Distinct Binding of Cholesterol and 5β-Cholestane-3α,7α,12α-triol to Cytochrome P450 27A1:  Evidence from Modeling and Site-Directed Mutagenesis Studies

Mast, Natalia; Murtazina, Dilyara A.; Liu, Hong; Graham, Sandra E.; Björkhem, Ingemar; Halpert, James R.; Peterson, Julian A.; Pikuleva, Irina A.

doi:10.1021/bi052654w

Cited by 25 publications

(53 citation statements)

References 23 publications

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“…This phenomenon may be an artifact of our heterologous expression system, as mutagenesis of analogous hydrophilic domains has not been precluded for other CYPs, including CYP51 [35] and the more closely related cholesterol metabolizing CYPs, CYP27A1 [36,37] and CYP7A1 [42]. Our modeling and biochemical studies suggests that proper organization of the B'-helix of CYP24A1, through contacts with the heme, F, G and I-helices, and the F-G and B'-C loops is required to maintain structural integrity of the reactive heme center.…”

Section: Discussionmentioning

confidence: 99%

Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: Insights into substrate specificity and membrane bound structure–function

Annalora

Bobrovnikov-Marjon

Serda

et al. 2007

Archives of Biochemistry and Biophysics

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Cytochrome P450C24A1 (CYP24A1), a peripheral inner mitochondrial membrane hemoprotein and candidate oncogene, regulates the side-chain metabolism and biological function of vitamin D and many of its related analog drugs. Rational mutational analysis of rat CYP24A1 based on hybrid (2C5/ BM-3) homology modeling and affinity labeling studies clarified the role of key domains (Nterminus, A', A, and F-helices, β3a strand, & β5 hairpin) in substrate binding and catalysis. The scope of our study was limited by an inability to purify stable mutant enzyme targeting soluble domains (B', G, and I-helices) and suggested greater conformational flexibility among CYP24A1's membraneassociated domains. The most notable mutants developed by modeling were V391T and I500A, which displayed defective binding function and profound metabolic defects for 25-hydroxylated vitamin D 3 substrates similar to a non-functional F-helix mutant (F249T) that we previously reported. Val-391 (β3a strand) and Ile-500 (β5 hairpin) are modeled to interact with Phe-249 (F-helix) in a hydrophobic cluster that directs substrate binding events through interactions with the vitamin D cis-triene moiety. Prior affinity labeling studies identified an amino-terminal residue (Ser-57) as a putative active-site residue that interacts with the 3β-OH group of the vitamin D A-ring. Studies with 3-epi and 3-deoxy-1,25(OH) 2 D 3 analogs confirmed interactions between the 3β-OH group and Ser-57 effect substrate recognition and trafficking while establishing that the trans conformation of A-ring hydroxyl groups (1α & 3β) is obligate for high-affinity binding to rat CYP24A1. Our work suggests that CYP24A1's amphipathic nature allows for monotopic membrane insertion, whereby a pw2d-like substrate access channel is formed to shuttle secosteroid substrate from the membrane to the active-site. We hypothesize that CYP24A1 has evolved a unique amino-terminal membrane § Deceased

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Section: Discussionmentioning

confidence: 99%

Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: Insights into substrate specificity and membrane bound structure–function

Annalora

Bobrovnikov-Marjon

Serda

et al. 2007

Archives of Biochemistry and Biophysics

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“…This does not necessarily mean a lack of binding, but could be a case of compound positioning in the enzyme active site at a distance to the heme iron (Isin and Guengerich, 2008). Bicalutamide, the strongest CYP27A1 inhibitor in the screening enzyme assay, was used for optimization of the conditions of the binding assay, which was carried out as described (Mast et al, 2006) at 30°C in a 1-ml solution of 50 mM KP i (pH 7.2) containing 0.4 mM CYP27A1, 1 mM EDTA, 0.01% CYMAL-7, 10% glycerol, and 0.1 M NaCl. Drugs were added from 1-5 mM stocks in the same vehicle as used for studies of drug effects on CYP27A1 activity.…”

Section: Methodsmentioning

confidence: 99%

Marketed Drugs Can Inhibit Cytochrome P450 27A1, a Potential New Target for Breast Cancer Adjuvant Therapy

