Substrate and reaction specificity of Mycobacterium tuberculosis cytochrome P450 CYP121: Insights from biochemical studies and crystal structures

Article abstract of DOI:10.1074/jbc.M112.443853

Cytochrome P450 CYP121 is essential for the viability of Mycobacterium tuberculosis. Studies in vitro show that it can use the cyclodipeptide cyclo(L-Tyr-L-Tyr) (cYY) as a substrate. We report an investigation of the substrate and reaction specificities of CYP121 involving analysis of the interaction between CYP121 and 14 cYY analogues with various modifications of the side chains or the diketopiperazine (DKP) ring. Spectral titration experiments show that CYP121 significantly bound only cyclodipeptides with a conserved DKP ring carrying two aryl side chains in L-configuration. CYP121 did not efficiently or selectively transform any of the cYY analogues tested, indicating a high specificity for cYY. The molecular determinants of this specificity were inferred from both crystal structures of CYP121-analog complexes solved at high resolution and solution NMR spectroscopy of the analogues. Bound cYY or its analogues all displayed a similar set of contacts with CYP121 residues Asn⁸⁵, Phe¹⁶⁸, and Trp¹⁸². The propensity of the cYY tyrosyl to point toward Arg³⁸⁶ was dependent on the presence of the DKP ring that limits the conformational freedom of the ligand. The correct positioning of the hydroxyl of this tyrosyl was essential for conversion of cYY. Thus, the specificity of CYP121 results from both a restricted binding specificity and a fine-tuned P450 substrate relationship. These results document the catalytic mechanism of CYP121 and improve our understanding of its function in vivo. This work contributes to progress toward the design of inhibitors of this essential protein of M. tuberculosis that could be used for antituberculosis therapy.

Full text of DOI:10.1074/jbc.M112.443853

Protein Structure and Folding:
Substrate and Reaction Specificity of
Mycobacterium tuberculosis Cytochrome
P450 CYP121: INSIGHTS FROM
BIOCHEMICAL STUDIES AND
CRYSTAL STRUCTURES
Matthieu Fonvielle, Marie-Hélène Le Du,
Olivier Lequin, Alain Lecoq, Mickaël Jacquet,
Robert Thai, Steven Dubois, Guillaume
Grach, Muriel Gondry and Pascal Belin
J. Biol. Chem. 2013, 288:17347-17359.
doi: 10.1074/jbc.M112.443853 originally published online April 25, 2013
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 24, pp. 17347–17359, June 14, 2013
© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Substrate and Reaction Specificity of Mycobacterium
tuberculosis Cytochrome P450 CYP121
INSIGHTS FROM BIOCHEMICAL STUDIES AND CRYSTAL STRUCTURES*
Received for publication, January 3, 2013, and in revised form, April 23, 2013 Published, JBC Papers in Press, April 25, 2013, DOI 10.1074/jbc.M112.443853
Matthieu Fonvielle‡1,2, Marie-He´le`ne Le Du§1, Olivier Lequin¶, Alain Lecoq‡, Mickae¨l Jacquet‡, Robert Thai‡,
Steven Dubois‡, Guillaume Grach‡3, Muriel Gondry‡, and Pascal Belin‡4
From the ‡Commissariat a` l’Energie Atomique et aux Energies Alternatives (CEA), iBiTec-S, Service d’Inge´nierie Mole´culaire des
Prote´ines, 91191 Gif-sur-Yvette Cedex, France, the §CEA, iBiTec-S, Service de Bioe´nerge´tique, Biologie Structurale et Me´canismes,
Laboratoire de Biologie Structurale et Radiobiologie, 91191 Gif-sur-Yvette Cedex, France, and the ¶Universite´ Pierre et Marie
Curie-Paris 06, UMR 7203 CNRS-UPMC-ENS, Laboratoire des Biomole´cules, 4 Place Jussieu, 75252 Paris Cedex 05, France
Background: Little is known about the substrate specificity of the essential P450 CYP121 from Mycobacterium tuberculosis.
Results: CYP121 substrate analogues either display impaired binding to CYP121 or are poorly transformed by CYP121.
Conclusion: CYP121 is a highly specific P450.
Significance: This work contributes to the understanding of the function and the catalytic mechanism of CYP121 and provides
novel data for the design of inhibitors.
Cytochrome P450 CYP121 is essential for the viability of gress toward the design of inhibitors of this essential protein of
Mycobacterium tuberculosis. Studies in vitro show that it can M. tuberculosis that could be used for antituberculosis therapy.
use the cyclodipeptide cyclo(L-Tyr-L-Tyr) (cYY) as a substrate.
We report an investigation of the substrate and reaction speci-
Cytochrome P450 enzymes (P450s)5 constitute a superfamily
of heme-containing proteins found in nearly all organisms from
bacteria to humans (1). Most catalyze mixed function oxida-
tions of a wide range of endogenous and xenobiotic substrates;
a very large number of substrates and reactions have been
described (2, 3). Nevertheless, it is widely accepted that there is
a common catalytic cycle in which the substrate plays impor-
tant roles. The substrate generally induces the low-to-high spin
transition that facilitates the continuation of the cycle and may
influence the nature of the reaction catalyzed (4). Some P450s
have broad substrate and reaction specificity, whereas others
are highly selective enzymes catalyzing regio- and stereospe-
cific reactions. The characterization of substrate and reaction
specificity of P450s would help to elucidate their biological
functions and decipher the catalytic mechanism, with major
implications for drug design and biocatalysis.
ficities of CYP121 involving analysis of the interaction between
CYP121 and 14 cYY analogues with various modifications of the
side chains or the diketopiperazine (DKP) ring. Spectral titra-
tion experiments show that CYP121 significantly bound only
cyclodipeptides with a conserved DKP ring carrying two aryl
side chains in L-configuration. CYP121 did not efficiently or
selectively transform any of the cYY analogues tested, indicating
a high specificity for cYY. The molecular determinants of this
specificity were inferred from both crystal structures of
CYP121-analog complexes solved at high resolution and solu-
tion NMR spectroscopy of the analogues. Bound cYY or its ana-
logues all displayed a similar set of contacts with CYP121 resi-
dues Asn85, Phe168, and Trp182. The propensity of the cYY
tyrosyl to point toward Arg386 was dependent on the presence of
the DKP ring that limits the conformational freedom of the
ligand. The correct positioning of the hydroxyl of this tyrosyl
was essential for conversion of cYY. Thus, the specificity of
CYP121 results from both a restricted binding specificity and a
fine-tuned P450 substrate relationship. These results document
the catalytic mechanism of CYP121 and improve our under-
standing of its function in vivo. This work contributes to pro-
CYP121 from Mycobacterium tuberculosis is one of the most
studied P450s from this human pathogen (5). The increasing
number of deaths associated with M. tuberculosis over the last
25 years has highlighted the limitations of currently available
anti-tuberculosis drugs. Novel antibiotics are required (6).
