Structural analysis of the gene encoding rat cholesterol alpha-hydroxylase, the key enzyme for bile acid biosynthesis

The gene encoding cholesterol 7 alpha-hydroxylase (P450VIIA) was isolated from rat genomic DNA. The gene spanned about 11 kilobases and contained six exons. Blotting analysis of genomic DNA and complete matching of restriction maps of several isolated genomic clones indicated that there appeared to be only one gene in the rat genome. The putative transcription initiation site was present 61 base pairs upstream from the ATG codon. The typical TATA sequence and CCAAT promoter element were found at 24 and 47 base pairs upstream from the transcription initiation site, respectively. Alignment of several P450 proteins showed that the cholesterol 7 alpha-hydroxylase gene shared location of introns with none of the other P450 genes except for intron 5, which was in the same position as intron 10 of the gene encoding P450IVA1. The alignment also indicated that the distal helix of cholesterol 7 alpha-hydroxylase contained an asparagine in place of the well conserved threonine that is postulated to be involved in the O2 binding site. Unusual residues, Asn-126 and Thr-442, were also found at the sites where all other P450s have positively charged amino acids, which are considered to be involved in interaction with heme propionate. These replacements may be related to the unique function and unusual lability of the hydroxylase. Analysis of evolutionary distance between the cholesterol 7 alpha-hydroxylase gene and other known P450 genes indicated that yeast P450LIA is most closely related to P450VIIA. This finding suggests that the cholesterol 7 alpha-hydroxylase gene is an evolutionarily old P450 gene.