Heterologous expression and characterization of the sterol 14α-demethylase CYP51F1 from Candida albicans

Article abstract of DOI:10.1016/j.abb.2011.02.002

Lanosterol 14α-demethylase (CYP51F1) from Candida albicans is known to be an essential enzyme in fungal sterol biosynthesis. Wild-type CYP51F1 and several of its mutants were heterologously expressed in Escherichia coli, purified, and characterized. It exhibited a typical reduced CO-difference spectrum with a maximum at 446 nm. Reconstitution of CYP51F1 with NADPH-P450 reductase gave a system that successfully converted lanosterol to its demethylated product. Titration of the purified enzyme with lanosterol produced a typical type I spectral change with K_d = 6.7 μM. The azole antifungal agents econazole, fluconazole, ketoconazole, and itraconazole bound tightly to CYP51F1 with K_d values between 0.06 and 0.42 μM. The CYP51F1 mutations F105L, D116E, Y132H, and R467K frequently identified in clinical isolates were examined to determine their effect on azole drug binding affinity. The azole K_d values of the purified F105L, D116E, and R467K mutants were little altered. A homology model of C. albicans CYP51F1 suggested that Tyr132 in the BC loop is located close to the heme in the active site, providing a rationale for the modified heme environment caused by the Y132H substitution. Taken together, functional expression and characterization of CYP51F1 provide a starting basis for the design of agents effective against C. albicans infections.

Full text of DOI:10.1016/j.abb.2011.02.002

Archives of Biochemistry and Biophysics 509 (2011) 9–15
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics
journal homepage: www.elsevier.com/locate/yabbi
Heterologous expression and characterization of the sterol 14a-demethylase
CYP51F1 from Candida albicans
Hyoung-Goo Park a, Im-Soon Lee a, Young-Jin Chun b, Chul-Ho Yun c, Jonathan B. Johnston d,
Paul R. Ortiz de Montellano d, Donghak Kim a,
⇑
a Department of Biological Sciences, Konkuk University, Seoul 143-701, Republic of Korea
b College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea
c School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea
d Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Lanosterol 14a-demethylase (CYP51F1) from Candida albicans is known to be an essential enzyme in fun-
Received 18 November 2010
and in revised form 31 January 2011
Available online 17 February 2011
gal sterol biosynthesis. Wild-type CYP51F1 and several of its mutants were heterologously expressed in
Escherichia coli, purified, and characterized. It exhibited a typical reduced CO-difference spectrum with a
maximum at 446 nm. Reconstitution of CYP51F1 with NADPH-P450 reductase gave a system that suc-
cessfully converted lanosterol to its demethylated product. Titration of the purified enzyme with lanos-
Keywords:
P450
CYP51
Lanosterol
Azole
Candida albicans
terol produced a typical type I spectral change with Kd = 6.7
fluconazole, ketoconazole, and itraconazole bound tightly to CYP51F1 with Kd values between 0.06 and
0.42 M. The CYP51F1 mutations F105L, D116E, Y132H, and R467K frequently identified in clinical iso-
lM. The azole antifungal agents econazole,
l
lates were examined to determine their effect on azole drug binding affinity. The azole Kd values of
the purified F105L, D116E, and R467K mutants were little altered. A homology model of C. albicans
CYP51F1 suggested that Tyr132 in the BC loop is located close to the heme in the active site, providing
a rationale for the modified heme environment caused by the Y132H substitution. Taken together, func-
tional expression and characterization of CYP51F1 provide a starting basis for the design of agents effec-
tive against C. albicans infections.
Ó 2011 Elsevier Inc. All rights reserved.
Introduction
increase in immunocompromised patients caused by HIV, cancer
chemotherapy, and organ/bone marrow transplantation, systemic
Cytochrome P450s (P450s, CYPs)1 are heme-thiolate-containing
enzymes that catalyze reactions involving both endogenous and
exogenous substrates [1,2]. These enzymes are found throughout
nature and in microorganisms play important roles, particularly in
the biosynthesis of antibiotics and other biologically active mole-
cules [3]. Genomic analysis of Candida albicans suggests that it con-
tains 10 putative P450 enzymes, including CYP51, CYP52 and CYP61.
