Lanosterol 14 alpha-demethylase: Difference between revisions

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[[File:Lanosterol skeletal.svg|thumb|Lanosterol]]
[[File:Lanosterol skeletal.svg|thumb|Lanosterol]]


'''Lanosterol 14α-demethylase''' (or '''CYP51A1''') is a [[cytochrome P450]] [[enzyme]] that is involved in the conversion of [[lanosterol]] to [[4,4-dimethylcholesta-8(9),14,24-trien-3β-ol]].<ref>"Metabocard for 4,4-Dimethylcholesta-8,14,24-trienol (HMDB01023)." Human Metabolome Database. Web. 25 Feb. 2014. <http://www.hmdb.ca/metabolites/HMDB01023>.</ref> The [[cytochrome P450]] [[isoenzyme]]s are a conserved group of [[protein]]s that serve as key players in the [[metabolism]] of [[organic compound|organic substance]]s and the [[biosynthesis]] of important [[steroid]]s, [[lipid]]s, and [[vitamin]]s in [[eukaryotes]].<ref>Lepesheva, Galina I., and Michael R. Waterman. "Sterol 14α-Demethylase Cytochrome P450 (CYP51), a P450 in All Biological Kingdoms." ''Biochim Biophys Acta''. 2008. 1770(3): 467-77.</ref> As a member of this family, lanosterol 14α-demethylase is responsible for an essential step in the biosynthesis of [[sterols]]. In particular, this protein catalyzes the removal of the C-14α-methyl group from [[lanosterol]] (Lepesheva et al.). This demethylation step is regarded as the initial checkpoint in the transformation of [[lanosterol]] to other [[sterols]] that are widely used within the cell (Lepesheva et al.).
'''Lanosterol 14α-demethylase''' ('''CYP51A1''') is a [[cytochrome P450]] [[enzyme]] that is involved in the conversion of [[lanosterol]] to 4,4-dimethylcholesta-8(9),14,24-trien-3β-ol.<ref>{{cite web | title = Metabocard for 4,4-Dimethylcholesta-8,14,24-trienol (HMDB01023) | url = http://www.hmdb.ca/metabolites/HMDB01023 | work = Human Metabolome Database | date =  February 2014 }}</ref> The [[cytochrome P450]] [[isoenzyme]]s are a conserved group of [[protein]]s that serve as key players in the [[metabolism]] of [[organic compound|organic substance]]s and the [[biosynthesis]] of important [[steroid]]s, [[lipid]]s, and [[vitamin]]s in [[eukaryotes]].<ref name="Lepesheva_2007">{{cite journal | vauthors = Lepesheva GI, Waterman MR | title = Sterol 14alpha-demethylase cytochrome P450 (CYP51), a P450 in all biological kingdoms | journal = Biochimica et Biophysica Acta | volume = 1770 | issue = 3 | pages = 467–77 | date = March 2007 | pmid = 16963187 | pmc = 2324071 | doi = 10.1016/j.bbagen.2006.07.018 }}</ref> As a member of this family, lanosterol 14α-demethylase is responsible for an essential step in the biosynthesis of [[sterols]]. In particular, this protein catalyzes the removal of the C-14α-[[methyl group]] from [[lanosterol]].<ref name="Lepesheva_2007" /> This demethylation step is regarded as the initial checkpoint in the transformation of [[lanosterol]] to other [[sterols]] that are widely used within the cell.<ref name="Lepesheva_2007" />


[[File:Ergosterol structure.svg|thumb|Ergosterol]]
[[File:Ergosterol structure.svg|thumb|Ergosterol]]
Although lanosterol 14α-demethylase is present in a wide variety of organisms, this enzyme is studied primarily in the context of [[fungi]], where it plays an essential role in mediating membrane permeability.<ref>Daum G, Lees ND, Bard M, Dickson R. "Biochemistry, Cell Biology and Molecular Biology of Lipids of Saccharomyces cervisiae". ''Yeast''. 1998. 14(16):1471-1510.</ref> In [[fungi]], CYP51 catalyzes the demethylation of [[lanosterol]] to create an important precursor that is eventually converted into [[ergosterol]] (Lepesheva et al.). This steroid then makes its way throughout the cell, where it alters the permeability and rigidity of plasma membranes much as cholesterol does in animals.<ref>Becher, Rayko, and Stefan G. R. Wirsel. "Fungal Cytochrome P450 Sterol 14α-demethylase (CYP51) and Azole Resistance in Plant and Human Pathogens." ''Applied Microbiology and Biotechnology.'' 2012. 95(4): 825-40.</ref> Because ergosterol constitutes a fundamental component of fungal membranes, many [[antifungal medications]] have been developed to inhibit 14α-demethylase activity and prevent the production of this key compound (Becher et al.).
Although lanosterol 14α-demethylase is present in a wide variety of organisms, this enzyme is studied primarily in the context of [[fungi]], where it plays an essential role in mediating membrane permeability.<ref name="Daum_1998">{{cite journal | vauthors = Daum G, Lees ND, Bard M, Dickson R | title = Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae | journal = Yeast | volume = 14 | issue = 16 | pages = 1471–510 | date = December 1998 | pmid = 9885152 | doi = 10.1002/(SICI)1097-0061(199812)14:16<1471::AID-YEA353>3.0.CO;2-Y }}</ref> In [[fungi]], CYP51 catalyzes the demethylation of [[lanosterol]] to create an important precursor that is eventually converted into [[ergosterol]].<ref name="Lepesheva_2007" /> This steroid then makes its way throughout the cell, where it alters the permeability and rigidity of plasma membranes much as cholesterol does in animals.<ref name="Becher_2012">{{cite journal | vauthors = Becher R, Wirsel SG | title = Fungal cytochrome P450 sterol 14α-demethylase (CYP51) and azole resistance in plant and human pathogens | journal = Applied Microbiology and Biotechnology | volume = 95 | issue = 4 | pages = 825–40 | date = August 2012 | pmid = 22684327 | doi = 10.1007/s00253-012-4195-9 }}</ref> Because ergosterol constitutes a fundamental component of fungal membranes, many [[antifungal medications]] have been developed to inhibit 14α-demethylase activity and prevent the production of this key compound.<ref name="Becher_2012" />


