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Functional expression and characterisation of human cytochrome P45017α in Pichia pastoris

Paper ID Volume ID Publish Year Pages File Format Full-Text
25362 43570 2007 10 PDF Available
Title
Functional expression and characterisation of human cytochrome P45017α in Pichia pastoris
Abstract

Human cytochrome P45017α (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 448 nm 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.

Keywords
CYP17, steroid 17α-hydroxylase cytochrome P450; CO, carbon monoxide; A4, androstenedione; DHEA, dehydroepiandrosterone; P5, pregnenolone; P4, progesterone; 17-OHP5, 17-hydroxypregnenolone; 17-OHP4, 17-hydroxyprogesterone; 16-OHP4, 16-hydroxyprogesterone;
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Functional expression and characterisation of human cytochrome P45017α in Pichia pastoris
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 129, Issue 4, 10 May 2007, Pages 635–644
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us