Interest in CYP121 as a potential drug target followed the early
discovery that CYP121 has strong affinity for several azole mol-
ecules that were also identified as effective antimycobacterial
compounds (7–10). McLean et al. (11) showed that the cyp121
gene is essential for mycobacterial growth, reinforcing the idea
that CYP121 could be a potential therapeutic target. The deter-
mination of the crystal structure of CYP121 in complex with
* This work was supported by the Commissariat a` l’Energie Atomique et aux
Energies Alternatives (CEA), CNRS, and grants from the Re´gion Ile-de-
France. The Service d’Inge´nierie Mole´culaire des Prote´ines is a member of
the Laboratory of Excellence LERMIT.
The atomic coordinates and structure factors (codes 4ICT, 4IQ9, 4IQ7, 4IPW, and
4IPS) have been deposited in the Protein Data Bank (http://wwpdb.org/).
1 Both authors contributed equally to this work.
2 Recipient of a CEA postdoctoral fellowship. Present address: Centre de
Recherche des Cordeliers, LRMA, Equipe 12, Universite´ Pierre et Marie 5 The abbreviations used are: P450, cytochrome P450 enzyme; cYY, cyclo(L-
Curie-Paris 6, UMR S 872, Paris, F-75006 France.
Tyr-L-Tyr); DKP, diketopiperazine; RP-HPLC, reverse phase HPLC; Ks, appar-
ent spectral dissociation constant; cYF, cyclo(L-Tyr-L-Phe); cYW, cyclo(L-Tyr-
L-Trp); cYA, cyclo(L-Tyr-L-Ala); cY-DOPA, c(L-Tyr-l-3,4-dihydroxyphenylalanine);
rms, root mean squared; RT, retention time; amu, atomic mass unit.
3 Recipient of a Re´gion Ile-de-France postdoctoral fellowship.
4 To whom correspondence should be addressed. Tel.: 169082576; Fax:
169089071; E-mail: pascal.belin@cea.fr.
JUNE 14, 2013•VOLUME 288•NUMBER 24
JOURNAL OF BIOLOGICAL CHEMISTRY 17347

CYP121 Specificity
FIGURE 1. Chemical structures of compounds described in this study.
fluconazole at 1.9 Å revealed a novel mode of azole binding to tometry, x-ray crystallography, enzymatic assays, mass spec-
P450, with a nitrogen atom coordinating the heme iron through trometry, and solution NMR spectroscopy to characterize the
the sixth iron ligand. However, no molecules, other than azoles, binding to and transformation by CYP121 of a series of sub-
that bind to CYP121 have been found, and initial attempts to strate analogues (Fig. 1).
identify ligands by testing compounds for their ability to induce
EXPERIMENTAL PROCEDURES
heme iron spin transition were unsuccessful (9).
Recently, we showed that CYP121 catalyzes an unusual reac-
Chemicals—Chemicals were from Sigma-Aldrich unless oth-
tion by forming a C–C bond between the two tyrosyl side chains erwise stated. All chemicals were of the highest purity available.
of the cyclodipeptide cyclo(L-Tyr-L-Tyr) (cYY; Fig. 1, 1) result-
Synthetic Chemistry Methods—Compounds 1, 4, 12, 13, and
ing in a novel chemical entity called mycocyclosin (Fig. 1, 2) (12, 14 were obtained from Bachem. Compounds 2, 3, 5, 6, and 7
13). This identification was based in part on the characteriza- were synthesized previously in our laboratory (12, 14). Com-
tion of the activity of Rv2275, encoded by a gene associated in pounds 8, 9, 10, 11, and 15 were chemically synthesized by
an operon-like structure with cyp121 (14, 15). Rv2275 uses adapting previously published procedures (17–21). The synthe-
charged tRNAs to synthesize cYY and several other cyclodipep- sis strategies are shown in Fig. 2.
tides. The identification of cYY was unexpected, because the
Amino acid derivatives were obtained from Bachem. They
x-ray crystal structure of CYP121 solved at atomic resolution were used as received without further purification. All non-
revealed a large active site cavity that could accommodate bulky aqueous reactions were carried out under an atmosphere of
hydrophobic substrates (16). Accordingly, the x-ray structure argon in flame- or oven-dried glassware with magnetic stirring.
of cYY-bound CYP121 solved at 1.4 Å resolution showed a sin- Thin layer chromatography analyses were performed on Merck
gle ligand molecule that only partially occupies the binding cav- Silica Gel 60 F254 plates, and components were visualized by
ity (12). The protein conformation in this complex is very sim- illumination with UV light or by staining with a potassium per-
ilar to that of ligand-free or fluconazole-bound CYP121. This manganate solution (1 g with 2 g of K2CO3 in 200 ml of water).
suggested the absence of significant conformational change Flash column chromatography was performed using Merck
upon ligand binding, consistent with the previously described Geduran Si60 (40–63 ␮m). All compounds were purified by
rigidity of the active site (12, 16). Our work also revealed unex- RP-HPLC (Discovery Bio Wide Pore C18 column, 250 ϫ 10
pected structural features of substrate-bound P450 with the mm, 5 ␮m). Purity was determined by analytical RP-HPLC
presence of a network of hydrogen bonds above the heme and (Atlantis dC18, 4.6 ϫ 250 mm, 5 ␮m; Ͼ95%). High resolution
between the substrate and residues of the active site and water mass spectra were recorded on a 4800 MALDI-TOF mass spec-
molecules, including the sixth heme iron ligand. One hydroxyl trometer as described previously, using 0.1–0.3 mM solutions of
of cYY, in particular, approaches the heme and participates in compound in 50% CH3CN (DMSO Ͻ 1‰) (14). Compounds
this network. According to these structural observations and were also characterized by NMR spectroscopy (see below).
ϩ
the reaction catalyzed, we proposed a role for the two side
High resolution mass spectra (m/z): 8, [MH] calculated for
ϩ
chains of cYY in transformation. Nevertheless, the molecular C18H19N2O5, 343.1294, found 343.1304. 9, [MH] calculated
ϩ
determinants of the substrate involved in the catalytic cycle for C18H19N2O4, 327.1345, found 327.1364. 10, [MH] calcu-
ϩ
remain to be determined. The aim of this work was to identify lated for C18H19N2O4, 327.1345, found 327.1348. 11, [MH]
the substrate determinants involved in the reaction and sub- calculated for C18H23N2O2, 299.1760, found 299.1757. 15,
ϩ
strate specificity of CYP121. We used UV-visible spectropho- [MH] calculated for C18H19N2O4, 313.1552, found 313.1552.
