Expression of human CYP27B1 in E. coli and characterisation in phospholipid vesicles (Activity towards native and novel substrates)

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[Truncated abstract] 25-Hydroxyvitamin D3-1α-hydroxylase (CYP27B1) is the enzyme that catalyses the final step of vitamin D3 activation, which is the 1α-hydroxylation of 25- hydroxyvitamin D3 to give 1α,25-dihydroxyvitamin D3. Despite the importance of CYP27B1, the human enzyme has been poorly characterised to date. The principal aim of this study was to express human CYP27B1 in Escherichia coli, isolate the enzyme in an active form and characterise its kinetic properties on a range of substrates. Human CYP27B1 containing a C-terminal 6-His-tag was successfully expressed in Escherichia coli using a co-expression system with GroEL/ES. Expression levels were low and the enzyme proved to be labile, but partial-purification of the active enzyme was achieved. Catalytic activity of purified human CYP27B1, as well as the mouse enzyme, was measured on substrates incorporated into phospholipid vesicles made up of dioleoyl phosphatidylcholine and cardiolipin. Some substrates, including 25-hydroxyvitamin D3, caused inhibition of the enzyme when present at high concentrations (typically >5×Km, depending on the substrate). The presence of cardiolipin in the vesicles had a marked effect on CYP27B1 activity, decreasing the Km for 25-hydroxyvitamin D3. In addition to 25-hydroxyvitamin D3, human CYP27B1 was also found to be active on 25-hydroxyvitamin D2 and the intermediates of the CYP24A1-mediated inactivation pathway, including, 24R,25-dihydroxyvitamin D3, 24-oxo-25- hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3. All these substrates showed comparable kcat values to that for 25-hydroxyvitamin D3 with 24-oxo-23,25- dihydroxyvitamin D3 exhibiting a high Km. Purified human and mouse CYP27B1 were also tested on biologically active vitamin D derivatives produced by CYP11A1...
Original languageEnglish
QualificationDoctor of Philosophy
Publication statusUnpublished - 2013


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