It also encodes one NADPH-P450 reductase (NPR) that can deliver
electrons to both P450 enzymes and heme oxygenase (http://
www.candidagenome.org/) [4].
infections of C. albicans remain a serious clinical problem [7,8]. A
frequent occurrence of numeric and structural chromosomal shuf-
fling in the C. albicans genome and the ability to switch its colony
phenotype are its best-known adaptation characteristics [9].
The CYP51 family is very special, as there is a strict functional
conservation of enzyme activity among its members in all biological
kingdoms except for some nematodes and insects that lack CYP51
and therefore obtain sterols from their diet [10,11]. They all catalyze
the same three sequential oxidative steps that remove the 14a-
methyl group from post-squalene sterol precursors [10]. Azole anti-
fungal agents are widely used in the treatment of fungal infections
and fungal CYP51 enzymes are considered as their primary target
[12]. However, the mechanism of antifungal action at the molecular
level has not yet been fully clarified. Azole inhibitionof CYP51in fun-
gi causes accumulation of membrane-disrupting methylated ergos-
terol precursors that prevent fungal growth [13]. Azole drugs bind as
a sixth ligand to the heme iron atom of CYP51 to give a Type II spec-
trum with a maximum at 430 nm and a minimum at 410 nm [14].
This interaction involves coordination of the iron with N3 of an imid-
azole ring or N4 of a triazole ring in the azoles.
Candida albicans is a well known pathogenic fungus of opportu-
nistic oral and vaginal infections in humans [5,6]. Due to the recent
⇑
Corresponding author. Address: Department of Biological Sciences, Konkuk
University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, South Korea. Fax: +82 2
3436 5432.
E-mail address: donghak@konkuk.ac.kr (D. Kim).
Abbreviations used: P450 (or CYP), cytochrome P450; PCR, polymerase chain
1
reaction; TB, terrific broth; DTT, dithiothreitol; NPR, NADPH-P450 reductase; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.
0003-9861/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.abb.2011.02.002

10
H.-G. Park et al. / Archives of Biochemistry and Biophysics 509 (2011) 9–15
Azole-resistant strains have emerged as a serious problem in
amplified using PCR with forward and reverse primers (50-AAACA
GGATCCATCGATGCTTAGGAGGTCATATGGCTATTGTTGAAAC-03, 50-
ATTATTTCTAGACCGGAAGCTTTTAGTGATGGTGATGGTGATGAAACA
TACAAGTTTCTCT-03) and the amplified PCR fragment was cloned
into the pCW(Ori+) vector using the BamHI and XbaI restriction
sites. The cloned vectors were verified by nucleotide sequencing
analysis and restriction digestion. C. albicans translates CTG as
Ser instead of Leu, the amino acid for which CTG is a universal co-
don [21]. The open reading frame of CYP51F1 contains a CTG
encoding a Ser that must be corrected by site-directed mutagenesis
to enable expression of the correct recombinant protein in E. coli.
Site-directed mutagenesis to change Leu263 to a Ser was carried
out using Quick-Change mutagenesis (Stratagene, La Jolla, CA) as
previously described [20]. Four CYP51F1 variant clones containing
F105L, Y132H, D116E, and R467K substitutions were constructed
by site-directed mutagenesis.
the clinical use of azole drugs in the treatment to C. albicans infec-
tions. Amino acid substitutions in CYP51F1 gene sequences have
been found in azole-resistant C. albicans isolates [14,15]. Interest-
ingly, in the studies of isolates from patients during the emergence
of resistance, increased transcriptional levels of CYP51F1 and the
drug transporters CDR and MDR1 were observed in addition to
the mutations in the CYP51F1 amino acid sequence [16,17]. Varia-
tions in the CYP51F1 gene are thought to be associated with resis-
tance to azole antifungal agents, but few detailed biochemical
studies at the protein level have so far addressed this problem.
