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 Ca(2+)-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 degrees 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.
Stampolidis
Pavlos
P
Institute of Biological Sciences, Cledwyn Building, Aberystwyth University, Aberystwyth, Ceredigion SY23 3DD, United Kingdom.
Kaderbhai
Naheed N
NN
Kaderbhai
Mustak A
MA
D001483:Base Sequence; D017931:DNA Primers; D004220:Disulfides / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D002479:Inclusion Bodies / Q000378:metabolism*; D008854:Microscopy, Electron; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000302:isolation & purification / Q000378:metabolism*; D019897:Periplasm / Q000201:enzymology*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D017931:DNA Primers; D004220:Disulfides; D011994:Recombinant Proteins; C023929:lupanine hydroxylase; D000587:Oxidoreductases Acting on CH-NH Group Donors