==Evolution==
==Evolution==
The structural and functional properties of the [[cytochrome P450]] superfamily have been subject to extensive diversification over the course of evolution (Becher et al.). Recent estimates indicate that there are currently 10 [[Class (biology)|classes]] and 267 [[Family (biology)|families]] of CYP proteins.<ref>Hannemann F, Bichet A, Ewen KM, Bernhardt R. "Cytochrome P450 Systems—Biological Variations of Electron Transport Chains. ''Biochim Biophys Acta Gen Subj''. 2007, 1770(3):330–344</ref> It is believed that 14α-demethylase or CYP51 diverged early in the cytochrome's [[evolutionary history]] and has preserved its function ever since; namely, the removal of the 14α-methyl group from sterol [[substrate (biochemistry)|substrates]] (Becher et al.).
The structural and functional properties of the [[cytochrome P450]] superfamily have been subject to extensive diversification over the course of evolution.<ref name="Becher_2012" /> Recent estimates indicate that there are currently 10 [[Class (biology)|classes]] and 267 [[Family (biology)|families]] of CYP proteins.<ref name="Hannemann_2007">{{cite journal | vauthors = Hannemann F, Bichet A, Ewen KM, Bernhardt R | title = Cytochrome P450 systems--biological variations of electron transport chains | journal = Biochimica et Biophysica Acta | volume = 1770 | issue = 3 | pages = 330–44 | date = March 2007 | pmid = 16978787 | doi = 10.1016/j.bbagen.2006.07.017 }}</ref> It is believed that 14α-demethylase or CYP51 diverged early in the cytochrome's [[evolutionary history]] and has preserved its function ever since; namely, the removal of the 14α-methyl group from sterol [[substrate (biochemistry)|substrates]].<ref name="Becher_2012" />


Although CYP51's mode of action has been well [[conserved sequence|conserved]], the protein's sequence varies considerably between biological kingdoms.<ref>Lepesheva GI, Waterman MR. "CYP51—The Omnipotent P450." ''Mol Cell Endocrinol''. 2004, 215:165–170</ref> CYP51 sequence comparisons between kingdoms reveal only a 22-30% similarity in amino acid composition.<ref>Lepesheva GI, Waterman MR. "Structural Basis for Conservation in the CYP51 Family." ''Biochim Biophys Acta Proteins Proteom.'' 2011, 1814:88–93</ref>
Although CYP51's mode of action has been well [[conserved sequence|conserved]], the protein's sequence varies considerably between biological kingdoms.<ref name="Lepesheva_2004">{{cite journal | vauthors = Lepesheva GI, Waterman MR | title = CYP51--the omnipotent P450 | journal = Molecular and Cellular Endocrinology | volume = 215 | issue = 1–2 | pages = 165–70 | date = February 2004 | pmid = 15026190 | doi = 10.1016/j.mce.2003.11.016 }}</ref> CYP51 sequence comparisons between kingdoms reveal only a 22-30% similarity in amino acid composition.<ref name="Lepesheva_2011">{{cite journal | vauthors = Lepesheva GI, Waterman MR | title = Structural basis for conservation in the CYP51 family | journal = Biochimica et Biophysica Acta | volume = 1814 | issue = 1 | pages = 88–93 | date = January 2011 | pmid = 20547249 | pmc = 2962772 | doi = 10.1016/j.bbapap.2010.06.006 }}</ref>


==Enzyme structure==
==Enzyme structure==
[[File:Structure of lanosterol 14 α-demethylase (CYP51).png|thumb|Structure of lanosterol 14α-demethylase (CYP51), as identified by Podust et al.]]
[[File:Structure of lanosterol 14 α-demethylase (CYP51).png|thumb|Structure of lanosterol 14α-demethylase (CYP51), as identified by Podust et al.]]