In this study, we have successfully cloned, overexpressed, and
purified CYP51F1 and several of its variants from C. albicans and
characterized these proteins in terms of their spectroscopic and
catalytic properties.
Materials and methods
Enzyme expression and purification
Chemicals and enzymes
Expression and purification of the CYP51F1 enzyme were car-
ried out as previously described with some modifications [22].
Briefly, the E. coli strains transformed with pCW(Ori+) vectors were
Lanosterol, econazole, fluconazole, itraconazole, ketoconazole,
sodium dithionite, glucose-6-phosphate, glucose-6-phosphate
dehydrogenase, and NADP+ were purchased from Sigma (St. Louis,
MO) or Aldrich Chemical Co. (Milwaukee, WI). Other chemicals
were of the highest grade commercially available. Escherichia coli
DH5a cells were purchased from Invitrogen (Carlsbad, CA). Recom-
binant rat NPR was expressed in E. coli and purified as previously
reported [18].
inoculated into TB medium containing 100 lg/ml ampicillin and
1.0 mM IPTG. The expression cultures were grown at 37 °C for
3 h and then at 28 °C with shaking at 200 rpm for 24 h in 1 l Fern-
bach flasks. Bacterial inner membrane fractions containing
CYP51F1 were isolated and prepared from 1 l TB (with ampicillin,
100 lg/ml) expression cultures of E. coli DH5a. Purification of
CYP51F1 enzyme using a Ni2+-nitrilotriacetate column was as pre-
viously described [22,23]. Briefly, membranes were solubilized at
4 °C overnight in 100 mM potassium phosphate buffer (pH 7.4)
containing 20% (w/v) glycerol, 0.5 M NaCl, 10 mM b-mercap-
toethanol, and 1.5% (w/v) CHAPS. The solubilized fraction was then
loaded onto a Ni2+-nitrilotriacetate column (Qiagen, Valencia, CA)
and the purified protein was obtained with the elution buffer con-
Construction of expression plasmids and site-directed mutagenesis
The general approach has been described previously [19,20].
The genomic DNA from C. albicans was kindly provided by Profes-
sor Won Ki Huh at Seoul National University. The open reading
frame for CYP51F1, and an added 6xHis-C-terminal tag, were
CaCYP51
HsCYP51
MtCYP51
TcCYP51
MAIVETVID- GI-------- ---------- NYFLSLSVTQ QISILLGVPF VYNLVWQYLY SL---RKDRA PLVFYWIPWF GSAASYGQQP YEFFESCRQ-
MAAAAGMLLL GLLQAGGSVL GQAMEKVTGG NLL-SM-LLI ACAFTLSLVY LIRLAAGHLV QLPA-GVKSP PYIFSPIPFL GHAIAFGKSP IEFLENAYE-
MSAVAL---- ---------- ---------- ---------- ---------- ---------- ---------- PRVSGGHDEH GHLEEFRTDP IGLMQRVRD-