2015

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Cytochrome P450 CYP27A1 is the only enzyme in humans converting cholesterol to 27-hydroxycholesterol, an oxysterol of multiple functions, including tissue-specific modulation of estrogen and liver X receptors. Both receptors seem to mediate adverse effects of 27-hydroxycholesterol in breast cancer when the levels of this oxysterol are elevated. The present work assessed druggability of CYP27A1 as a potential antibreast cancer target. We selected 26 anticancer and noncancer medications, most approved by the Food and Drug Administration, and evaluated them first in vitro for inhibition of purified recombinant CYP27A1 and binding to the enzyme active site. Six strong CYP27A1 inhibitors/binders were identified. These were the two antibreast cancer pharmaceuticals anastrozole and fadrozole, antiprostate cancer drug bicalutamide, sedative dexmedetomidine, and two antifungals ravuconazole and posaconazole. Anastrozole was then tested in vivo on mice, which received subcutaneous drug injections for 1 week. Mouse plasma and hepatic 27-hydroxycholesterol levels were decreased 2.6-and 1.6-fold, respectively, whereas plasma and hepatic cholesterol content remained unchanged. Thus, pharmacologic CYP27A1 inhibition is possible in the whole body and individual organs, but does not negatively affect cholesterol elimination. Our results enhance the potential of CYP27A1 as an antibreast cancer target, could be of importance for the interpretation of Femara versus Anastrozole Clinical Evaluation Trial, and bring attention to posaconazole as a potential complementary anti-breast cancer medication. More medications on the US market may have unanticipated off-target inhibition of CYP27A1, and we propose strategies for their identification.

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“…The same workers performed mutagenesis studies that established the important residues involved in ferredoxin interaction. Although there is currently no crystal structure of CYP27A1, numerous homology models have been proposed for the enzyme predicted from other CYPs ( 19,43 ). Until the recent emergence of crystal structures of CYP2R1 and CYP24A1, these models offered the best structural insights into general structure and substrate-binding pockets of vitamin D-related CYPs.…”

Section: Cyp27a1mentioning

confidence: 99%

Cytochrome P450-mediated metabolism of vitamin D

Jones

Prosser

Kaufmann

2014

Journal of Lipid Research

395

292

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signal transduction system, the DBP-knockout mouse is normocalcemic and exhibits normal tissue distribution of vitamin D metabolites and vitamin D action despite exhibiting very low blood levels of 1,25-(OH) 2 D 3 . This unexpected phenotype raised questions about DBP's exact role: essential transporter or buffer against toxicity ( 7,8 ). Work performed on the 25-hydroxylases over the past four decades in humans and a variety of animal species has revealed that several cytochrome P450 enzymes 2 (CYP), such as CYP2R1, CYP27A1, CYP3A4, CYP2D25, and perhaps others, are capable of 25-hydroxylation of vitamin D 3 or related compounds. These CYPs can be referred to as vitamin D 3 -25-hydroxylases, and it is CYP2R1 that is emerging as the physiologically relevant enzyme ( 9 ). On the other hand, there is no ambiguity over the second step of 1 ␣ -hydroxylation or the 25-OH-D 3 -1 ␣ -hydroxylase enzyme responsible, which is carried by a single cytochrome P450 2 named CYP27B1 ( 10-12 ). The inactivation of vitamin D is carried out by the mitochondrial enzyme, 25-hydroxyvitamin D 3 -24-hydroxylase, fi rst described in the early 1970s and initially believed to be involved solely in the renal 24-hydroxylation of 25-OH-D 3 ( 13 ). Work performed over the last 35 years has shown that 24-hydroxylase enzyme activity is the result of CYP24A1 ( 5, 14 ). CYP24A1 catalyzes the conversion of both 25-OH-D 3 and 1,25-(OH) 2 D 3 into a series of 24-and 23-hydroxylated products targeted for excretion along well-established pathways culminating in the water-soluble biliary metabolite calcitroic acid or a 26,23-lactone.This article assembles the most currently pertinent literature on the activating and inactivating enzymes of vitamin D metabolism and highlights protein structure and enzymatic properties, crystal structures, gene organization, and The activation of vitamin D 3 is accomplished by sequential steps of 25-hydroxylation to produce the main circulating form, 25-hydroxyvitamin D (25-OH-D 3 ), followed by 1 ␣ -hydroxylation to the hormonal form, 1 ␣ ,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ) ( 1 ) ( Fig. 1 ). The initial step of 25-hydroxylation occurs in the liver ( 2 ), and the second step occurs both in the kidney and extrarenal sites ( 3, 4 ). The fat-soluble vitamin D and its metabolites are transported from one tissue to another on the vitamin D-binding protein (DBP), which shows different affi nity for the individual metabolites ( 5 ). The cell-surface receptor megalin-cubilin is thought to facilitate the endocytosis of a DBP-bound 25-OH-D 3 into a number of cell types, especially kidney cells ( 6 ). While it is widely believed that DBP is an essential component of the vitamin D

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Distinct Binding of Cholesterol and 5β-Cholestane-3α,7α,12α-triol to Cytochrome P450 27A1: Evidence from Modeling and Site-Directed Mutagenesis Studies

Cited by 25 publications

References 23 publications

Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: Insights into substrate specificity and membrane bound structure–function

Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: Insights into substrate specificity and membrane bound structure–function

Marketed Drugs Can Inhibit Cytochrome P450 27A1, a Potential New Target for Breast Cancer Adjuvant Therapy

Cytochrome P450-mediated metabolism of vitamin D

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