Although the structure of 14α-demethylase may vary substantially from one organism to the next, [[sequence alignment]] analysis reveals that there are six regions in the protein that are highly [[conserved sequence|conserved]] in [[eukaryotes]] (Lepesheva et al.). These include residues in the B' helix, B'/C loop, C helix, I helix, K/β1-4 loop, and β-strand 1-4 that are responsible for forming the surface of the substrate binding cavity (Becher et al.). [[Homology modeling]] reveals that [[substrate (biochemistry)|substrates]] migrate from the surface of the protein to the enzyme's buried [[active site]] through a channel that is formed in part by the A' [[alpha helix]] and the β4 loop.<ref>Hargrove TY, Wawrzak Z, Liu JL, Nes WD, Waterman MR, Lepesheva GI. "Substrate Preferences and Catalytic Parameters Determined by Structural Characteristics of Sterol 14alpha-demethylase (CYP51) from Leishmania infantum." ''J Biol Chem''. 2011, 286:26838–26848.</ref><ref>Podust LM, von Kries JP, Eddine AN, Kim Y et al. "Small-Molecule Scaffolds for CYP51 Inhibitors Identified by High-Throughput Screening and Defined by X-ray Crystallography." ''Antimicrob Agents Chemother''. 2007, 51(11): 3915-23.</ref> Finally, the [[active site]] contains a [[heme]] [[prosthetic group]] in which the iron is tethered to a thiolate ligand on a conserved cysteine residue (Lepesheva et al.). This group also binds diatomic oxygen at the sixth coordination site, which is eventually incorporated onto the substrate (Lepesheva et al.).
Although the structure of 14α-demethylase may vary substantially from one organism to the next, [[sequence alignment]] analysis reveals that there are six regions in the protein that are highly [[conserved sequence|conserved]] in [[eukaryotes]].<ref name="Lepesheva_2011" /> These include residues in the B' helix, B'/C loop, C helix, I helix, K/β1-4 loop, and β-strand 1-4 that are responsible for forming the surface of the substrate binding cavity.<ref name="Becher_2012" /> [[Homology modeling]] reveals that [[substrate (biochemistry)|substrates]] migrate from the surface of the protein to the enzyme's buried [[active site]] through a channel that is formed in part by the A' [[alpha helix]] and the β4 loop.<ref name="Hargrove_2011">{{cite journal | vauthors = Hargrove TY, Wawrzak Z, Liu J, Nes WD, Waterman MR, Lepesheva GI | title = Substrate preferences and catalytic parameters determined by structural characteristics of sterol 14alpha-demethylase (CYP51) from Leishmania infantum | journal = The Journal of Biological Chemistry | volume = 286 | issue = 30 | pages = 26838–48 | date = July 2011 | pmid = 21632531 | pmc = 3143644 | doi = 10.1074/jbc.M111.237099 }}</ref><ref name="Podust_2007">{{cite journal | vauthors = Podust LM, von Kries JP, Eddine AN, Kim Y, Yermalitskaya LV, Kuehne R, Ouellet H, Warrier T, Alteköster M, Lee JS, Rademann J, Oschkinat H, Kaufmann SH, Waterman MR | display-authors = 6 | title = Small-molecule scaffolds for CYP51 inhibitors identified by high-throughput screening and defined by X-ray crystallography | journal = Antimicrobial Agents and Chemotherapy | volume = 51 | issue = 11 | pages = 3915–23 | date = November 2007 | pmid = 17846131 | pmc = 2151439 | doi = 10.1128/AAC.00311-07 }}</ref> Finally, the [[active site]] contains a [[heme]] [[prosthetic group]] in which the iron is tethered to a thiolate ligand on a conserved cysteine residue.<ref name="Lepesheva_2011" /> This group also binds diatomic oxygen at the sixth coordination site, which is eventually incorporated onto the substrate.<ref name="Lepesheva_2011" />


==Enzyme mechanism==
==Enzyme mechanism==
[[File:Lanosterol Demethylation Pathway.png|thumb|Three-step demethylation of lanosterol, mediated by lanosterol 14α-demethylase.]]
[[File:Lanosterol Demethylation Pathway.png|thumb|Three-step demethylation of lanosterol, mediated by lanosterol 14α-demethylase.]]


The enzyme-catalyzed [[demethylation]] of [[lanosterol]] is believed to occur in three steps, each of which requires one molecule of diatomic oxygen and one molecule of [[NADPH]] (or some other [[reducing equivalent]]).<ref>Vanden Bossche H, Koymans L. "Cytochromes P450 in Fungi." Mycoses. 1998, 41:32–38</ref> During the first two steps, the 14α-methyl group undergoes typical [[Cytochrome P450|cytochrome]] monooxygenation in which one oxygen atom is incorporated by the substrate and the other is reduced to water, resulting in the sterol's conversion to a carboxyalcohol and then a carboxyaldehyde (Lepesheva et al.). The aldehyde then departs as [[formic acid]] and a double bond is simultaneously introduced to yield the demethylated product (Lepesheva et al.).
The enzyme-catalyzed [[demethylation]] of [[lanosterol]] is believed to occur in three steps, each of which requires one molecule of diatomic oxygen and one molecule of [[NADPH]] (or some other [[reducing equivalent]]).<ref name="Vanden_Bossche_1998">{{cite journal | vauthors = Vanden Bossche H, Koymans L | title = Cytochromes P450 in fungi | journal = Mycoses | volume = 41 Suppl 1 | issue = | pages = 32–8 | date = 1998 | pmid = 9717384 | doi = | url = }}</ref> During the first two steps, the 14α-methyl group undergoes typical [[Cytochrome P450|cytochrome]] monooxygenation in which one oxygen atom is incorporated by the substrate and the other is reduced to water, resulting in the sterol's conversion to a carboxyalcohol and then a carboxyaldehyde.<ref name="Lepesheva_2011" /> The aldehyde then departs as [[formic acid]] and a double bond is simultaneously introduced to yield the demethylated product.<ref name="Lepesheva_2011" />