MFIEAIV--- ---------- ---------- ---------- -----LALTA LILYSVYSVK SFNTTRPTDP PVYPVTVPFL GHIVQFGKNP LEFMQRCKRD
*
*
*
CaCYP51
HsCYP51
MtCYP51
TcCYP51
KYGDVFSFML LGKIMTVYLG PKGHEFVFNA KLSDVSAEDA YKHLTTPVFG KGVIYDCPNS RLMEQKKFAK FALTTDSFKR YVPKIREEIL NYFVTDESFK
KYGPVFSFTM VGKTFTYLLG SDAAALLFNS KNEDLNAEDV YSRLTTPVFG KGVAYDVPNP VFLEQKKMLK SGLNIAHFKQ HVSIIEKETK EYFES---W-
ECGDVGTFQL AGKQVVLLSG SHANEFFFRA GDDDLDQAKA YPF-MTPIFG EGVVFDASPE RRKEMLH--N AALRGEQMKG HAATIEDQVR RMIAD---W-
LKSGVFTISI GGQRVTIVGD PHEHSRFFSP RNEILSPREV YTI-MTPVFG EGVAYAAPYP RMREQLNFLA EELTIAKFQN FVPAIQHEVR KFMAEN--W-
CaCYP51
HsCYP51
MtCYP51
TcCYP51
LKEKT-HGVA NVMKTQPEIT IFTASRSLFG DEMRRIFDR- SFAQLYSDLD KGFTPINFVF P---NLPLPH YWRRDAAQKK ISATYMKEIK SRRER-GDID
---GE-SGEK NVFEALSELI ILTASHCLHG KEIRSQLNE- KVAQLYADLD GGFSHAAWLL PG--WLPLPS FRRRDRAHRE IKDIFYKAIQ KRRQS-Q---
---GE-AGEI DLLDFFAELT IYTSSACLIG KKFRDQLDG- RFAKLYHELE RGTDPLAYVD P---YLPIES FRRRDEARNG LVALVADIMN GRIAN-PPTD
---KEDEGVI NLLEDCGAMI INTACQCLFG EDLRKRLNAR HFAQLLSKME SSLIPAAVFM PWLLRLPLPQ SARCREARAE LQKILGEIIV AREKEEASKD
CaCYP51
HsCYP51
MtCYP51
TcCYP51
-PNRDLIDSL LIHSTYKDGV -KMTDQEIAN LLIGILMGGQ HTSASTSAWF LLHLGEKPHL --QDVIYQEV VELLKEKGGD -LNDLTYED- LQKLPSVNNT
EKIDDILQTL -LDATYKDGR -PLTDDEVAG MLIGLLLAGQ HTSSTTSAWM GFFLARDKTL --QKKCYLE- ---QKTVCGE NLPPLTYDQ- LKDLNLLDRC
KSDRDMLDVL -IAVKAETGT PRFSADEITG MFISMMFAGH HTSSGTASWT LIELMRHRDA --YAAVIDE- ---LDELYGD -GRSVSFHA- LRQIPQLENV
NNTSDLLGGL -LKAVYRDGT -RMSLHEVCG MIVAAMFAGQ HTSTITTSWS MLHLMHPKNK KWLDKLHKE- ---IDEFP-- --AQLNYDNV MDEMPFAERC
CaCYP51
HsCYP51
MtCYP51
TcCYP51
IKETLRMHMP LHSIFRKVTN PLRIPETNYI VPKGHYVLVS PGYAHTSERY FDNPEDFDPT RWDTAAAKAN SVSFNSSDEV DYGFGKVSKG VSSPYLPFGG
IKETLRLRPP IMIMMRMART PQTV--AGYT IPPGHQVCVS PTVNQRLKDS WVERLDFNPD RYLQDNPAS- ---------- --------G- EKFAYVPFGA
LKETLRLHPP LIILMRVAKG EFEV--QGHR IHEGDLVAAS PAISNRIPED FPDPHDFVPA RYEQPRQED- ---------- --------LL NRWTWIPFGA
VRESIRRDPP LLMVMRMVKA EVKV--GSYV VPKGDIIACS PLLSHHDEEA FPNPRLWDPE RDEKV----- ---------- ---------- -DGAFIGFGA
*
CaCYP51
HsCYP51
MtCYP51
TcCYP51
GRHRCIGEQF AYVQLGTILT TFVYNLRWTI DGYKV-PDPD YSSMVVLPTE PAE-IIWEKR ETCMF
GRHRCIGENF AYVQIKTIWS TMLRLYEFDL IDGYF-PTVN YTTMIHTPEN P-V-IRYKRR S---K
GRHRCVGAAF AIMQIKAIFS VLLREYEFEM AQPPESYRND HSKMVVQLAQ PAC-VRYRRR TG--V
GVHKCIGQKF ALLQVKTILA TAFREYDFQL LRDEV-PDPD YHTMVVGPTL NQCLVKYTRK KKLPS
Fig. 1. Sequence alignment of C. albicans CYP51F1 with other CYP51 enzymes. The amino acid sequences were aligned using the software T-Coffee (http://www.tcoffee.org).
