Dual metabolic pathway of 25‐hydroxyvitamin D₃ catalyzed by human CYP24

Abstract

<jats:p>Human 25‐hydroxyvitamin D<jats:sub>3</jats:sub> (25(OH)D<jats:sub>3</jats:sub>) 24‐hydroxylase (CYP24) cDNA was expressed in <jats:italic>Escherichia coli</jats:italic>, and its enzymatic and spectral properties were revealed. The reconstituted system containing the membrane fraction prepared from recombinant <jats:italic>E. coli</jats:italic> cells, adrenodoxin and adrenodoxin reductase was examined for the metabolism of 25(OH)D<jats:sub>3</jats:sub>, 1α,25(OH)<jats:sub>2</jats:sub>D<jats:sub>3</jats:sub> and their related compounds. Human CYP24 demonstrated a remarkable metabolism consisting of both C‐23 and C‐24 hydroxylation pathways towards both 25(OH)D<jats:sub>3</jats:sub> and 1α,25(OH)<jats:sub>2</jats:sub>D<jats:sub>3</jats:sub>, whereas rat CYP24 showed almost no C‐23 hydroxylation pathway [Sakaki, T. Sawada, N. Nonaka, Y. Ohyama, Y. &amp; Inouye, K. (1999) <jats:italic>Eur. J. Biochem</jats:italic>. <jats:bold>262</jats:bold>, 43–48]. HPLC analysis and mass spectrometric analysis revealed that human CYP24 catalyzed all the steps of the C‐23 hydroxylation pathway from 25(OH)D<jats:sub>3</jats:sub> via 23<jats:italic>S</jats:italic>,25(OH)<jats:sub>2</jats:sub>D<jats:sub>3</jats:sub>, 23<jats:italic>S</jats:italic>,25,26(OH)<jats:sub>3</jats:sub>D<jats:sub>3</jats:sub> and 25(OH)D<jats:sub>3</jats:sub>‐26,23‐lactol to 25(OH)D<jats:sub>3</jats:sub>‐26,23‐lactone in addition to the C‐24 hydroxylation pathway from 25(OH)D<jats:sub>3</jats:sub> via 24<jats:italic>R</jats:italic>,25(OH)<jats:sub>2</jats:sub>D<jats:sub>3</jats:sub>, 24‐oxo‐25(OH)D<jats:sub>3</jats:sub>, 24‐oxo‐23<jats:italic>S</jats:italic>,25(OH)<jats:sub>2</jats:sub>D<jats:sub>3</jats:sub> to 24,25,26,27‐tetranor‐23(OH)D<jats:sub>3</jats:sub>. On 1α,25(OH)<jats:sub>2</jats:sub>D<jats:sub>3</jats:sub> metabolism, similar results were observed. These results strongly suggest that the single enzyme human CYP24 is greatly responsible for the metabolism of both 25(OH)D<jats:sub>3</jats:sub> and 1α,25(OH)<jats:sub>2</jats:sub>D<jats:sub>3</jats:sub>. We also succeeded in the coexpression of CYP24, adrenodoxin and NADPH‐adrenodoxin reductase in <jats:italic>E. coli</jats:italic>. Addition of 25(OH)D<jats:sub>3</jats:sub> to the recombinant <jats:italic>E. coli</jats:italic> cell culture yielded most of the metabolites in both the C‐23 and C‐24 hydroxylation pathways. Thus, the <jats:italic>E. coli</jats:italic> expression system for human CYP24 appears quite useful in predicting the metabolism of vitamin D analogs used as drugs.