==Biological function==
==Biological function==
The biological role of this protein is also well understood. The [[demethylated]] products of the CYP51 reaction are vital intermediates in pathways leading to the formation of [[cholesterol]] in humans, [[ergosterol]] in fungi, and other types of [[sterols]] in plants (Lepesheva et al.). These [[sterols]] localize to the [[plasma membrane]] of cells, where they play an important structural role in the regulation of membrane fluidity and permeability and also influence the activity of enzymes, ion channels, and other cell components that are embedded within (Daum et al.).<ref>Abe F, Usui K, Hiraki T. "Fluconazole Modulates Membrane Rigidity, Heterogeneity, and Water Penetration into the Plasma Membrane in ''Saccharomyces cerevisiae''." ''Biochemistry''. 2009, 48:8494–8504</ref><ref>"Itraconazole (DB01167)." ''DrugBank''. 25 Feb. 2014. <http://www.drugbank.ca/drugs/DB01167>.</ref> With the proliferation of immuno-suppressive diseases such as [[HIV/AIDS]] and [[cancer]], patients have become increasingly vulnerable to opportunistic [[fungal infections]] (Richardson et al.). Seeking new means to treat such infections, drug researchers have begun targeting the 14α-demethylase enzyme in fungi; destroying the fungal cell's ability to produce ergosterol causes a disruption of the plasma membrane, thereby resulting in cellular leakage and ultimately the death of the pathogen (''DrugBank'').
The biological role of this protein is also well understood. The [[demethylated]] products of the CYP51 reaction are vital intermediates in pathways leading to the formation of [[cholesterol]] in humans, [[ergosterol]] in fungi, and other types of [[sterols]] in plants.<ref name="Lepesheva_2011" /> These [[sterols]] localize to the [[plasma membrane]] of cells, where they play an important structural role in the regulation of membrane fluidity and permeability and also influence the activity of enzymes, ion channels, and other cell components that are embedded within.<ref name="Daum_1998" /><ref name="Abe_2009">{{cite journal | vauthors = Abe F, Usui K, Hiraki T | title = Fluconazole modulates membrane rigidity, heterogeneity, and water penetration into the plasma membrane in Saccharomyces cerevisiae | journal = Biochemistry | volume = 48 | issue = 36 | pages = 8494–504 | date = September 2009 | pmid = 19670905 | doi = 10.1021/bi900578y }}</ref><ref>{{cite web | title = Itraconazole (DB01167) | work = DrugBank | url = http://www.drugbank.ca/drugs/DB01167 }}</ref> With the proliferation of immuno-suppressive diseases such as [[HIV/AIDS]] and [[cancer]], patients have become increasingly vulnerable to opportunistic [[fungal infections]] (Richardson et al.). Seeking new means to treat such infections, drug researchers have begun targeting the 14α-demethylase enzyme in fungi; destroying the fungal cell's ability to produce ergosterol causes a disruption of the plasma membrane, thereby resulting in cellular leakage and ultimately the death of the pathogen (''DrugBank'').


