Functional expression and characterisation of human cytochrome P45017alpha in Pichia pastoris.
Human cytochrome P45017alpha (CYP17), present in mammalian adrenal and gonadal tissues, catalyses both steroid 17-hydroxylation and C17,20 lyase reactions, producing intermediates for the glucocorticoid and androgenic pathways, respectively. The characterisation of this complex enzyme was initially hampered due to low level in vivo expression of CYP17. Heterologous expression systems have contributed greatly to our current knowledge of CYP17's dual catalytic activity. However, due to the hydrophobic nature of this membrane-bound protein, primarily truncated and modified forms of CYP17 are currently being expressed heterologously. Although the N-terminally modified enzyme has been well characterised, protein structure and function studies still necessitate the expression of unmodified, wild-type CYP17. We report here the expression of a catalytically active, unmodified human CYP17 in the industrial methylotrophic yeast, Pichia pastoris. A typical P450 carbon monoxide difference spectrum, with an absorption maximum at 448nm and a substrate-induced type I spectrum were recorded using a detergent-solubilised cellular fraction containing CYP17. The expressed enzyme catalysed the conversion of progesterone to 17-hydroxyprogesterone as well as 16-hydroxyprogesterone, a product unique to human and chimpanzee CYP17. This is the first report showing the heterologous expression of a fully functional human steroidogenic cytochrome P450 enzyme in P. pastoris.
Kolar
Norbert W
NW
Department of Biochemistry, University of Stellenbosch, Matieland, South Africa.
Swart
Amanda C
AC
Mason
J Ian
JI
Swart
Pieter
P
D006801:Humans; D007700:Kinetics; D010843:Pichia / Q000201:enzymology*; D011374:Progesterone / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D011994:Recombinant Proteins; D011374:Progesterone; D013254:Steroid 17-alpha-Hydroxylase