17348 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 288•NUMBER 24•JUNE 14, 2013

CYP121 Specificity
FIGURE 2. Synthetic schemes for 8, 9, and 10 (A), 11 (B), and 15 (C). A, cyclodipeptides were synthesized by liquid phase peptide synthesis by adapting
previously described procedures (19). a, amino acids with side chains R1 and R2 and protecting groups P1 (Fmoc (fluorenylmethyloxycarbonyl) for synthesis of
8 or Boc (tert-butyloxycarbonyl) for synthesis of 9 and 10), P2 (acetonid for synthesis of 8 and tBu (tert-butyl) for synthesis of 9 and 10), and P3 (tBu) were
incubated in N,NЈ-dicyclohexylcarbodiimide and triethylamine for 1 h at 4 °C and then overnight at room temperature, followed by overnight dessication; b
(only for the synthesis of 8), dissolution in dimethylformamide/dichloromethane (1:1), 2 h at room temperature, evaporation to dryness; c, dissolution in
HCO2H/TFA/H2O (9:0.5:0.5), 2 h at room temperature, evaporation to dryness; d, dissolution in butanol-1/toluene (9:1) or butanol-1 (synthesis of 9 and 10),
reflux for 2 h, purification by RP-HPLC. B, the synthesis of 11 was adapted from previously published procedures (17). a, piperazine, triethylamine, and
1-(benzyloxy)-4-(chloromethyl)benzene (1:2.2:10) were stirred in tetrahydrofuran for 18 h at room temperature, evaporated to dryness, and subjected to
chromatography on SiO2 in ethyl acetate/hexane (7:3, v/v); b, dissolution in methanol/ethanol (8:2, v/v), hydrogenation with 5 mg 10% palladium on carbon,
drying under vacuum, purification by RP-HPLC. C, the synthesis of 15 was adapted from previous publications describing reductive amination (18, 20, 21) and
liquid phase peptide synthesis (19). a, incubation in dimethylformamide/AcOH for 0.5 h at room temperature, cooled to 0 °C, NaBH3CN, 16 h at room
temperature, evaporation under reduced pressure at temperature Ͻ40 °C; b, dissolution in methanol, 10% palladium on carbon overnight under 3.3 bars of H2,
evaporation to dryness; c, dissolution in HCOOH, 2 h at room temperature with stirring, evaporation under reduced pressure (temperature Ͻ40 °C), addition
of triethylamine and dry dimethylformamide and stirring for 16 h at room temperature, evaporation under reduced pressure, purification by RP-HPLC.
Stock Solutions of Compounds—Compounds were dissolved day. In these conditions, the amount of 1 in the samples was less
in DMSO at the highest concentration that could be achieved. than 1‰ as estimated by LC-MS/MS using a calibration curve
Concentrations were determined by UV-visible spectropho- with 1. In solutions of 13 and 14 (derivatives of linear dipeptide
tometry after dilution of the stock solution in 100 mM potas- YY by acetylation of the primary amine and amidation of
sium phosphate, pH 7.4, with the final DMSO concentration the carboxylic acid, respectively), 1 was detected only in trace
below 10%. For peptide compounds, the molar extinction coef- quantities (Ͻ0.3‰).
ficients of phenylalanine, tryptophan, and tyrosine were used.
CYP121 Expression and Purification—Wild type CYP121
For 8, 11, and 15, molar extinction coefficients were found to be was produced in Escherichia coli and purified as described pre-
3580 MϪ1 cmϪ1 at 279 nm, 1570 MϪ1 cmϪ1 at 271 nm, and 1795 viously (9, 12). Purified CYP121 had an Rz value (ratio of A416 nm
M
Ϫ1 cmϪ1 at 276 nm, respectively. Clear stock solutions of 3, 5, to A280 nm) of Ͼ1.8. The purity was Ͼ95% as estimated by SDS-
and 6 could only be obtained at concentrations up to 15–20, PAGE analysis. The concentration of CYP121 was determined
40–50, and 60–70 mM, respectively. Other compounds were by using the ⑀416 of 110 mMϪ1 cmϪ1 (11). Purified CYP121 (ϳ1
dissolved at concentrations of around 200 mM without diffi- mM) was stored in 50 mM Tris-HCl, 1 mM EDTA (pH 7.2) at
culty. Stock solutions were conserved at Ϫ20 °C and were Ϫ80 °C.
thawed at room temperature before use. RP-HPLC analysis of
Binding of Compounds to CYP121—Ligand binding to
dilutions of these stock solutions revealed that purity was Ͼ99% CYP121 was analyzed at 20 °C by spectral titration using a dou-
(estimated from peak areas determined on chromatograms ble-beam Uvikon 943 spectrophotometer and 1-cm path length
recorded at 220 nm). However, the solution of 12 (linear dipep- quartz cells as described previously (12). The final DMSO con-
tide YY) was found to change over time, with the appearance of centration was kept under 1% (v/v). For 3 and 15, light scatter-
a major contaminant; LC-MS/MS analysis indicated the pres- ing appeared during titration, resulting in large variations of Ks
ence of a high concentration of 1 (cyclic dipeptide cYY, between and ⌬Absmax values between the different assays. To limit the
5 and 10%). This was attributed to the formation of an intramo- effects of light scattering on Ks determinations, ligand solution
lecular peptide bond, possibly favored by the presence of was added to the reference cuvette instead of DMSO. Plots
DMSO. To limit the formation of 1, stock solutions of 12 were appeared hyperbolic for all compounds showing binding to
prepared in DMSO extemporaneously and were used the same CYP121, and data were fitted to the equation, ⌬Abs ϭ(⌬Absmax ϫ
JUNE 14, 2013•VOLUME 288•NUMBER 24
JOURNAL OF BIOLOGICAL CHEMISTRY 17349

CYP121 Specificity
TABLE 1
Data collection and refinement statisticsa
Ligand bound to CYP121
3
4
7
8
15
Protein Data Bank entry
4ICT
4IQ9
4IQ7
4IPW
4IPS
Data collection
Beam line
ID23
0.933
P6522
ID23
0.933
P6522
ID23
ID23
0.933
P6522
PX1 (Pilatus)
0.8726
Wavelength (Å)
Space group
0.933
P6522
P6522
Resolution limits (Å)
Rmerge
1.8 (1.9–1.8)
0.152 (0.541)
296,243 (47,663)
44,879 (6445)
9.6 (3.4)
1.4 (1.48–1.4)
0.092 (0.346)
1,099,644 (100,350)
93,373 (13,226)
18.1 (4.4)
1.9 (2.0–1.9)
0.146 (0.435)
206,187 (32,849)
37,824 (5442)
10.4 (3.9)
98.4 (99.6)
5.5 (6.0)
1.4 (1.48–1.4)
0.099 (0.285)
389,209 (33,474)
86,350 (10,768)
11.5 (2.8)
1.2 (1.27–1.2)
0.042 (0.281)
2,918,439 (343,052)
274,037 (43,130)
32.65 (5.38)
99.2 (96.4)
Total no. of reflections
No. of unique reflections
Mean (I)/sd(I)
Completeness (%)
Multiplicity
99.8 (100.0)
6.6 (7.4)
99.8 (99.0)
92.5 (80.7)
11.8 (7.6)
4.5 (3.1)
10.65 (7.95)
Refinement
R factor
0.1776
0.2285
0.8910
3735
0.1582
0.1794
0.930
3989
0.1500
0.1884
0.937
3751
0.1566
0.1824
0.929
3868
0.1582
0.1710
0.932
3792
Rfree
Figure of merit
Total no. of atoms
No. of water molecules
Root mean square deviation
Bond lengths (Å)
Bond angles (degrees)
Average B factors (Å2):
All atoms
588
695
605
655
596
0.010
1.196
0.010
1.00
0.010
0.99
0.012
1.06
0.010
1.01
24.9
22.0
39.3
18.3
31.7
17.2
21.7
18.4
37.0
12.8
25.5
16.2
13.1
30.6
8.9
16.1
13.5
29.5
10
Protein
14.0
Water
31.6
Heme
9.6
Ligand
14.3/16.6
10.1
16.9
a Data in parentheses indicate the last resolution shell.