[[Azole]]s are currently the most popular class of [[antifungals]] used in both agricultural and medical settings (Becher et al.). These compounds bind as the sixth ligand to the [[heme]] group in CYP51, thereby altering the structure of the [[active site]] and acting as [[noncompetitive inhibitors]].<ref>Mullins JGL, Parker JE, Cools HJ, Togawa RC, Lucas JA, et al. "Molecular Modeling of the Emergence of Azole Resistance in ''Mycosphaerella graminicola''." ''PLoS ONE''. 2011, 6(6): e20973. doi:10.1371/journal.pone.0020973</ref> The effectiveness of [[imidazoles]] and [[triazoles]] (common [[azole]] subclasses) as inhibitors of 14α-demethylase have been confirmed through several experiments. Some studies test for changes in the production of important downstream [[ergosterol]] intermediates in the presence of these compounds.<ref>Tuck SF, Patel H, Safi E, Robinson CH. "Lanosterol 14 Alpha-Demethylase (P45014DM): Effects of P45014DM Inhibitors on Sterol Biosynthesis Downstream of Lanosterol." ''J Lipid Res''. 1991, 32(6): 893-902</ref> Other studies employ [[spectrophotometry]] to quantify azole-CYP51 interactions (Becher et al.). Coordination of [[azoles]] to the prosthetic [[heme]] group in the enzyme's active site causes a characteristic shift in CYP51 [[absorbance]], creating what is commonly referred to as a type II difference spectrum.<ref>Vanden Bossche H, Marichal P, Gorrens J, Bellens D, Verhoeven H, Coene MC, Lauwers W, Janssen PAJ. "Interaction of Azole Derivatives with Cytochrome P-450 Isozymes in Yeast, Fungi, Plants and Mammalian-cells. ''Pestic Sci''. 1987, 21:289–306</ref><ref>Yoshida Y, Aoyama Y. "Interaction of Azole Antifungal Agents with Cytochrome P-45014DM Purified from ''Saccharomyces cerevisiae'' Microsomes." ''Biochem Pharmacol''. 1987, 36:229–235</ref>
[[Azole]]s are currently the most popular class of [[antifungals]] used in both agricultural and medical settings.<ref name="Becher_2012" /> These compounds bind as the sixth ligand to the [[heme]] group in CYP51, thereby altering the structure of the [[active site]] and acting as [[noncompetitive inhibitors]].<ref name="Mullins_2011">{{cite journal | vauthors = Mullins JG, Parker JE, Cools HJ, Togawa RC, Lucas JA, Fraaije BA, Kelly DE, Kelly SL | title = Molecular modelling of the emergence of azole resistance in Mycosphaerella graminicola | journal = PLOS One | volume = 6 | issue = 6 | pages = e20973 | date = 2011 | pmid = 21738598 | pmc = 3124474 | doi = 10.1371/journal.pone.0020973 | bibcode = 2011PLoSO...620973M }}</ref> The effectiveness of [[imidazoles]] and [[triazoles]] (common [[azole]] subclasses) as inhibitors of 14α-demethylase have been confirmed through several experiments. Some studies test for changes in the production of important downstream [[ergosterol]] intermediates in the presence of these compounds.<ref name="Tuck_1991">{{cite journal | vauthors = Tuck SF, Patel H, Safi E, Robinson CH | title = Lanosterol 14 alpha-demethylase (P45014DM): effects of P45014DM inhibitors on sterol biosynthesis downstream of lanosterol | journal = Journal of Lipid Research | volume = 32 | issue = 6 | pages = 893–902 | date = June 1991 | pmid = 1940622 | doi = | url = }}</ref> Other studies employ [[spectrophotometry]] to quantify azole-CYP51 interactions.<ref name="Becher_2012" /> Coordination of [[azoles]] to the prosthetic [[heme]] group in the enzyme's active site causes a characteristic shift in CYP51 [[absorbance]], creating what is commonly referred to as a type II difference spectrum.<ref name = "Vanden_Bossche_1987">{{cite journal | vauthors = Vanden Bossche H, Marichal P, Gorrens J, Bellens D, Verhoeven H, Coene MC, Lauwers W, Janssen PA |title=Interaction of azole derivatives with cytochrome P-450 isozymes in yeast, fungi, plants and mammalian cells |journal=Pesticide Science |date=1987 |volume=21 |issue=4 |pages=289–306 |doi=10.1002/ps.2780210406}}</ref><ref name="Yoshida_1987">{{cite journal | vauthors = Yoshida Y, Aoyama Y | title = Interaction of azole antifungal agents with cytochrome P-45014DM purified from Saccharomyces cerevisiae microsomes | journal = Biochemical Pharmacology | volume = 36 | issue = 2 | pages = 229–35 | date = January 1987 | pmid = 3545213 | doi = | url = }}</ref>


