helix-turn-helix DNA-binding motif and a carboxy-terminal domain involved in effector recognition.

Its exact function is not known, but it was inferred by sequence similarity. phnF represses phnCDE, in phosphorus replete conditions.

phnGHIJKLM

It forms the C-P lyase core complex and is responsible for the coupling of phosphonate to ATP and C-P bond cleavage. In this complex, the PhnI, a nucleosidase capable of deglycosylating ATP and GTP to ribose 5-triphosphate, is supported by phnG, phnH, and phnL in the catalysis of the transfer of phosphonate moiety to a 5′-triphosphate alkyl phosphonate intermediate, according to the following reaction:

After that, PhnM catalyses the pyrophosphate release through the reaction:

Following, phnJ is allowed to cleave the C-P lyase bond via an S-adenosyl methionine (SAM)-dependent radical mechanism, thus converting the resulting ribose cyclic phosphate into PRPP. This reaction is possible because of the interaction of phnK trough the via its α helices 3 and 4 with PhnJ, exposing its active site.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1178
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1178
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1178
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1178
  
• 1000
  COMPATIBLE WITH RFC[1000]