Generation of a homology model for the human cytochrome P450, CYP24A1, and the testing of putative substrate binding residues by site-directed mutagenesis and enzyme activity studies

Masuda, Sonoko; Prosser, David E.; Guo, Yu-Ding; Kaufmann, Martin; Jones, Glenville

doi:10.1016/j.abb.2006.11.030

Cited by 33 publications

(34 citation statements)

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“…After consideration of the homology model of hCYP24A1 (21) and the sequence alignments of a wider range of CYP24A1 species orthologs, our studies focused on three residues that differ, including Ala-326 ( Fig. 2), Asn-448, and Gln-471 (SI Fig.…”

Section: Resultsmentioning

confidence: 99%

“…A substrate docking study was undertaken with the CYP24A1 homology model (21), to determine the proximity of the Ala-326 residue in relation to the 25-hydroxylated side chain of the sub- HPLC and LC-MS-based characterization of the catabolic products of 1␣,25-( strate. As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The studies reported here show that, when this alanine 326 in the hCYP24A1 is changed to a glycine, as it is in opossum and guinea pig, the 24-hydroxylation pattern primarily giving rise to calcitroic acid is dramatically altered to a 23-hydroxylation pattern, in which the terminal metabolite is 1␣,25-(OH) 2 D 3 -26,23-lactone, a pattern that closely resembles that observed with the opossum CYP24A1. Such a radical change in hydroxylation pattern does not occur when other residues in hCYP24A1 are mutated (21), making a strong case for the fact that the side chain of Ala-326 is a major determinant of the depth of substrate penetration into the substrate-binding pocket and the alignment of the hydroxylation site above the heme Fe. Indeed, homology modeling of hCYP24A1 (21), and its related CYP, CYP27A1 (25), places the I-helix closely abutting the substrate binding pocket and the residue Ala-326 facing the terminal methyl groups of the vitamin D side chain, thus independently supporting the conclusion from our mutagenesis studies that Ala-326 occupies a critical position.…”

Section: Discussionmentioning

confidence: 99%

“…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 have used homology modeling to reveal the general structure of the protein and highlight active-site residues for mutagenesis studies (21,22). Masuda et al (21) have shown the importance of residues in the BЈ-helix, BЈ/C loop, F-helix, and ␤-5 hairpin of the human enzyme.…”

mentioning

confidence: 99%

“…Recent studies of rat and human CYP24A1 have used homology modeling to reveal the general structure of the protein and highlight active-site residues for mutagenesis studies (21,22). Masuda et al (21) have shown the importance of residues in the BЈ-helix, BЈ/C loop, F-helix, and ␤-5 hairpin of the human enzyme.…”

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confidence: 99%

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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

Prosser

Kaufmann

O’Leary

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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

Prosser

Kaufmann

O’Leary

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Understanding Cytochrome P450 Enzyme Substrate Inhibition and Prospects for Elimination Strategies

Zhang,

Wang

et al. 2024

ChemBioChem

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Cytochrome P450 (CYP450) enzymes, which are widely distributed and pivotal in various biochemical reactions, catalyze diverse processes such as hydroxylation, epoxidation, dehydrogenation, dealkylation, nitrification, and bond formation. These enzymes have been applied in drug metabolism, antibiotic production, bioremediation, and fine chemical synthesis. Recent research revealed that CYP450 catalytic kinetics deviated from the classic Michaelis–Menten model. A notable substrate inhibition phenomenon that affects the catalytic efficiency of CYP450 at high substrate concentrations was identified. However, the substrate inhibition of various reactions catalyzed by CYP450 enzymes have not been comprehensively reviewed. This review describes CYP450 substrate inhibition examples and atypical Michaelis–Menten kinetic models, and provides insight into mechanisms of these enzymes. We also reviewed 3D structure and dynamics of CYP450 with substrate binding. Outline methods for alleviating substrate inhibition in CYP450 and other enzymes, including traditional fermentation approaches and protein engineering modifications. The comprehensive analysis presented in this study lays the foundation for enhancing the catalytic efficiency of CYP450 by deregulating substrate inhibition.

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Human Cytochrome P450 Enzymes

Guengerich

2015

Cytochrome P450

133

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Generation of a homology model for the human cytochrome P450, CYP24A1, and the testing of putative substrate binding residues by site-directed mutagenesis and enzyme activity studies

Cited by 33 publications

References 45 publications

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

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

Understanding Cytochrome P450 Enzyme Substrate Inhibition and Prospects for Elimination Strategies

Human Cytochrome P450 Enzymes

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Generation of a homology model for the human cytochrome P450, CYP24A1, and the testing of putative substrate binding residues by site-directed mutagenesis and enzyme activity studies

Cited by 33 publications

References 45 publications

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

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

Understanding Cytochrome P450 Enzyme Substrate Inhibition and Prospects for Elimination Strategies

Human Cytochrome P450 Enzymes

Contact Info

Product

Resources

About

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

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