Single A326G mutation converts human CYP24A1 from 25-OH-D ₃ -24-hydroxylase into -23-hydroxylase, generating 1α,25-(OH) ₂ D ₃ -26,23-lactone

Abstract

Studies of 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) have demonstrated that it is a bifunctional enzyme capable of the 24-hydroxylation of 1␣,25-(OH) 2D3, leading to the excretory form, calcitroic acid, and 23-hydroxylation, culminating in 1␣,25-(OH) 2D3-26,23-lactone. The degree to which CYP24A1 performs either 23-or 24-hydroxylation is species-dependent. In this paper, we show that the human enzyme that predominantly 24-hydroxylates its substrate differs from the opossum enzyme that 23-hydroxylates it at only a limited number of amino acid residues. Mutagenesis of the human form at a single substrate-binding residue (A326G) dramatically changes the regioselectivity of the enzyme from a 24-hydroxylase to a 23-hydroxylase, whereas other modifications have no effect. Ala-326 is located in the I-helix, close to the terminus of the docked 25-hydroxylated side chain in a CYP24A1 homology model, a result that we interpret indicates that substitution of a glycine at 326 provides extra space for the side chain of the substrate to move deeper into the pocket and place it in a optimal stereochemical position for 23-hydroxylation. We discuss the physiological ramifications of these results for species possessing the A326G substitution, as well as implications for optimal vitamin D analog design. (Fig. 1). Studies using CYP24A1-knockout (8) and VDR-knockout mice (8, 9) demonstrate the total absence of calcitroic acid and 1␣,25-(OH) 2 D 3 -26,23-lactone formation when the vitamin Dinducible, VDR-mediated CYP24A1 is not expressed. The in vivo result of CYP24A1 ablation is decreased viability, with 50% of mice not surviving beyond weaning because of hypercalcemia and nephrocalcinosis (8, 10). Although it has been well established that calcitroic acid is the major biliary product of 1␣,25-(OH) 2 D 3 in the rat (11, 12), there have been claims that intermediates of the C24 oxidation pathway retain some biological activity (13) and that the terminal product of 23-hydroxylation, namely 1␣,25-(OH) 2 D 3 -26,23-lactone, belongs to a family of potent VDR antagonists with potential for use in the treatment of Paget's disease (14, 15).The degree to which CYP24A1 performs either of these pathways varies with the species. It had been shown in the mid-1980s by using isolated kidney mitochondria from a variety of species, that the 24-hydroxylase and 23-hydroxylase activities copurify (16). Although both pathways are used in humans, certain species, such as the guinea pig (17, 18), were shown to 23-hydroxylate, whereas other species, such as the rat, primarily 24-hydroxylate the substrate, 25-OH-D 3 (16). These studies suggested that the two enzyme activities might have different kinetic parameters, but it was not clear whether the activities resided on a single or distinct polypeptide chains. With the cloning and expression of the recombinant protein from different species (19,20), it became clear that a single polypeptide chain was capable of both C23-and C24-hydroxylation activities (5-7).Recent studies of rat and human CYP24A1 hav...

The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species

“…In 2007, Prosser et al demonstrated that an active site mutation, A326G, converts human CYP24A1 into a complete C23 hydroxylase, likely by increasing the space available for substrate binding (20). However, there is also mounting evidence that redox partner interactions influence the substrate binding and recognition regions of P450 enzymes (21,22,26,30).…”

“…While calcitroic acid is excreted in urine, 1,25(OH) 2 D3-26, 23-lactone has been demonstrated to modulate vitamin D signaling by binding the vitamin D receptor (18,19). Intriguingly, this first step in the deactivation pathway is also regioselective according to species, with the human isoform hydroxylating at both C23 and C24 sites at an approximate 2:8 ratio while rat CYP24A1 is a pure C24 hydroxlase and opossum CYP24A1 is a pure C23 hydroxylase (20).…”

Cytochrome P450-mediated metabolism of vitamin D

“…In the VDR-null mouse, we see a complete breakdown of this autoregulation process because CYP27B1 is not suppressed by excessive 1,25-(OH) 2 D 3 by guest, on April 28, 2019 www.jlr.org Downloaded from to a calcitroic-acid product while more primitive organisms have Gly326 and show predominantly 23-hydroxylation to give a 26,23-lactone product. The functional signifi cance of two distinct pathways in different species is unknown ( 18 ).…”

“…The substrate-binding pocket is formed by several secondary structures folded around the distal face of the heme-group so that the substrate can be brought to within 3.2 Å of the iron atom for hydroxylation. An analysis of the heme-ligand geometry of 49 substrate-bound crystal structures revealed the hydroxylation target carbons actually adopt a spatially conserved orientation to the heme iron, and this can be triangulated for use in docking studies ( 18 ).…”

“…Attempts to identify the key substrate-binding residues were originally guided by homology models (18)(19)(20)(21)(22) based upon 10-20 available crystal structures from unrelated soluble prokaryotic CYPs. Recently, the study of the active site of vitamin D-related CYPs has been further advanced by the emergence of X-ray crystallography-derived models vant vitamin D 3 -25-hydroxylase.…”