The residues corresponding to the conserved heme binding sites in the CYP51 family are shown with boxes. The alignment scores of C. albicans CYP51F1 to human,
Mycobacterium tuberculosis, and Trypanosoma cruzi CYP51’s were 86, 87, and 85, respectively.

H.-G. Park et al. / Archives of Biochemistry and Biophysics 509 (2011) 9–15
11
(kDa)
Spectroscopic characterization
240
140
The CO-P450 complexes were generated by passing CO gas
through solutions of the P450. Then, sodium dithionite was added
to reduce the purified ferric CYP51F1 enzymes. UV–visible spectra
were collected on a CARY 100 Varian spectrophotometer in
100 mM potassium phosphate buffer (pH 7.4) at room
temperature.
100
70
61.5 kDa
Spectral binding titrations
50
35
Purified CYP51F1 wild-type and variant enzymes were diluted
to 2 lM in 100 mM potassium phosphate buffer (pH 7.4) and di-
vided between two glass cuvettes. Spectra (350–500 nm) were re-
corded with subsequent additions of substrate (lanosterol) or azole
compounds (in methanol stock) using a CARY 100 Varian spectro-
photometer [24]. The difference in absorbance between the wave-
length maximum and minimum was plotted versus the substrate
concentration [25,26]. The data were fit to the typical hyperbolic
equation for lanosterol and the quadratic equation for azole drugs
using nonlinear regression analysis with Graph-Pad Prism software
(Graph-Pad, San Diego, CA).
25
20
Fig. 2. SDS–PAGE of the purified CYP51F1 protein.
Lanosterol demethylation assay
Lanosterol demethylation by CYP51F1 was determined using a
P450/NPR/phospholipid reconstituted system. The reaction mix-
ture included 100 pmol purified P450 enzyme, 200 pmol rat NPR,
and DLPC (45
lM) in 0.50 ml of 100 mM potassium phosphate buf-
fer (pH 7.4), along with lanosterol (200
lM). An NADPH-generating
system [26] was used to start reactions. Incubations were generally
done for 30 min at 37 °C and terminated by addition of l ml of
CH2Cl2. The CH2C2 extract was dried under N2 and then converted
to trimethylsilyl derivatives by incubation with 50 ll of N,O-
bis(trimethylsilyl)trifluoroacetamide at 70 °C for 10–15 min. The
derivatized samples were allowed to cool, vortexed, and trans-
ferred to sealed Teflon-capped glass vials for either manual or
autoinjection into the GC-MS. Analyses were performed on an Agi-
lent 6850 gas chromatograph coupled to an Agilent 5973 Network
Mass Selective Detector using electron ionization (EI) as previously
described [27].
Homology modeling
A molecular model of C. albicans CYP51F1 structure was con-
structed using the X-ray crystal structure of Mycobacterium tuber-
culosis CYP51 (PDB 1EA1) as a template. The coordinates of the
model were obtained using the SWISS-MODEL software from the
Swiss Institute of Bioinformatics (http://www.swissmodel.exp-
asy.org/).
Results
Sequence alignment with other P450s
The ERG11 gene from C. albicans (Genebank: X13296,
orf19_922)2 encodes CYP51F1 (lanosterol 14
a-demethylase), a
member of the cytochrome P450 family that functions in ergosterol
biosynthesis. Phylogenetically, CYP51 enzymes are highly conserved
throughout nature from bacteria, through fungi, to humans. Amino
acid sequence alignment of CYP51F1 from C. albicans with CYP51
Fig. 3. Spectral analysis of the purified CYP51F1 protein. (A) The Fe2+ꢀCO vs Fe2+
difference spectrum and (B) the absolute spectra of the Fe3+, Fe2+, and Fe2+ꢀCO forms
were recorded.
