Part:BBa_K4821004

RdhANp_M132P_K12

This sequence codes for a modified RdhANp that has been docked to the para chlorine position in PCB 132 and codon-optimized for expression in E. coli K-12. This enzyme contains the following mutations: F294I K295L E298L K310A R312T P314Y M315F S423T S425A M426L N455L A456M H457D V556I A563G. It was designed as an improvement on part BBa_K4821002, the codon-optimized wild-type RdhANp dehalogenase to possess higher catalytic activity against PCB132.

Usage and Biology

This is a modified RdhANp with the potential to dechlorinate PCB132 at the para position for more efficient breakdown of the compound to acetyl CoA by the biphenyl degradation pathway (bph gene cluster). Mechanistically, Y426 in RdhANp protonates the chlorine located at 3.5A from the oxygen of Y426 and 5.0A from the cobalamin to catalyze a dechlorination reaction where the chlorine would coordinate with the Co on the cobalamin.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1628
  Illegal BamHI site found at 312
  Illegal BamHI site found at 419
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]