Targeting of active human cytochrome P4501A1 (CYP1A1) to the periplasmic space of Escherichia coli.
Native human cytochrome P4501A1 (CYP1A1) was appended at its amino terminus to the secretory signal of Escherichia coli alkaline phosphatase. The chimeric P450 construct was placed under the transcriptional control of the native phoA promoter in a prokaryotic expression vector. Induction of the hemoprotein by heterologous expression in E. coli following growth in a phosphate-limited medium resulted in abundant synthesis of recombinant CYP1A1 as detected by reduced CO-difference spectra. Furthermore, the signal-appended CYP1A1 was translocated across the bacterial inner membrane by the sec-dependent pathway and processed to yield authentic, heme-incorporated P450 within the periplasmic space. In vitro and whole-cell metabolic activity studies showed that the periplasmically-located CYP1A1 competently catalysed NADPH-dependent benzo[a]pyrene 3-hydroxylation and 7-ethoxyresorufin O-deethylation. The means to localise cytochromes P450 in the periplasm offers an ability to produce high levels of protein, attributable to the less hostile nature of the compartment, and therein the enzymes for posttranslational assembly of heme with the translocated protein.
Kaderbhai
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MA
AberBiocentre, Institute of Biological Sciences, Edward Llwyd Building, Aberystwyth, Wales, SY23 3DA, United Kingdom.
Ugochukwu
C C
CC
Lamb
D C
DC
Kelly
S L
SL
D000469:Alkaline Phosphatase; D002451:Cell Compartmentation; D018844:Cyclin-Dependent Kinases / Q000235:genetics; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006801:Humans; D019897:Periplasm / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology*; D021381:Protein Transport
D029968:Escherichia coli Proteins; D021382:Protein Sorting Signals; D019363:Cytochrome P-450 CYP1A1; D018844:Cyclin-Dependent Kinases; D000469:Alkaline Phosphatase; C114089:phoA protein, E coli