Prolonged use of [[azoles]] as [[antifungals]] has resulted in the emergence of [[drug resistance]] among certain fungal strains (Becher et al.). Mutations in the [[coding region]] of CYP51 genes, overexpression of CYP51, and overexpression of membrane efflux transporters can all lead to resistance to these antifungals.<ref>Vanden Bossche H, Dromer F, Improvisi I, Lozano-Chiu M, Rex JH, Sanglard D. "Antifungal Drug Resistance in Pathogenic Fungi". ''Med Mycol''. 1998, 36(1): 119–28.</ref><ref>Leroux P, Albertini C, Gautier A, Gredt M, Walker AS. "Mutations in the CYP51 Gene Correlated with Changes in Sensitivity to Sterol 14Alpha-Demethylation Inhibitors in Field Isolates of ''Mycosphaerella graminicola''." ''Pest Manag Sci''. 2007, 63:688–698</ref><ref>Sanglard D, Ischer F, Koymans L, Bille J. "Amino Acid Substitutions in the Cytochrome P-450 Lanosterol 14Alpha-Demethylase (CYP51A1) from Azole-resistant ''Candida albicans'' Clinical Isolates Contribute to Resistance to Azole Antifungal Agents. ''Antimicrob Agents Chemother.'' 1998, 42:241–253</ref><ref>Cannon RD, Lamping E, Holmes AR, Niimi K, Baret PV, Keniya MV, Tanabe K, Niimi M, Goffeau A, Monk BC. "Efflux-mediated Antifungal Drug Resistance. ''Clin Microbiol Rev.'' 2009, 22:291– 321</ref> Consequently, the focus of [[azole]] research is beginning to shift towards identifying new ways to circumvent this major obstacle (Becher et al.).
Prolonged use of [[azoles]] as [[antifungals]] has resulted in the emergence of [[drug resistance]] among certain fungal strains.<ref name="Becher_2012" /> Mutations in the [[coding region]] of CYP51 genes, overexpression of CYP51, and overexpression of membrane efflux transporters can all lead to resistance to these antifungals.<ref name="Vanden_Bossche_1998b">{{cite journal | vauthors = Vanden Bossche H, Dromer F, Improvisi I, Lozano-Chiu M, Rex JH, Sanglard D | title = Antifungal drug resistance in pathogenic fungi | journal = Medical Mycology | volume = 36 Suppl 1 | issue = | pages = 119–28 | date = 1998 | pmid = 9988500 | doi = | url = }}</ref><ref name="Leroux_2007">{{cite journal | vauthors = Leroux P, Albertini C, Gautier A, Gredt M, Walker AS | title = Mutations in the CYP51 gene correlated with changes in sensitivity to sterol 14 alpha-demethylation inhibitors in field isolates of Mycosphaerella graminicola | journal = Pest Management Science | volume = 63 | issue = 7 | pages = 688–98 | date = July 2007 | pmid = 17511023 | doi = 10.1002/ps.1390 }}</ref><ref name="Sanglard_1998">{{cite journal | vauthors = Sanglard D, Ischer F, Koymans L, Bille J | title = Amino acid substitutions in the cytochrome P-450 lanosterol 14alpha-demethylase (CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents | journal = Antimicrobial Agents and Chemotherapy | volume = 42 | issue = 2 | pages = 241–53 | date = February 1998 | pmid = 9527767 | pmc = 105395 | doi = | url = }}</ref><ref name="Cannon_2009">{{cite journal | vauthors = Cannon RD, Lamping E, Holmes AR, Niimi K, Baret PV, Keniya MV, Tanabe K, Niimi M, Goffeau A, Monk BC | title = Efflux-mediated antifungal drug resistance | journal = Clinical Microbiology Reviews | volume = 22 | issue = 2 | pages = 291–321, Table of Contents | date = April 2009 | pmid = 19366916 | pmc = 2668233 | doi = 10.1128/CMR.00051-08 }}</ref> Consequently, the focus of [[azole]] research is beginning to shift towards identifying new ways to circumvent this major obstacle.<ref name="Becher_2012" />


==See also==
==See also==
* [[Steroidogenic enzyme]]
* [[Steroidogenic enzyme]]
* [[Fungicide]]
* [[Azole antifungals]]
* [[Antifungal]]


==References==
==References==
Line 57: Line 56:
==External links==
==External links==
* {{MeshName|cytochrome+P-450+CYP51}}
* {{MeshName|cytochrome+P-450+CYP51}}


{{Cytochrome P450}}
{{Cytochrome P450}}

Latest revision as of 04:51, 28 November 2018

Cytochrome P450, Family 51, Subfamily A, Polypeptide 1
Identifiers
SymbolCYP51A1
Alt. symbolsCYP51, P45014DM
Entrez1595
HUGO2649
OMIM601637
RefSeqNM_000786
UniProtQ16850
Other data
EC number1.14.13.70
LocusChr. 7 q21.2-21.3
File:Lanosterol skeletal.svg
Lanosterol

Lanosterol 14α-demethylase (CYP51A1) is a cytochrome P450 enzyme that is involved in the conversion of lanosterol to 4,4-dimethylcholesta-8(9),14,24-trien-3β-ol.[1] The cytochrome P450 isoenzymes are a conserved group of proteins that serve as key players in the metabolism of organic substances and the biosynthesis of important steroids, lipids, and vitamins in eukaryotes.[2] As a member of this family, lanosterol 14α-demethylase is responsible for an essential step in the biosynthesis of sterols. In particular, this protein catalyzes the removal of the C-14α-methyl group from lanosterol.[2] This demethylation step is regarded as the initial checkpoint in the transformation of lanosterol to other sterols that are widely used within the cell.[2]

File:Ergosterol structure.svg
Ergosterol

Although lanosterol 14α-demethylase is present in a wide variety of organisms, this enzyme is studied primarily in the context of fungi, where it plays an essential role in mediating membrane permeability.[3] In fungi, CYP51 catalyzes the demethylation of lanosterol to create an important precursor that is eventually converted into ergosterol.[2] This steroid then makes its way throughout the cell, where it alters the permeability and rigidity of plasma membranes much as cholesterol does in animals.[4] Because ergosterol constitutes a fundamental component of fungal membranes, many antifungal medications have been developed to inhibit 14α-demethylase activity and prevent the production of this key compound.[4]

Evolution

The structural and functional properties of the cytochrome P450 superfamily have been subject to extensive diversification over the course of evolution.[4] Recent estimates indicate that there are currently 10 classes and 267 families of CYP proteins.[5] It is believed that 14α-demethylase or CYP51 diverged early in the cytochrome's evolutionary history and has preserved its function ever since; namely, the removal of the 14α-methyl group from sterol substrates.[4]

Although CYP51's mode of action has been well conserved, the protein's sequence varies considerably between biological kingdoms.[6] CYP51 sequence comparisons between kingdoms reveal only a 22-30% similarity in amino acid composition.[7]

Enzyme structure

File:Structure of lanosterol 14 α-demethylase (CYP51).png
Structure of lanosterol 14α-demethylase (CYP51), as identified by Podust et al.