2
CYP51F1 from C. albicans is the ERG11 gene (Genebank: X13296, orf19_922)
taining 400 mM imidazole. Eluted fraction containing P450 was
dialyzed at 4 °C against 100 mM potassium phosphate buffer (pH
7.4) containing 20% (v/v) glycerol, 0.1 mM EDTA.
encoding lanosterol 14a-demethylase and it has an amino acid sequence similarity
score of 86% with that of human CYP51A1 (NM_000786) lanosterol 14?-demethylase
(http://drnelson.uthsc.edu/cytochromeP450.html).

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H.-G. Park et al. / Archives of Biochemistry and Biophysics 509 (2011) 9–15
Fig. 4. GC–MS of products formed in the reaction of CYP51F1 with lanosterol. (A) GC–MS total ion trace of the reaction products, with the inset showing an expanded view of
the substrate (lanosterol) and FF-MAS (P1); (B) CYP51F1 reactions and controls were also analyzed in selected ion monitoring (SIM) mode. The SIM spectra for m/z 498 and m/
z 482 correspond to lanosterol and FF-MAS, respectively. For each SIM spectrum the RED trace corresponds to CYP51F1 incubations while the black spectrum represents
control reactions. (C) Elecron impact mass-spectrum of hydroxylanosterol TMS-ether (P2). The molecular ion [M]+ꢂ is found at m/z 586, M – CH3 at m/z 571, and the signature
ion at m/z 103 corresponds to loss of CH2OTMS from a primary alcohol.
of other species reveals high sequence similarities with scores from
85% to 87% identity (Fig. 1) [28]. Good sequence alignment with hu-
man, M. tuberculosis, and Trypanosoma cruzi CYP51 suggests that the
gene product ERG11 from C. albicans is likely to have lanosterol 14a-
demethylase catalytic activity.
enzyme per liter of culture medium (data not shown). After ultra-
centrifugation, the expressed P450 protein was observed in the
membrane fraction. CHAPS successfully solubilized the mem-
brane-bound P450 protein and the solubilized protein was purified
on a Ni-column. The resulting protein migrated on SDS–PAGE as a
single band at 61.5 kDa, as expected for the open reading frame of
the CYP51F1 gene with a His-tag (Fig. 2). Protein sequencing of
the purified protein by mass-spectrometry indicated that the puri-
fied protein was the expected CYP51F1 from C. albicans (Supple-
mentary Fig. 1).
Expression and purification of CYP51F1
The reduced CO-binding spectrum in the whole cell culture
showed the typical P450 expression level of ꢁ50 nmol P450 holo-

H.-G. Park et al. / Archives of Biochemistry and Biophysics 509 (2011) 9–15
13
Spectral properties of CYP51F1
The reduced CO-difference spectrum of purified CYP51F1 had a
maximum absorption at 446 nm (Fig. 3A). Examination of the
absolute spectrum showed that the ferric form of the protein
was in the low spin state with a Soret band at 419 nm, while the
ferrous protein had a broad absorption peak around 420 nm
(Fig. 3B). The smaller
a-, and b-bands of the ferric P450 were at
568 and 536 nm, respectively (Fig. 3B). On reduction with sodium
dithionite, the
respectively.
a-, and b-bands shifted to 559 and 530 nm
Catalytic activities of CYP51F1
Purified CYP51F1 reconstituted with rat NPR supported the cat-
alytic turnover of lanosterol to yield three GC–MS detectable prod-
ucts (Fig. 4A). The first product peak (P1) in the chromatogram was
determined as the final 14a demethylated metabolite (FF-MAS, fol-
licular fluid-meiosis activating steroid) of lanosterol, although it
was partially masked by the tail of the lanosterol peak. Selected
ion monitoring (SIM) scans for m/z = 498 (lanosterol TMS ether)
and also m/z = 482 (FF-MAS TMS ether) confirmed that peak P1
did in fact correspond to FF-MAS (Fig. 4B). The second product peak
(P2) was consistent with the product of oxidation of the methyl to
Fig. 5. Titration of CYP51F1 with lanosterol. Increasing concentrations of lanosterol
were added to both the sample and reference cuvettes. The inset shows the plot of
D
A390–420 nm vs. concentration of lanosterol.
