Molecular cloning, expression analysis and functional characterization of chicken cytochrome P450 27A1: A novel mitochondrial vitamin D3 25-hydroxylase

Abstract

Vitamin D3 is hydroxylated by cytochrome P450 (CYP) before exerting biological effects. The chicken CYP involved in vitamin D3 25-hydroxylation has yet to be cloned, and little is known about its functional characteristics, tissue distribution, and cellular expression. We identified a novel, full-length CYP27A1 gene cloned from chicken hepatocyte cDNA that encodes a putative protein of 518 amino acids. Swiss modeling revealed that chicken CYP27A1 has a classic open-fold form. Multisequence homology alignment determined that CYP27A1 contains conserved motifs for substrate recognition and binding. Quantitative real-time PCR analysis in 2-mo-old Partridge Shank broilers demonstrated that CYP27A1 mRNA levels were highest in the liver, followed by the thigh muscles, the breast muscles, and kidneys. The transcripts of CYP27A1 in breast muscles were significantly higher in males than in females. A subcellular localization analysis demonstrated that CYP27A1 was mainly expressed in the mitochondria. In vitro enzyme assays suggested that recombinant CYP27A1 hydroxylates vitamin D3 at the C-25 position to form 25-hydroxyvitamin D3 (25(OH)D3). The Km and Vmax values for CYP27A1-dependent vitamin D3 25-hydroxylation were estimated to be 4.929 μM and 0.389 mol min-1 mg-1 protein, respectively. In summary, these results suggest that CYP27A1 encodes a mitochondrial CYP that plays an important physiologic role in the 25-hydroxylation of vitamin D3 in chickens, providing novel insights into vitamin D3 metabolism in this species.