Although the structure of 14α-demethylase may vary substantially from one organism to the next, sequence alignment analysis reveals that there are six regions in the protein that are highly conserved in eukaryotes.[7] These include residues in the B' helix, B'/C loop, C helix, I helix, K/β1-4 loop, and β-strand 1-4 that are responsible for forming the surface of the substrate binding cavity.[4] Homology modeling reveals that substrates migrate from the surface of the protein to the enzyme's buried active site through a channel that is formed in part by the A' alpha helix and the β4 loop.[8][9] Finally, the active site contains a heme prosthetic group in which the iron is tethered to a thiolate ligand on a conserved cysteine residue.[7] This group also binds diatomic oxygen at the sixth coordination site, which is eventually incorporated onto the substrate.[7]

Enzyme mechanism

File:Lanosterol Demethylation Pathway.png
Three-step demethylation of lanosterol, mediated by lanosterol 14α-demethylase.

The enzyme-catalyzed demethylation of lanosterol is believed to occur in three steps, each of which requires one molecule of diatomic oxygen and one molecule of NADPH (or some other reducing equivalent).[10] During the first two steps, the 14α-methyl group undergoes typical cytochrome monooxygenation in which one oxygen atom is incorporated by the substrate and the other is reduced to water, resulting in the sterol's conversion to a carboxyalcohol and then a carboxyaldehyde.[7] The aldehyde then departs as formic acid and a double bond is simultaneously introduced to yield the demethylated product.[7]

Biological function

The biological role of this protein is also well understood. The demethylated products of the CYP51 reaction are vital intermediates in pathways leading to the formation of cholesterol in humans, ergosterol in fungi, and other types of sterols in plants.[7] These sterols localize to the plasma membrane of cells, where they play an important structural role in the regulation of membrane fluidity and permeability and also influence the activity of enzymes, ion channels, and other cell components that are embedded within.[3][11][12] With the proliferation of immuno-suppressive diseases such as HIV/AIDS and cancer, patients have become increasingly vulnerable to opportunistic fungal infections (Richardson et al.). Seeking new means to treat such infections, drug researchers have begun targeting the 14α-demethylase enzyme in fungi; destroying the fungal cell's ability to produce ergosterol causes a disruption of the plasma membrane, thereby resulting in cellular leakage and ultimately the death of the pathogen (DrugBank).

Azoles are currently the most popular class of antifungals used in both agricultural and medical settings.[4] These compounds bind as the sixth ligand to the heme group in CYP51, thereby altering the structure of the active site and acting as noncompetitive inhibitors.[13] The effectiveness of imidazoles and triazoles (common azole subclasses) as inhibitors of 14α-demethylase have been confirmed through several experiments. Some studies test for changes in the production of important downstream ergosterol intermediates in the presence of these compounds.[14] Other studies employ spectrophotometry to quantify azole-CYP51 interactions.[4] Coordination of azoles to the prosthetic heme group in the enzyme's active site causes a characteristic shift in CYP51 absorbance, creating what is commonly referred to as a type II difference spectrum.[15][16]

Prolonged use of azoles as antifungals has resulted in the emergence of drug resistance among certain fungal strains.[4] Mutations in the coding region of CYP51 genes, overexpression of CYP51, and overexpression of membrane efflux transporters can all lead to resistance to these antifungals.[17][18][19][20] Consequently, the focus of azole research is beginning to shift towards identifying new ways to circumvent this major obstacle.[4]

