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

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.