Human CYP27A1 is a mitochondrial cytochrome P450, which is principally found in the liver and plays important roles in the biological activation of vitamin D3 and in the biosynthesis of bile acids. We have applied a systematic analysis of hydrogen bonding patterns in 11 prokaryotic and mammalian CYP crystal structures to construct a homology-based model of CYP27A1. Docking of vitamin D3 structures into the active site of this model identified potential substrate contact residues in the F-helix, the β-3 sheet, and the β-5 sheet. Site-directed mutagenesis and expression in COS-1 cells confirmed that these positions affect enzymatic activity, in some cases shifting metabolism of 1α-hydroxyvitamin D3 to favor 25- or 27-hydroxylation. The results suggest that conserved hydrophobic residues in the β-5 hairpin help define the shape of the substrate binding cavity and that this structure interacts with Phe-248 in the F-helix. Mutations directed toward the β-3a strand suggested a possible heme-binding interaction centered on Asn-403 and a structural role for substrate contact residues Thr-402 and Ser-404.

Abbreviations used: CYP27A1, cytochrome P450 25-hydroxylase; CTX, cerebrotendinous xanthomatosis; CYP, cytochrome P450; CYP24A1, 1α,25-dihydroxyvitamin D3-inducible cytochrome P450 24-hydroxylase; CYP27B1, cytochrome P450 1α-hydroxylase; HPLC, high performance lic|uid chromatography; VDDR-1, vitamin D-dependent rickets-type I; 1α-OH-D^sub 3^, 1α-hydroxyvitamin D3; 1α,25-(OH)^sub 2^D^sub 3^, 1α,25-dihydroxyvitamin D3 25-OH-D^sub 3^, 25-hydroxyvitamin D3; RMSD, root mean-square deviation; congenital adrenal hyperplasia, CAH.

The use of vitamin D3 by the body is mediated through a series of mitochondrial cytochrome P450 enzyme reactions in which the parent vitamin is transformed sequentially by the hepatic 25-hydroxylase (CYP27A1) into 25-OH-D^sub 3^ and the renal 1α-hydroxylase (CYP27B1) into the active hormone, 1α,25-(OH)^sub 2^D^sub 3^. Subsequent catabolism in vitamin D target tissues by the 1α,25-(OH)^sub 2^D^sub 3^-inducible 24-hydroxylase (CYP24A1) produces the biliary excretory product calcitroic acid (1). The active hormone and its precursors are transported between tissues complexed to vitamin D-binding protein. In the nucleus of cells in vitamin D target tissues, 1α,25-(OH)^sub 2^D3 binds vitamin D receptor to transactivate, the expression of specific gene products affecting calcium homeostasis and the proliferation and differentiation of certain specialized cell types (2). Thus the structure of the vitamin D molecule helps define crucial interactions with at least five proteins, thereby influencing transport, activation, biological action, catabolism, pharmacokinetics, and the precise sequence of these events (3). However, research into the design of vitamin D prodrugs has shown that the activation sequence can be manipulated, and that a nonphysiological, 1α-hydroxylated vitamin D analog can be 25-hydroxylated and activated in a single cytochrome-mediated step (4).

The circulating level of 25-OH-D^sub 3^ is a measure of vitamin D status (5), but the CYP enzymes regulating its formation are still not completely defined. The best studied 25-hydroxylase, CYP27A1, is well conserved across species and is a bifunctional enzyme that also expresses 27-hydroxylase activity toward cholesterol and related sterols in two separate bile acid biosynthetic pathways (6). Despite being the affected target in cerebrotendinous xanthomatosis (CTX) (7,8), a lipid storage disorder characterized by the accumulation of abnormal metabolites of bile acid precursors (9), the only indication of vitamin D deficiency associated with the loss of CYP27A1 is an increased risk of osteoporosis and bone fractures and in low 25-OH-D^sub 3^ levels that fail to normalize with bile-acid replacement therapy (10). This suggests a redundant or compensatory 25-hydroxylase activity that is in agreement with one of the earliest studies of vitamin D 25-hydroxylation, which inferred the existence of a high specificity, low capacity (microsomal) enzyme and a low specificity, high capacity (mitochondrial) enzyme (11). Two of the candidates for the human microsomal 25-hydroxylase include CYP3A4, a hepatic drug metabolizing enzyme with broad substrate specificity (12,13), and CYP2R1, recently shown to have activity (14) and to be associated with a mild form of rickets (15). A similar microsomal 25-hydroxylase redundancy is seen in the rat enzymes CYP2J3 (16), CYP2R1, and CYP2C11 (17), the pig CYP2D25 (18), and the mouse CYP2R1 (19).

Since there are no crystal structures of mitochondrial cytochrome P450s, any model for CYP27A1 must be based upon one or more existing prokaryotic or microsomal CYP crystal structures. Although an important model of bovine mitochondrial CYP11A1 (side-chain cleavage enzyme, P450scc) (20) is available in the Protein Data Bank, there is disagreement concerning the existence of an ad hoc surface loop between the E- and F-helices hypothesized to mediate membrane binding in it since subsequent models of bovine CYP11A1 (21), human CYP11A1 (22), human CYP11B1 and CYP11B2 (23), and all cytochrome P450 crystal structures, including 14α-sterol demethylase (CYP51) from Mycobacterium tuberculosis (24,25) and the mammalian enzymes CYP2B4 (26), CYP2C5 (27,28), CYP2C8 (29), CYP2C9 (30), and CYP3A4 (31,32) show no evidence of this surface loop. Thus the quality of a previous model for CYP27A1 based on the original bovine model (7) remains in doubt.