[L])/(Ks ϩ [L]), where ⌬Absmax is the overall maximal absor- such that B factor values would be of the same order for each
bance variation, and Ks is the apparent spectral dissociation
constant.
conformation. At the end of the refinement process, one addi-
tional run of BUSTER5 was performed after removal of the
ligand molecules from the coordinate files, to calculate unbi-
ased 2Fo Ϫ Fc electron density omit maps. Table 1 reports fur-
ther information on x-ray analysis statistics.
For the determination of the Ks value of cYY for CYP121 in
the presence of another ligand, the inhibitor ligand was added
to the reference and sample cuvettes, the base line was
recorded, CYP121 was added to the reference cuvette, and the
titration with cYY was performed as above. The final DMSO
concentration remained under 1.5% (v/v). Data were analyzed
as above. All experiments were performed in triplicate at least,
and results are expressed as means Ϯ S.D.
Turnover Experiments—CYP121 conversion of compounds
was assessed in the presence of an electron transport chain
constituted of spinach ferredoxin and spinach ferredoxin
ϩ
NADP reductase as described previously (12). The final
DMSO concentration was kept at 1%, because preliminary
experiments performed with final DMSO concentrations rang-
ing from 1 to 5% indicated the absence of detectable effects on
cYY transformation by CYP121 at concentrations below 5%
(data not shown). Samples collected at various times were acid-
ified and analyzed by LC-MS/MS as described previously (12).
Control experiments in which CYP121 was omitted were con-
ducted and analyzed as above. In these control experiments, the
DMSO concentration was 1% (v/v).
Crystallization and Structure Determination of Ligand-bound
CYP121—Ligand-bound CYP121 crystals were grown at 6 °C in
a few days by the sitting drop method as described previously
(purified CYP121 at 230 ␮M and ligand concentration around 1
mM in 100 mM NaMES at pH 5.0–5.5 and 1.6–2.2 M
(NH4)2SO4). Crystals were cryoprotected in a solution contain-
ing 20% glycerol in the crystallization solution and cryocooled
in liquid nitrogen. Diffraction data were collected at the Euro-
pean Synchrotron Radiation Facility or at SOLEIL (see Table 1
for details), processed with MOSFLM, and scaled with SCALA
from the CCP4 package (22). The structures were refined with
REFMAC (23) and BUSTER5 (24), using the coordinates of
native CYP121 as the input entry (Protein Data Bank entry
1N40) (25), and manually corrected using COOT 0.6 (26).
Ligands were built at the end of the refinement processes. The
quality of the electron density was compatible with the mole-
cule being completely built for complexes of CYP121 with 3, 8,
and 15. In the case of CYP121 bound to 4 and CYP121 bound to
7, two alternate conformations were modeled on the basis of
the electron density map. Their respective occupancies were
adjusted to 0.25 (conformation 1) and 0.75 (conformation 2) for
Solution NMR Spectroscopy for Conformational Analysis—A
Bruker Avance III spectrometer equipped with a TCI cryo-
probe and operating at a 1H frequency of 500 MHz was used for
NMR experiments. Spectra were recorded at 30 °C in DMSO-
d6, in CD3OD, or in 90% H2O, 10% D2O (Eurisotop). 1H and 13
C
resonances were assigned through the analysis of one-dimen-
sional 1H, one-dimensional 13C DEPTQ (distortionless en-
1
hancement by polarization transfer), two-dimensional H-1H
1
COSY, two-dimensional H-1H ROESY (rotating frame NOE
spectroscopy), two-dimensional 1H-13C HSQC (heteronuclear
single quantum correlation), and two-dimensional 1H-13C
HMBC (heteronuclear multiple-bond correlation). 1H and 13
C
4 and to 0.34 (conformation a) and 0.66 (conformation b) for 7, chemical shifts were referenced to the DMSO solvent signal (␦
17350 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 24•JUNE 14, 2013

CYP121 Specificity
TABLE 2
NMR assignments for conformational analysis of the compounds in solution
RESULTS
2.50 and 39.5 ppm, respectively). NMR data were processed and
analyzed with Bruker TOPSPIN version 3.1 software.
Rationale for the Choice of cYY Analogues—The cYY ana-
logues were chosen to be relevant to various issues: determining
the specificity of CYP121, investigating its in vivo function, and
providing information pertinent to drug design. Mycocyclosin
2, the major product of the reaction, was also included to study
any possible end product inhibition of the reaction. CYP121
and the cyclodipeptide synthase Rv2275 are genetically linked,
so we tested cyclodipeptides synthesized by Rv2275 because
they may be natural substrates. They include cYF (3), cYW (4),
cyclo(L-Tyr-L-Leu) (5), cyclo(L-Tyr-L-Met) (6), and cYA (7) (14,
28). We also included the cyclodipeptide cY-DOPA (8). This
cyclodipeptide differs from cYY by only one additional
The conformational analysis was based on the measurement
3
of homonuclear JH␣-H␤ coupling constants on one-dimen-
1
3
3
sional H spectra and heteronuclear JH␣-C␥ and JH␤-CO cou-
pling constants measured on phase-sensitive two-dimensional
HMBC experiments. Using the Karplus curves obtained for
these three vicinal coupling constants (27), the populations of
␹1 rotamers and stereospecific assignment of H␤ methylenic
protons could be obtained unambiguously. In particular, the
3JH␣-C␥ coupling constant provided direct estimates of the
ϩ
3
gauche rotamer population, and JH␤-CO coupling constants
allowed H␤ methylenic protons to be assigned stereospecifically.
NMR assignments of the compounds are presented in Table 2.
