CYP24A1 is the multicatalytic cytochrome P450 responsible for the catabolism of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] via the C23- and C24-oxidation pathways. The importance of this enzyme was recently highlighted in epidemiological studies which show that CYP24A1 gene polymorphisms are linked to serum vitamin D levels. Despite its importance, the human isoform has been poorly characterised to date. The principal aim of this project was to express and purify active human CYP24A1 and to determine the kinetics of each step in the C24-oxidation pathway of 1,25(OH)2D3 catabolism that it catalyses. A further aim was to characterise its metabolism of CYP11A1-derived 20S-hydroxyvitamin D3 [20(OH)D3] and 20S,23-dihydroxyvitamin D3 [20,23(OH)2D3] which are biologically active vitamin D3 analogues which display low calcaemic activity and have therapeutic potential.
Human CYP24A1 containing a C-terminal 6 His-tag was successfully expressed in Escherichia coli using a coexpression system with GroEL/ES, and was partially purified it in an active state that permitted detailed characterisation of its catalytic properties. Rat CYP24A1 was also expressed and purified for some comparative studies. To permit kinetic analysis of each step of the C24-oxidation pathway, the intermediates, 1,24,25-trihydroxyvitamin D3, 24-oxo-1,25-dihydroxyvitamin D3, 24-oxo-1,23,25-trihydroxyvitamin D3 and tetranor-1,23-dihydroxyvitamin D3 were enzymatically produced from 1,25(OH)2D3 using the highly expressed rat CYP24A1. Substrates for CYP24A1 were incorporated into phospholipid vesicles made up of dioleoyl phosphatidylcholine and cardiolipin as this resemble the inner mitochondrial membrane where the P450 resides in vivo. Both 1,25(OH)2D3 and tetranor-1,23(OH)2D3 were found to partition strongly into the phospholipid bilayer when in aqueous medium. Changes to the phospholipid concentration did not affect the kinetic parameters for the metabolism of 1,25(OH)2D3 by CYP24A1, indicating that it is the concentration of substrates in the membrane phase (mol substrate/mol phospholipid) that determines their rate of metabolism. Human CYP24A1 exhibited Km values for the different C24-intermediates ranging from 0.34 to 15 mmol/mol phospholipid, with 24-oxo-1,23,25(OH)3D3 displaying the lowest and 1,24,25(OH)3D3 displaying the highest. The kcat values varied by up to 3.8-fold with 1,24,25(OH)3D3 displaying the highest kcat (34 min-1) and 24-oxo-1,23,25(OH)3D3 the lowest. The data show that the cleavage of the side chain of 24-oxo-1,23,25(OH)3D3 occurs with the highest catalytic efficiency (kcat/Km) and produces 23-oxo-tetranor-1(OH)D3, and not tetranor-1,23(OH)2D3, as the primary product. These kinetic analyses also show that intermediates of the C24-oxidation pathway effectively compete with precursor substrates for binding to the active site of the enzyme, which manifests as accumulation of intermediates indicating that they dissociate after each catalytic step.
|Qualification||Doctor of Philosophy|
|Publication status||Unpublished - Jul 2015|