0.08
0.06
0.04
0.02
0.00
-0.02
0.08
0.08
0.04
0.02
0.06
0.04
0.04
0
6
12
0
6
12
azole, μΜ
azole, μΜ
0.02
0.00
-0.02
Fluconazole
Econazole
-0.04
-0.04
350
400
450
500
350
400
450
500
0.06
0.08
0.06
0.04
0.02
0.00
-0.02
0.05
0.025
0.04
0.02
0
6
12
azole, μΜ
0.00
0.08
0.04
-0.02
Ketoconazole
Itraconazole
0
6
12
azole, μΜ
-0.04
350
400
450
500
350
400
450
500
wavelength, nm
Fig. 6. Titration of CYP51F1 with azole compounds. Purified CYP51F1 (2 lM) was dissolved in 100 mM potassium phosphate buffer (pH 7.4) and the absorption spectrum of
the ferric form (350–500 nm) was recorded with subsequent additions of the azole compound fluconazole, econazole, ketoconazole, or itraconazole. The binding affinities of
the azoles calculated from the plots are shown in Table 1.

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H.-G. Park et al. / Archives of Biochemistry and Biophysics 509 (2011) 9–15
Table 1
Binding affinities of azole agents to the C. albicans CYP51F1 wild-type and mutant proteins.
Azole agents
Kd (lM)
Wild type
F105L
D116E
Y132H
R467 K
Econazole
0.20 0.04
0.06 0.01
0.19 0.06
0.42 0.08
0.10 0.03
0.02 0.01
0.16 0.04
0.17 0.04
0.14 0.03
0.03 0.01
0.15 0.03
0.43 0.08
0.42 0.06
0.15 0.06
NDa
0.13 0.03
0.06 0.01
0.18 0.04
0.66 0.13
Fluconazole
Itraconazole
Ketoconazole
0.54 0.07
a
Not determined.
molecule by the lanosterol to give a low-spin hexa coordinated
P450 heme (Fig. 5). The calculated Kd value is 6.7 0.7
l
M
(Fig. 5). Furthermore, purified CYP51F1 produced a typical type II
spectral change, with an increase at 430 nm and a decrease at
410 nm, on binding of azole drugs (Fig. 6), as expected for the bind-
ing of a strong ligand to the heme iron atom [29]. The calculated Kd
values for the azole drugs are between 0.06 and 0.42 lM (Table 1).
Mutations that occur frequently in the CYP51F1 gene (F105L,
D116E, Y132H, and R467K) in clinically isolated azole-resistant C.
albicans strains were analyzed to determine their effect on the
binding affinity of azole drugs [15]. Recombinant mutant enzymes
were expressed and purified as already described. There were no
notable differences in the reduced-CO difference and absolute
spectra (data not shown). Three of the mutant proteins bound
azole agents to give typical type II spectra (Supplementary Figs. 2–
5). However, binding of azoles to the Y132H mutant showed the
expected decrease at 410 nm but little increase at 430 nm (Supple-
mentary Fig. 4). Unexpectedly, the Kd values for the binding of
azoles to the purified F105L, D116E, and R467K mutants were little
altered (Table 1). The binding of lanosterol to the purified F105L,
D116E, and R467K mutants occurred with a type I spectral change
and binding affinities similar to those for the wild-type enzyme
(Supplementary Fig. 6). Titration of the Y132H mutant with lanos-
terol gave the expected decrease at 425 nm, but the distinct in-
crease in the peak at 390 nm was not observed.