JUNE 14, 2013•VOLUME 288•NUMBER 24
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CYP121 Specificity
TABLE 3
UV-visible spectroscopic characterization of ligand binding to CYP121
a The final DMSO concentration in the cuvette was between 1 and 1.5% (1.5% for 12).
b nd, not determined. Ks could not be determined because of light scattering after the addition of the compound to the cuvette even at lower concentrations of the compound
or in the presence of 1% DMSO from the beginning of the titration.
hydroxyl positioned on one carbon atom involved in the C–C of cYY for CYP121 in the presence of 5 and 6 could not be
coupling catalyzed by CYP121. To evaluate the role of the chi- determined accurately because of light scattering (Table 3).
rality of tyrosine residues in cYY, we also investigated cyclo(L- However, spectral variations were observed upon the addition
Tyr-D-Tyr) and cyclo(D-Tyr-D-Tyr) (9 and 10, respectively). To of a micromolar concentration of cYY to a CYP121 solution in
study the relationship with CYP121 of compounds that carry the presence of 5 and 6 at 216 and 264 ␮M, respectively. Com-
two tyrosyl side chains anchored on a scaffold other than the pounds cYF (3), cYW (4), and cY-DOPA (8), which have two
DKP ring, we included a compound that carries the two tyrosyls aromatic side chains, exhibited significant binding to CYP121
on a piperazine scaffold (11), linear YY dipeptides with free or with Ks values in the same range as those for cYY. These three
blocked N and C termini (12, 13, and 14), and a compound that compounds showed substrate-like binding with low to high
differs from cYY by the reduction to CH2 of one of the keto spin transition as indicated by a peak around 387–390 nm and a
functions of the DKP ring (15).
trough around 417–420 nm on difference spectra. Binding of
Cyclodipeptides with Two Aryl Side Chains in L-Configura- cY-DOPA to CYP121 was indistinguishable from that of cYY,
tion Bind Efficiently to CYP121—Binding of compounds to except for a slightly lower Ks value. There were small differ-
CYP121 was assessed in ligand titration experiments by UV- ences in wavelength values between spectra for cYY binding
visible spectroscopy. Because the ligands studied are poorly and those for cYF and cYW binding. The intensity of the spin
soluble in water, we used stock solutions in DMSO. The transition for these two compounds was significantly lower
addition of 1% DMSO to a CYP121 solution slightly dimin- than that for cYY (75 and 61% lower, respectively). The Eadie-
ished the intensity of the Soret band (Ͻ1%) without observ- Hofstee representation of the data indicated the singularity of
able effect on ␭max. Furthermore, binding constants of CYP121 cYW binding to CYP121 (data not shown). Data for cYW bind-
for cYY were similar for final DMSO concentrations of 1 or 2% ing appear to fit two straight lines of different slopes better than
(19.4 Ϯ 0.6 and 21.6 Ϯ 1.2 ␮M, respectively). Therefore, the final a single straight line, suggesting the possibility of two binding
DMSO concentration was kept under 1% in ligand titration sites. There was similar, but weaker, evidence of two binding
experiments.
sites in the data for cYY and cYF but not for cY-DOPA. No
In addition, for ligands exhibiting no spectral variation upon evidence for binding of 9 or 10 to CYP121 was obtained, in
addition to a solution of CYP121, their effects at a high concen- either titration experiments or inhibition studies, indicating
tration on the affinity of CYP121 for its substrate cYY were that changing the stereochemistry of one or two C␣ atoms of
evaluated in inhibition experiments. In these assays, the DMSO cYY greatly impaired binding.
concentration remained Յ1.5%. Data obtained in these two
We then studied the role of the DKP ring with a range of
molecules carrying the two tyrosyl side chains presented either
experiments are summarized in Table 3.
Very little binding or no binding was observed for com- on a linear peptide (12, 13, and 14) or on another cyclic scaffold
pounds comprising one aliphatic side chain, cyclo(L-Tyr-L-Leu) (11 and 15). No binding was observed for 11 that possesses a
(5), cyclo(L-Tyr-L-Met) (6), and cYA (7). The binding constant non-peptide scaffold, in either titration experiments or inhibi-
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CYP121 Specificity
TABLE 4
tion studies. For the linear peptides, direct binding was detected
only for 14 but with the Ks value being 38 times as high as that
for cYY. Furthermore, type II binding was observed, indicating
a heme coordination through a nitrogen atom. The possibility
of 12 binding to CYP121 cannot be excluded, because a 40%
increase of the Ks value of cYY for CYP121 was observed in
inhibition studies. Nevertheless, a large amount of 12 was used
(Ͼ20 fold the Ks value), suggesting that the binding, if there was
any, was much weaker than that of cYY for CYP121. The reduc-
tion of one keto function of the DKP ring of cYY to CH2 greatly
impaired binding to CYP121, with a 34-fold increase of the Ks
value. Type I binding similar to that involving cYY was
observed, but the calculated value for overall absorbance vari-
ation was significantly lower, suggesting a different binding
type.
Metabolites detected in a CYP121 activity assay using compound 1, 3,
4, 7, 8, or 15 as substrate
CYP121 Specifically Transforms cYY—Mycocyclosin 2 was
identified as the major product of the activity of CYP121 on cYY
1 (12), but the formation of minor products was not addressed.
To provide a more complete description, we reinvestigated the
transformation of cYY by CYP121, focusing on minor products.
We used tandem mass spectrometry coupled to reverse phase
HPLC (LC-MS/MS) to analyze the reaction after a 1-h incuba-
tion (Table 4, substrate ϭ 1). In addition to mycocyclosin 2,
several other metabolites were found in small amounts (each
Ͻ2%). Analysis of the MS2 spectra of the metabolites charac-
ϩ
terized by the [MH] ion at m/z 302.1 and 316.0 was mostly
uninformative about the nature of these compounds. The
metabolite characterized by a retention time of 30.8 min and a
ϩ
[MH] ion at m/z 325.0 displays an MS2 spectrum sharing sim-
ilarities with that of mycocyclosin (data not shown). The MS2
ϩ
spectrum of the metabolite with an [MH] ion at m/z 341.0
revealed the same neutral losses as those observed for mycocy-
closin (data not shown), suggesting a possible hydroxylation of
mycocyclosin during the reaction. Finally, a targeted search for
metabolites at m/z 343.0 Ϯ 0.5 (ϩ16 mass increase relative to
cYY) was unsuccessful, such that hydroxylation of cYY was not
detected.
We next investigated the transformation by CYP121 of com-
pounds inducing the low to high spin transition (cYF, cYW,
cYA, cY-DOPA, and 15) (Fig. 3 and Table 4). Compound 4
(cYW) was the only substrate analog that was significantly con-
sumed by CYP121 in activity assays; ϳ50% of the initial com-
pound remained after a 1-h incubation with CYP121, whereas
95% remained in the absence of CYP121. More than 10 differ-
ent metabolites appearing during the reaction catalyzed by
CYP121 are observed, but no major metabolite could be iden-
tified (Table 4). Analysis of ion current chromatograms
revealed molecular weight differences of ϩ16, ϩ32, ϩ4, and Ϫ2
relative to cYW. These results indicate the lack of specificity of
CYP121 for metabolite formation from cYW. Some of these
a The retention times in min (RT) and m/z values for the [MH] ion are indicated
ϩ
in parentheses.
b Reported metabolites were not detected in an assay in the absence of CYP121,
apart from the remaining substrate and from the assay conducted with 4 (see
“Results”).
c Peak areas were obtained from chromatograms recorded at 214 nm.
d m/z values correspond to the [MH] ion. Question marks indicate that no value
ϩ
could be obtained.
e Identity was attributed when retention time, m/z value, and MS2 spectra were
identical to those of one of the compounds described in this study. nd, identity
of the metabolite could not be determined.
