Periplasmically-exported lupanine hydroxylase undergoes transition from soluble to functional inclusion bodies in Escherichia coli

P. Stampolidis, Naheed N. Kaderbhai, Mustak A. Kaderbhai

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7 Citations (SciVal)


Pseudomonas lupanine hydroxylase is a periplasmic-localised, two domain quinocytochrome c enzyme. It requires numerous post-translocation modifications involving signal peptide processing, disulphide bridge formation and, heme linkage in the carboxy-terminal cytochrome c domain to eventually generate a Ca2+-bound quino-c hemoprotein that hydroxylates the plant alkaloid, lupanine. An exported, functional recombinant enzyme was generated in Escherichia coli by co-expression with cytochrome c maturation factors. Increased growth temperatures ranging from 18 to 30 °C gradually raised the enzyme production to a peak together with its concomitant aggregation as red solid particles, readily activatable in a fully functional form by mild chaotropic treatment. Here, we demonstrate that the exported lupanine hydroxylase undergoes a cascade transition from a soluble to “non-classical” inclusion body form when build-up in the periplasm exceeded a basal threshold concentration. These periplasmic aggregates were distinct from the non-secreted, signal-sequenceless counterpart that occurred as misfolded, non-functional concatamers in the form of classical inclusion bodies. We discuss our findings in the light of current models of how aggregation of lupanine hydroxylase arises in the periplasmic space.
Original languageEnglish
Pages (from-to)8-15
Number of pages8
JournalArchives of Biochemistry and Biophysics
Issue number1
Publication statusPublished - 23 Jan 2009


  • Protein export
  • Periplasmic space
  • Amorphous protein aggregation
  • LH
  • Inclusion bodies
  • Disulphide-bond formation
  • Pyrroloquinoline quinine
  • Post-translational modifications
  • Quinocytochrome c


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