Discussion
We previously reported purification and characterization of
CYP52A1, the Alk8 gene product from C. albicans [30], that oxidizes
dodecanoic acid to the
x-hydroxylated metabolite as a major
product and the ( -1)-hydroxylated compound as a minor one. A
x
functional heme oxygenase from C. albicans, the product of the
CaHmx1 gene, has also been characterized [20]. Recently, the NPR
of C. albicans, coded by the NCP1 gene, was characterized [31].
The purified C. albicans NPR was shown to be an orthologous
reductase that supports both the cytochrome P450 and heme oxy-
genase activities. Our present biochemical characterization of
CYP51F1 from C. albicans complements these earlier studies and
broadens our understanding of the critical system of catalytic
hemoproteins in C. albicans.
Fig. 7. Positions of the mutated amino acid residues in CYP51F1. (A) Ribbon
diagram of the C. albicans CYP51F1 model using the X-ray crystal structure of M.
tuberculosis CYP51 (PDB 1EA1) as a template and (B) the positions of the Y132,
D116, and R476 mutations are indicated with colors. Heme and fluconazole are
shown in blue and green, respectively.
the alcohol, the expected first step of lanosterol demethylation
(Fig. 4C). Its fragmentation pattern was consistent with the TMS-
ether. The third product (P3) could not be identified. It had a mass
44 Da higher than that of the alcohol but did not fit with any of the
intermediates expected in the multi-step demethylation
mechanism.
The ERG11 gene in C. albicans encodes CYP51F1, a putative sterol
14a-demethylase, that is required for the biosynthesis of ergosterol.
Indeed, purified CYP51F1 converts lanosterol to its demethylated
product through initial formation of the alcohol metabolite, but
the conversion rate is relatively low (Fig. 4). The aldehyde interme-
diate expected in the oxidation sequence was not detected, possibly
because it is too rapidly oxidized to the acid intermediate. One ca-
veat in the assignment of oxidized products is that the identification
of the site of hydroxylation is still putative since it was based only on
mass spectral evidence. However, the mass spectrometric evidence
is commonly used for these types of metabolites when the metabo-
lite standards are not available [27,32,33]. HPLC analysis of the en-
zyme reactions were performed to obtain the enzyme oxidation
Binding of lanosterol and azole agents to CYP51F1 wild type and
variants
Titration of purified CYP51F1 with lanosterol showed a typical
type I spectral change with an increase at 390 nm and decrease
at 420 nm, consistent with displacement of the iron-bound water

H.-G. Park et al. / Archives of Biochemistry and Biophysics 509 (2011) 9–15
15
rate. A new peak appeared in the chromatogram, assuming the puta-
tive lanosterol oxidation product (Supplementary Fig. 8A). But, the
identification of the product mass was failed due to no ionization
in LC-mass spectrometry. The rate of oxidation of lanosterol based
on this HPLC analysis was estimated with assuming the new peak
as the lanosterol oxidation product. The calculated kcat value was
& Family Affairs, Republic of Korea (A084005), and by National
Institutes of Health Grant GM25515 to P.R.O.M.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.abb.2011.02.002.
approximately 0.19 minꢃ1 with a Km value of 20
lM for the wild-
type CYP51F1 enzyme (Supplementary Fig. 8B). In addition, the
putative oxidation product peak disappeared when the lanosterol
oxidation reaction was performed with azole compounds, suggest-
ing that the oxidation activity of purified enzyme was inhibited by
azole drugs (Supplementary Fig. 8A).
Site-specific mutations in the CYP51F1 gene have been consid-
ered as one of the main mechanisms for azole resistance in clini-
cally isolated C. albicans strains. So far, about 140 mutations have
been reported in the literature, but few of them have been specif-
ically associated with azole resistance [34]. Multiple mechanisms
of resistance have been proposed, including a reduction of intracel-
lular azole concentration or an increased CYP51 expression [14].
Changes in CYP51F1 expression have been detected in resistant
strains, but their role in resistance is unclear.
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Acknowledgments
This study was supported by a grant to D.K. from the Korea
Healthcare Technology R&D Project, Ministry for Health, Welfare