scholarly journals Expression, Purification, and Characterization of Aspergillus fumigatus Sterol 14-α Demethylase (CYP51) Isoenzymes A and B

2010 ◽  
Vol 54 (10) ◽  
pp. 4225-4234 ◽  
Author(s):  
Andrew G. S. Warrilow ◽  
Nadja Melo ◽  
Claire M. Martel ◽  
Josie E. Parker ◽  
W. David Nes ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Type I ◽  
Purification And Characterization ◽  
Prosthetic Group ◽  
Binding Properties ◽  
Fluconazole Resistance ◽  
Characteristic Type ◽  
Heme Prosthetic Group ◽  
Binding Spectra

ABSTRACT Aspergillus fumigatus sterol 14-α demethylase (CYP51) isoenzymes A (AF51A) and B (AF51B) were expressed in Escherichia coli and purified. The dithionite-reduced CO-P450 complex for AF51A was unstable, rapidly denaturing to inactive P420, in marked contrast to AF51B, where the CO-P450 complex was stable. Type I substrate binding spectra were obtained with purified AF51B using lanosterol (Ks , 8.6 μM) and eburicol (Ks , 22.6 μM). Membrane suspensions of AF51A bound to both lanosterol (Ks , 3.1 μM) and eburicol (Ks , 4.1 μM). The binding of azoles, with the exception of fluconazole, to AF51B was tight, with the Kd (dissociation constant) values for clotrimazole, itraconazole, posaconazole, and voriconazole being 0.21, 0.06, 0.12, and 0.42 μM, respectively, in comparison with a Kd value of 4 μM for fluconazole. Characteristic type II azole binding spectra were obtained with AF51B, whereas an additional trough and a blue-shifted spectral peak were present in AF51A binding spectra for all azoles except clotrimazole. This suggests two distinct azole binding conformations within the heme prosthetic group of AF51A. All five azoles bound relatively weakly to AF51A, with Kd values ranging from 1 μM for itraconazole to 11.9 μM for fluconazole. The azole binding properties of purified AF51A and AF51B suggest an explanation for the intrinsic azole (fluconazole) resistance observed in Aspergillus fumigatus.

scholarly journals Azole Binding Properties of Candida albicans Sterol 14-α Demethylase (CaCYP51)

2010 ◽  
Vol 54 (10) ◽  
pp. 4235-4245 ◽  
Author(s):  
Andrew G. S. Warrilow ◽  
Claire M. Martel ◽  
Josie E. Parker ◽  
Nadja Melo ◽  
David C. Lamb ◽  
...  
Keyword(s):  
Carbon Monoxide ◽  
Candida Albicans ◽  
Inhibitory Concentration ◽  
Tight Binding ◽  
Type I ◽  
Wild Type ◽  
Binding Properties ◽  
Fluconazole Resistance ◽  
Binding Spectra

ABSTRACT Purified Candida albicans sterol 14-α demethylase (CaCYP51) bound the CYP51 substrates lanosterol and eburicol, producing type I binding spectra with Ks values of 11 and 25 μM, respectively, and a Km value of 6 μM for lanosterol. Azole binding to CaCYP51 was “tight” with both the type II spectral intensity (ΔA max) and the azole concentration required to obtain a half-ΔA max being proportional to the CaCYP51 concentration. Tight binding of fluconazole and itraconazole was confirmed by 50% inhibitory concentration determinations from CYP51 reconstitution assays. CaCYP51 had similar affinities for clotrimazole, econazole, itraconazole, ketoconazole, miconazole, and voriconazole, with Kd values of 10 to 26 μM under oxidative conditions, compared with 47 μM for fluconazole. The affinities of CaCYP51 for fluconazole and itraconazole appeared to be 4- and 2-fold lower based on CO displacement studies than those when using direct ligand binding under oxidative conditions. Econazole and miconazole were most readily displaced by carbon monoxide, followed by clotrimazole, ketoconazole, and fluconazole, and then voriconazole (7.8 pmol min−1), but itraconzole could not be displaced by carbon monoxide. This work reports in depth the characterization of the azole binding properties of wild-type C. albicans CYP51, including that of voriconazole, and will contribute to effective screening of new therapeutic azole antifungal agents. Preliminary comparative studies with the I471T CaCYP51 protein suggested that fluconazole resistance conferred by this mutation was through a combination of increased turnover, increased affinity for substrate, and a reduced affinity for fluconazole in the presence of substrate, allowing the enzyme to remain functionally active, albeit at reduced velocity, at higher fluconazole concentrations.

Escherichia coli DNA topoisomerase III: purification and characterization of a new type I enzyme

Biochemistry ◽  
1984 ◽  
Vol 23 (9) ◽  
pp. 1899-1906 ◽  
Author(s):  
Kalkunte S. Srivenugopal ◽  
Daniel Lockshon ◽  
David R. Morris
Keyword(s):  
Escherichia Coli ◽  
Type I ◽  
Purification And Characterization ◽  
Dna Topoisomerase ◽  
Topoisomerase Iii ◽  
New Type ◽  
Dna Topoisomerase Iii

Purification and characterization of simian foamy virus type I structural core polypeptides

1986 ◽  
Vol 87 (1-2) ◽  
pp. 87-96 ◽  
Author(s):  
A. B. Benzair ◽  
A. Rhodes-Feuillette ◽  
J. Lasneret ◽  
R. Emanoil-Ravier ◽  
J. Peries
Keyword(s):  
Virus Type ◽  
Foamy Virus ◽  
Type I ◽  
Purification And Characterization ◽  
Simian Foamy Virus

scholarly journals Purification and characterization of human T-cell leukemia virus type I protease produced inEscherichia coli