ϩ
f Metabolites with the [MH] ion at m/z 348, m/z 366, and m/z 382 were observed
in the same peak characterized by a retention time of 35.7 min.
metabolites were also detected in an assay conducted in the ues, and MS2 spectra identical to those of cYY and mycocyclo-
absence of CYP121, but in much lower amounts, suggesting the sin. This indicates that CYP121 may hydroxylate cYF, albeit at
propensity of cYW to be transformed.
very low rate.
The rate of transformation of cYF (3) was very slow, with
Transformation of cY-DOPA (8) by CYP121 was very slow;
ϳ98% of compound remaining after 1 h of incubation (100% in 91% of the compound remained after 1 h (100% in the control
the control reaction without CYP121). Analysis of the ion cur- without CYP121). A first metabolite appeared during the reaction
ϩ
rent chromatograms obtained for the 60 min samples indicated witharetentiontimeϳ2minlowerandanm/zvaluefor[MH] 16
the presence of two metabolites with retention time, m/z val- units higher than the initial product, suggesting a possible
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CYP121 Specificity
FIGURE 3. CYP121 activity on substrate analogues. The transformation by
CYP121 of cYY (1) and of substrate analogues (3, 4, 7, 8, and 15) was assessed
in coupled enzymatic reactions as described under “Experimental Proce-
dures” and analyzed by LC-MS/MS. Chromatograms were recorded at 214
nm, and the percentage of remaining substrate analog in the reaction was
estimated by measuring peak area. Results are presented for each compound
according to the time of the reaction.
FIGURE 4. Bound ligands in CYP121 crystal structures. 2Fo Ϫ Fc omit maps
at 1 ␴ level (blue mesh) of the ligand molecule found in 4ICT (A), 4IQ9 (B), 4IQ7
(C), 4IPW (D), and 4IPS (E) are shown. The ligands are superimposed as stick
models with carbon atoms in yellow or orange (alternate conformations for B
hydroxylation. A second peak appeared at a retention time of 31.5
min for which no m/z value could be determined. No compound and C), oxygen atoms in red, and nitrogen atoms in blue.
ϩ
with a m/z value for the [MH ] ion at 341 (2-unit decrease) was
observed, showingthatthemajorreactioncatalyzedbyCYP121on cYF, cYW, cYA, cY-DOPA, and 15, respectively (determined
cYY did not occur with cY-DOPA.
for 381, 359, 374, 354, and 359 equivalent C␣ atoms, respec-
For cYA (7), no new metabolite was detected upon incuba- tively). Furthermore, for each structure solved, atoms of the
tion with CYP121, consistent with the absence of consumption side chains of the residues that constitute the active site super-
observed (Fig. 3).
impose well on the equivalent atoms in native CYP121 (Protein
Conversion of 15 by CYP121 was very inefficient; 98% of the Data Bank entry 1N40).
compound remained after 1 h (100% remained in the control
In each structure, the ligand binds at the previously described
without CYP121). Three additional compounds appeared spe- cYY-binding site (Fig. 5). There was thus a common binding
cifically in the presence of CYP121, and each accounted for less mode, similar to that identified for cYY. It involves van der
than 1% of 15.
Waals interactions of one aryl side chain with Phe168 and
Crystal Structures of cYY Analog-bound CYP121 Reveal a Trp182, and stacking of the DKP ring against the 310 helix and BЈ
Common Binding Mode—The crystal structures of CYP121 helix with one of its carbonyls hydrogen-bonding the N␦2 of
bound to cYF, cYW, cYA, cY-DOPA, and 15 were determined. Asn85, except for conformation 2 of cYW (Fig. 5). For each
Data collection and refinement statistics are summarized in structure, the sixth ligand of the heme iron is not displaced and
Table 1. Independent of the nature of the molecule bound to is positioned between 2.3 and 2.7 Å from the iron (Table 5). As
CYP121, crystals were grown as previously described and were in the cYY-CYP121 complex, the iron remains in the plane of
found to belong to the same crystal form as crystals of native the heme. Details of the interactions established by each com-
CYP121 used for the atomic resolution of the CYP121 structure pound are given in Fig. 6. The major specific features of each
(16). The various structures were solved and refined at high compound bound to CYP121 are as follows.
resolution (1.2–1.9 Å resolution) such that the quality of the
The position adopted by 3 (cYF) in the CYP121 binding
structures is compatible with fine structural analysis. The omit site was with the tyrosyl residue pointing toward helices F
maps calculated from the data showed unambiguous density and G and the phenylalanyl moiety pointing toward the
corresponding to the compound bound to CYP121 (Fig. 4). One heme and facing the DKP ring in positions highly similar to
single molecule was modeled in all cases except for cYW, for those observed for cYY (Fig. 5A). The ring approaching the
which two alternate conformations are observed. Each overall heme, however, was closer to the heme iron; the distance
structure of ligand-bound CYP121 is highly similar to that of between the C⑀1 carbon atom and the iron is 5.3 Å (6.1 Å
native CYP121 (Protein Data Bank entry 1N40) with root mean between the equivalent atoms in the CYP121-cYY complex).
square deviations on equivalent C␣ atoms of 0.177, 0.112, Furthermore, no hydrogen-bonded water molecule network
0.169, 0.133, and 0.099 Å for complexes of CYP121 bound to connected the ligand and the protein at the oxygen binding
17354 JOURNAL OF BIOLOGICAL CHEMISTRY
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CYP121 Specificity
FIGURE 5. Crystal structures of CYP121 bound to 3 (A), 4 (B and C), 7 (D), 8 (E), and 15 (F). Detailed views of the active site are presented. The protein
backbone is in a schematic representation colored in wheat. Residues of the active site and the heme are in a stick representation colored in green with oxygen,
nitrogen, and iron atoms in red, blue, and orange, respectively. The ligand is in a stick representation with carbon, oxygen, and nitrogen atoms colored in yellow,
red, and blue, respectively. Water molecules are shown as red spheres. Red dashed lines, possible hydrogen bonds. The two conformations observed for the
tyrosyl side chain of 7 are noted as a and b in D. In each panel, the inset shows the superimposition of the CYP121-bound ligand (yellow carbons) over the
CYP121-bound cYY (white carbons), obtained by superimposition of each overall structure presented herein with the crystal structure of cYY-bound CYP121
(Protein Data Bank entry 3G5H).
Phe168, with a distance of 3.2 Å between the CH2 carbon atom
TABLE 5
of cYW and the C⑀2 carbon atom of Phe168. The ␹1 angle of
Distances between the heme iron and the fifth and sixth ligand in
CYP121 crystal structures
Phe168 was slightly different from that observed in native
Distances
CYP121 (Ϫ69.8 and Ϫ75.1°, respectively), resulting in the side
Protein Data
Bank entry
Fe–6th
ligand
chain being positioned slightly away from the ligand binding
cavity and closer to the Leu174 side chain. In the second confor-
mation, the tyrosyl ring occupies roughly the same place, but
the tryptophan and DKP rings interchange through a rotation
of about 180° along the elongation axis of the compound (Fig.
5C). As a consequence, the DKP ring is located above the heme,
with the oxygen atom of one carbonyl interacting with the iron
through two tightly hydrogen-bonded water molecules, WAT2
and WAT1 (distances between the heteroatoms of CϭO/
WAT2/WAT1 and iron are 2.7, 2.6, and 2.3 Å, respectively).