See also

References

  1. ↑ "Metabocard for 4,4-Dimethylcholesta-8,14,24-trienol (HMDB01023)". Human Metabolome Database. February 2014.
  2. ↑ 2.0 2.1 2.2 2.3 Lepesheva GI, Waterman MR (March 2007). "Sterol 14alpha-demethylase cytochrome P450 (CYP51), a P450 in all biological kingdoms". Biochimica et Biophysica Acta. 1770 (3): 467–77. doi:10.1016/j.bbagen.2006.07.018. PMC 2324071. PMID 16963187.
  3. ↑ 3.0 3.1 Daum G, Lees ND, Bard M, Dickson R (December 1998). "Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae". Yeast. 14 (16): 1471–510. doi:10.1002/(SICI)1097-0061(199812)14:16<1471::AID-YEA353>3.0.CO;2-Y. PMID 9885152.
  4. ↑ 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 Becher R, Wirsel SG (August 2012). "Fungal cytochrome P450 sterol 14α-demethylase (CYP51) and azole resistance in plant and human pathogens". Applied Microbiology and Biotechnology. 95 (4): 825–40. doi:10.1007/s00253-012-4195-9. PMID 22684327.
  5. ↑ Hannemann F, Bichet A, Ewen KM, Bernhardt R (March 2007). "Cytochrome P450 systems--biological variations of electron transport chains". Biochimica et Biophysica Acta. 1770 (3): 330–44. doi:10.1016/j.bbagen.2006.07.017. PMID 16978787.
  6. ↑ Lepesheva GI, Waterman MR (February 2004). "CYP51--the omnipotent P450". Molecular and Cellular Endocrinology. 215 (1–2): 165–70. doi:10.1016/j.mce.2003.11.016. PMID 15026190.
  7. ↑ 7.0 7.1 7.2 7.3 7.4 7.5 7.6 Lepesheva GI, Waterman MR (January 2011). "Structural basis for conservation in the CYP51 family". Biochimica et Biophysica Acta. 1814 (1): 88–93. doi:10.1016/j.bbapap.2010.06.006. PMC 2962772. PMID 20547249.
  8. ↑ Hargrove TY, Wawrzak Z, Liu J, Nes WD, Waterman MR, Lepesheva GI (July 2011). "Substrate preferences and catalytic parameters determined by structural characteristics of sterol 14alpha-demethylase (CYP51) from Leishmania infantum". The Journal of Biological Chemistry. 286 (30): 26838–48. doi:10.1074/jbc.M111.237099. PMC 3143644. PMID 21632531.
  9. ↑ Podust LM, von Kries JP, Eddine AN, Kim Y, Yermalitskaya LV, Kuehne R, et al. (November 2007). "Small-molecule scaffolds for CYP51 inhibitors identified by high-throughput screening and defined by X-ray crystallography". Antimicrobial Agents and Chemotherapy. 51 (11): 3915–23. doi:10.1128/AAC.00311-07. PMC 2151439. PMID 17846131.
  10. ↑ Vanden Bossche H, Koymans L (1998). "Cytochromes P450 in fungi". Mycoses. 41 Suppl 1: 32–8. PMID 9717384.
  11. ↑ Abe F, Usui K, Hiraki T (September 2009). "Fluconazole modulates membrane rigidity, heterogeneity, and water penetration into the plasma membrane in Saccharomyces cerevisiae". Biochemistry. 48 (36): 8494–504. doi:10.1021/bi900578y. PMID 19670905.
  12. ↑ "Itraconazole (DB01167)". DrugBank.
  13. ↑ Mullins JG, Parker JE, Cools HJ, Togawa RC, Lucas JA, Fraaije BA, Kelly DE, Kelly SL (2011). "Molecular modelling of the emergence of azole resistance in Mycosphaerella graminicola". PLOS One. 6 (6): e20973. Bibcode:2011PLoSO...620973M. doi:10.1371/journal.pone.0020973. PMC 3124474. PMID 21738598.
  14. ↑ Tuck SF, Patel H, Safi E, Robinson CH (June 1991). "Lanosterol 14 alpha-demethylase (P45014DM): effects of P45014DM inhibitors on sterol biosynthesis downstream of lanosterol". Journal of Lipid Research. 32 (6): 893–902. PMID 1940622.
  15. ↑ Vanden Bossche H, Marichal P, Gorrens J, Bellens D, Verhoeven H, Coene MC, Lauwers W, Janssen PA (1987). "Interaction of azole derivatives with cytochrome P-450 isozymes in yeast, fungi, plants and mammalian cells". Pesticide Science. 21 (4): 289–306. doi:10.1002/ps.2780210406.
  16. ↑ Yoshida Y, Aoyama Y (January 1987). "Interaction of azole antifungal agents with cytochrome P-45014DM purified from Saccharomyces cerevisiae microsomes". Biochemical Pharmacology. 36 (2): 229–35. PMID 3545213.
  17. ↑ Vanden Bossche H, Dromer F, Improvisi I, Lozano-Chiu M, Rex JH, Sanglard D (1998). "Antifungal drug resistance in pathogenic fungi". Medical Mycology. 36 Suppl 1: 119–28. PMID 9988500.
  18. ↑ Leroux P, Albertini C, Gautier A, Gredt M, Walker AS (July 2007). "Mutations in the CYP51 gene correlated with changes in sensitivity to sterol 14 alpha-demethylation inhibitors in field isolates of Mycosphaerella graminicola". Pest Management Science. 63 (7): 688–98. doi:10.1002/ps.1390. PMID 17511023.
  19. ↑ Sanglard D, Ischer F, Koymans L, Bille J (February 1998). "Amino acid substitutions in the cytochrome P-450 lanosterol 14alpha-demethylase (CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents". Antimicrobial Agents and Chemotherapy. 42 (2): 241–53. PMC 105395. PMID 9527767.
  20. ↑ Cannon RD, Lamping E, Holmes AR, Niimi K, Baret PV, Keniya MV, Tanabe K, Niimi M, Goffeau A, Monk BC (April 2009). "Efflux-mediated antifungal drug resistance". Clinical Microbiology Reviews. 22 (2): 291–321, Table of Contents. doi:10.1128/CMR.00051-08. PMC 2668233. PMID 19366916.

External links

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