FEBS Letters ◽  
1991 ◽  
Vol 293 (1-2) ◽  
pp. 106-110 ◽  
Author(s):  
Makoto Kobayashi ◽  
Yumiko Ohi ◽  
Tsuneo Asano ◽  
Takaki Hayakawa ◽  
Koichi Kato ◽  
...  
Keyword(s):  
T Cell ◽  
Leukemia Virus ◽  
Type I ◽  
Inescherichia Coli ◽  
Cell Leukemia ◽  
Purification And Characterization ◽  
Cell Leukemia Virus Type ◽  
Human T Cell ◽  
T Cell Leukemia Virus

Partial purification and characterization of the endotoxin from Aspergillus fumigatus

1961 ◽  
Vol 14 (4) ◽  
pp. 347-358 ◽  
Author(s):  
Evelyn M. Rau ◽  
Evelyn B. Tilden ◽  
Virgil L. Koenig
Keyword(s):  
Aspergillus Fumigatus ◽  
Partial Purification ◽  
Purification And Characterization

Overproduction, purification and characterization of FtmPT1, a brevianamide F prenyltransferase from Aspergillus fumigatus

Microbiology ◽  
2005 ◽  
Vol 151 (7) ◽  
pp. 2199-2207 ◽  
Author(s):  
Alexander Grundmann ◽  
Shu-Ming Li
Keyword(s):  
Aspergillus Fumigatus ◽  
Enzymic Activity ◽  
Divalent Metal Ions ◽  
Purification And Characterization ◽  
Turnover Number ◽  
Dimethylallyl Diphosphate ◽  
Dimethylallyltryptophan Synthase ◽  
Aromatic Substrates ◽  
Prenyl Diphosphate

A putative prenyltransferase gene, ftmPT1, was identified in the genome sequence of Aspergillus fumigatus. ftmPT1 was cloned and expressed in Escherichia coli, and the protein FtmPT1 was purified to near homogeneity and characterized biochemically. This enzyme was found to catalyse the prenylation of cyclo-l-trp-l-Pro (brevianamide F) at the C-2 position of the indole nucleus. FtmPT1 is a soluble monomeric protein, which does not contain the usual prenyl diphosphate binding site (N/D)DXXD found in most prenyltransferases, and which does not require divalent metal ions for its enzymic activity. K m values for brevianamide F and dimethylallyl diphosphate were determined as 55 and 74 μM, respectively. The turnover number was 5·57 s−1. FtmPT1 showed a high substrate specificity towards dimethylallyl diphosphate, but accepted different tryptophan-containing cyclic dipeptides. Together with dimethylallyltryptophan synthase of ergot alkaloid biosynthesis, FtmPT1 belongs to a new group of prenyltransferases with aromatic substrates.

Production, Purification, and Characterization of Exoglucanase by Aspergillus fumigatus

2013 ◽  
Vol 170 (4) ◽  
pp. 895-908 ◽  
Author(s):  
Raja Tahir Mahmood ◽  
Muhammad Javaid Asad ◽  
Nazia Mehboob ◽  
Maria Mushtaq ◽  
Muhammad Gulfraz ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Purification And Characterization

scholarly journals The type II "receptor" as a decoy target for interleukin 1 in polymorphonuclear leukocytes: characterization of induction by dexamethasone and ligand binding properties of the released decoy receptor.

1994 ◽  
Vol 179 (2) ◽  
pp. 739-743 ◽  
Author(s):  
F Re ◽  
M Muzio ◽  
M De Rossi ◽  
N Polentarutti ◽  
J G Giri ◽  
...  
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Interleukin 1 ◽  
Soluble Form ◽  
Type I ◽  
Type Ii ◽  
Membrane Expression ◽  
Binding Properties ◽  
Signaling Function ◽  
Human Polymorphonuclear Leukocytes

Whereas the signaling function of the interleukin 1 (IL-1) receptor type I (IL-1R I) has been well documented, the type II "receptor" has been suggested to act as a decoy target for this cytokine. Since IL-1 may represent a key target of the immunomodulatory and antiinflammatory properties of glucocorticoids (GC), the aim of this study was to investigate the effects of dexamethasone (Dex) on IL-1R expression in human polymorphonuclear leukocytes (PMN), which express predominantly the type II molecule (IL-1R II). We found that Dex augments the levels of steady state transcripts encoding the IL-1R I and, most prominently, those of IL-1R II. Dex induced both transcripts via transcription-dependent mechanisms and by prolongation of the mRNAs half-lives. Inhibition of protein synthesis superinduced basal and Dex-augmented IL-1R II mRNA, whereas it completely inhibited the induction by Dex of IL-1R I transcripts. Induction of IL-1R II mRNA by Dex was associated with augmented membrane expression and release of the type II IL-1 binding molecule. This effect was mediated by the GC receptor. Other steroids (17 beta-estradiol, progesterone, and testosterone) were ineffective. The concentrations of IL-1 alpha and IL-1 receptor antagonist required to displace the binding of IL-1 beta to the soluble form of the decoy molecule induced by Dex from PMN were, respectively, 100 and 2 times higher compared with IL-1 beta. The induction by Dex of the type II receptor, a decoy molecule for IL-1, may contribute to the immunosuppressive and antiinflammatory activities of Dex.

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