The tryptophan positions with the indole ring parallel to the
heme, facing the DKP ring and packing against the 310 helix.
Interestingly, the ionic interaction between one carbonyl of the
DKP and Asn85-N␦2 conserved in other structures was substi-
tuted by an ionic bond between the N⑀1 of the tryptophan and
the carbonyl of Val83.
Bound ligand
Resolution
S–Fe
Å
Å
None
1N40
3G5F
3G5H
4ICT
4IQ9
4IQ7
4IPW
4IPS
1.06
1.4
1.4
1.8
1.4
1.9
1.4
1.2
2.4
2.4
2.4
2.3
2.3
2.3
2.3
2.3
2.1
2.5
2.4
2.7
2.3
2.7
2.4
2.3
None
1
3
4
7
8
15
site of the heme. This structure highlights the importance of
the missing hydroxyl for establishment of the hydrogen-
bond network above the heme and for the positioning of the
substrate.
Two alternate conformations were observed at the same
CYP121 binding site for 4 (cYW; Fig. 5, B and C). Conformation
1 (occupancy of 0.25) was very similar to that of cYY, the so-
called canonical ligand conformation (Fig. 5B, inset). In this
conformation, the tyrosyl side chain points toward helices F
and G but does not occupy exactly the same position as those
observed for cYY or cYF. The tryptophan moiety faces the DKP
ring and is located above the heme. The N⑀1 of tryptophanyl is
the ligand atom closest to the heme iron, located at 5.6 Å. It
interacts with the iron through the two water molecules WAT2
and WAT1 (distances between the heteroatoms of NH/WAT2/
WAT1 and iron are 3.1, 2.7, and 2.3 Å, respectively). In this
In the structure of the CYP121-7 (cYA) complex, the DKP
ring and the C␤ carbon atom of the tyrosyl side chain are well
defined, but two alternative conformations of the tyrosyl side
chain could be modeled (Fig. 5D). In one of the conformations,
the tyrosyl points toward helices F and G similarly to the equiv-
alent group in cYY. In the second conformation, the tyrosyl side
chain points toward Phe280 facing the DKP ring. The hydroxyl
is positioned just above the heme at 3.0 Å from one nitrogen of
the guanidinium of Arg386, and it interacts with Gln385 through
conformation, the tryptophanyl comes in close proximity to one water molecule. Its position is very close to that of the
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CYP121 Specificity
FIGURE 6. Schematic representation of polar contacts (A) and non-polar contacts (B) made by ligands in the presence of CYP121. For compounds 4 and
7, the conformation is indicated in parentheses (1 and 2 for 4, a and b for 7). A, dashed lines indicate hydrogen bonds with interatomic distances given in Å. w,
water molecule. L6, sixth ligand of the heme iron. B, dashed lines, non-covalent interactions (van der Waals, hydrophobic) between atoms closer than 5 Å.
Lines starting from the center of an aromatic ring mean that each atom constituting the ring participates in non-covalent interactions. For clarity, the
names of the atoms of CYP121 residues are omitted. The distances in Å between the heme iron (Fe) and the closest ligand atoms are indicated beside
double arrows.
hydroxyl of cYY (the distance between the two hydroxyls is this tyrosyl ring occupied a position that was not observed for
0.8 Å) (Fig. 5D, inset). No electron density, however, was any other compound studied. The weaker electron density and
observed between cYA and the sixth ligand of the heme iron. the higher B factor of the ring (19 Å2 instead of 14.7 Å2 for the
In the crystal structure of CYP121 in complex with 8 (cY- tyrosyl ring pointing toward helices F and G) suggest that the
DOPA), the ligand positions at the same location as cYY (Fig. occupancy of the ring may not be 100%. Similarly, the tyrosyl
5E). The only difference with cYY is associated with the addi- ring is located close to Met62, at 2.9–3.1 Å and 4.1 Å from the
tional hydroxyl group; it is involved in two direct hydrogen minor (30%) and major (70%) alternate conformations of
bonds with Thr77-O␥1 (3.2 Å) and the oxygen atom of the car- Met62, respectively. This suggests that the major conformation
bonyl of Ala167 (3.1 Å). All of these observations are consistent for Met62 is the conformation with the tyrosyl ring of 15 occu-
with the spectral variations observed upon CYP121 titration pying its visible position, whereas the minor conformation for
with cY-DOPA.
Met62 is stable when this tyrosyl is elsewhere. Nevertheless, no
Compound 15 positions in the CYP121 binding site with one clear alternate conformation of 15 was visible in the electron
tyrosyl pointing toward helices F and G and the ketopiperazine density. In particular, there was no electron density indicating
ring at a position similar to that observed for the DKP ring of that the tyrosyl ring might adopt the canonical position facing
other ligands (Fig. 5F). The major difference with other ligands the piperazinone ring with the hydroxyl directed toward
involves the second tyrosyl that does not face the ketopipera- Arg386. At the oxygen-binding site, the network of hydrogen-
zine ring; the ␹1 angle associated with this tyrosine is 165.1°, bonded water molecules observed in the structure of cYY-
whereas the angle in the case of cYY is 63.8°. As a consequence, bound CYP121 is largely disrupted. In particular, there was a
17356 JOURNAL OF BIOLOGICAL CHEMISTRY
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CYP121 Specificity
large electron density bulb whose center was located 3.5 Å from pounds, the binding mode was essentially the same as that of
the sixth ligand of the heme iron.
cYY: the DKP ring facing the 310 helix with hydrogen bonding
Solution NMR Spectroscopy of Ligands Reveals Rapid Equi- between one carbonyl and the side chain of Asn85 and one aryl
librium between Different Conformations—Changes in ligand side chain positioned for ␲-stacking interactions with Phe168
conformation upon binding may make a large contribution to and Trp182 (see Fig. 3) (12). Solution NMR spectroscopic anal-
affinity (29). The compounds that we studied are particularly ysis of ligands indicated that the presence of only one aryl side
flexible, in part due to the rotation of the aryl side chains around chain anchored on the DKP ring (5, 6, and 7) favors a confor-
the C␣–C␤ atom, and consequently, elucidation of the confor- mation detrimental for efficient binding with the aryl side chain
mations these compounds adopt in solution may help to iden- facing the DKP ring. This preferential conformation was indeed
tify determinants of binding. Therefore, we used NMR spec- observed for compound 7 bound to CYP121 (see Fig. 3D). One
troscopy to analyze the conformations in solution of 3, 4, 5, 6, 7, consequence of this preferential conformation is that ␲-stack-
8, 9, 10, and 15. In particular, the populations of the different ␹1 ing interactions with Phe168 and Trp182 are prevented. The
rotamers of aromatic residues were determined from the binding of cyclodipeptides to CYP121 is also conditioned by the
3
3
vicinal homonuclear JH␣-H␤ and heteronuclear JH␣-C␥ and stereochemistry of the C␣ atom; compounds 9 and 10, which
3JH␤-CO coupling constants. The JH␣-H␤ coupling constants are stereoisomers of the CYP121 substrate cYY, failed to bind.
3
measured in one-dimensional spectra for 1, 5, 7, and 15 differed Solution NMR spectroscopy showed that the preferential con-
only slightly between solutions in water and in DMSO, indicat- formation of 9 corresponds to the tyrosyl side chains facing the
ing that the solvent has little influence on the conformation of two sides of the DKP ring; this causes substantial steric hin-
these compounds. The following conformational studies were drance to binding to CYP121. In the case of 10, for which both
therefore carried out mostly in DMSO because of the poor sol- C␣ atoms were in the D-configuration and there was no binding
ubility of these compounds in water. Cyclic dipeptides 5, 6, and to CYP121, the tyrosyl side chains are predicted to position on
7, each with an aliphatic residue, showed a major conformer for the same side of the DKP ring as for cYY. One noticeable dif-
ϩ
the tyrosyl side chain in solution (ϳ80% gauche , ϳ20% trans ference between 10 and cYY, however, is the positions of the
ϩ
␹1 rotamers). This major gauche conformation corresponds CϭO and NH functions of each amino acid in the DKP ring
to an orientation of the aromatic side chain above the DKP ring, with respect to the corresponding C␣–C␤ bond (see Fig. 1).
indicating a favorable interaction between the aromatic and The crystal structure of cYY-bound CYP121 shows one hydro-
DKP rings, as described previously (30). The conformational gen bond between one carbonyl of the DKP ring of cYY and the
preferences of the heterochiral compound 9 were similar, with side chain of Asn85. According to these observations, it appears
the major conformer corresponding to the two aromatic rings that 10 cannot bind to CYP121 in the same way as cYY with
pointing to the two faces of the DKP ring. In 1, 3, 4, 8, and 10, both the ␲-stacking interactions described above and hydrogen
the two aromatic side chains have similar ␹1 rotamer distribu- bonding between Asn85 and the DKP ring. Our results clearly
ϩ
tions and show a smaller gauche population (ϳ50% gaucheϩ, indicate that the integrity of the DKP ring is a key feature of
Ϫ
ϳ35% gauche , ϳ15% trans), indicating that the aromatic rings cyclodipeptide binding: its replacement (11), its opening (12,
compete for orientation toward the DKP ring. There is no pref- 13, and 14), or even the reduction of one of its keto functions
erence for tyrosyl, dihydroxyphenylalanyl, or tryptophanyl aro- (15) substantially reduced binding to CYP121. A comparison of
ϩ
matic rings in the gauche conformer.
the crystal structures of cYY-bound CYP121 and 15-bound
Conformational analysis of 15 indicated an equilibrium CYP121 does not reveal obvious contacts between the ligand
between two non-planar half-chair conformations of the piper- and the protein that could explain the 32-fold lower affinity of
azinone ring, as indicated by the vicinal coupling constants of 15 (Table 3 and Fig. 6). The conformations of the ligands in
the methylene group in the ring. The tyrosyl side chains do not solution may contribute to CYP121 binding. Indeed, solution
have preferred conformations.
NMR spectroscopy findings for the ligands lead us to suggest
that the DKP ring in cYY restricts the conformational space of
tyrosyl side chains, leading to conformations more favorable for
DISCUSSION
CYP121 is a P450 from M. tuberculosis that has drawn atten- CYP121 binding than those found in 15. This restricted chemical
tion for several reasons. The organization of its active site and space for efficient binding to CYP121 is consistent with the previ-
features of the reaction it catalyzes suggest novel mechanisms ously observed rigidity of the CYP121 active site cavity (16).
(12, 16), and it is also essential in vivo such that it is a potential
We show that, in addition to this specificity of binding,
target for the development of novel antimycobacterial agents CYP121 exhibits further stringent requirements for catalysis;
(11, 28, 31). Here, we report an investigation of the substrate four of the five compounds (3, 7, 8, and 14) that are able to bind
and reaction specificities of CYP121 involving synthesizing to CYP121 are transformed only poorly or not at all. For exam-
substrate analogues and studying their binding to and transfor- ple, the relationships of cYF (3) and cYA (7) with CYP121 illus-
mation by CYP121.
trate the requirement for not only the presence (cYF) but also
The major conclusion from these experiments is that the correct positioning (cYA) of the hydroxyl above the heme
CYP121 is a highly specific P450; it did not efficiently or selec- for efficient transformation. Its absence or incorrect position-
tively transform the analogues tested. We show that the molec- ing impairs the low to high spin transition concomitant with the
ular basis of this specificity is primarily the control of ligand modification of the hydrogen bond network involving the sixth
binding. Indeed, only three of the 14 substrate analogues tested ligand of the heme iron, showing the importance of this net-
efficiently bound to CYP121 (3, 4, and 8). For these three com- work to the heme iron spin state. This is consistent with previ-
JUNE 14, 2013•VOLUME 288•NUMBER 24
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CYP121 Specificity
ous observations suggesting a possible change in spin state for the reaction catalyzed by CYP121 suggests that the Rv2275/
CYP101A through modification of the pKa value of the sixth CYP121 tandem mostly synthesizes and transforms cYY in vivo.
Ϫ
ligand, influencing its OH /H2O equilibrium (32–34). In the This transformation occurs almost exclusively through C–C
case of CYP121, the loss of the hydrogen bond network may coupling with very few minor products. In particular, we were
have a similar effect, with the associated reduction of spin tran- unable to detect any hydroxylation of cYY. However, other
sition. The importance of spin transition in the P450 catalytic P450s can hydroxylate phenol to catechol (47–50), and the
cycle is associated with an increase of the heme iron oxidation/ positioning of cYY in the CYP121 active site is consistent with
reduction potential that may be necessary for rapid reduction of such a reaction (12). Interestingly, compound 8, a hydroxylated
P450s and the continuation of the catalytic cycle (4, 35). In the cYY, binds more efficiently than cYY to CYP121 but is poorly
case of CYP121, the lower spin transition observed when 3 or 7 transformed. Consequently, if CYP121 were able to synthesize
is bound cannot alone account for the very low transformation 8 by hydroxylation of cYY, it appears that the product of the
rate, because the spin transition remains relatively high (25 and reaction would inhibit the enzymatic activity. Mycocyclosin
42% of that observed with cYY, respectively). More likely, the (2), the natural product of CYP121 activity on cYY, shows no
hydroxyl group of cYY specifically favors the reaction and the such end product inhibition. These results are consistent with
nature of the transformation. Indeed, no intramolecular C–C CYP121 being essential for viability in vivo (11).
bond could be detected for 3 transformation, and the only
The cyp121 gene is essential for mycobacterial growth, so
product identified was hydroxylated 3. Arene hydroxylation by inhibiting CYP121 is a promising approach to treating tubercu-
P450 reactive species compound I implies that the ␲-system is losis (11, 31). Our findings contribute to the development of
initially attacked by the activated oxygen of compound I (36, inhibitors with potential clinical applications. We demonstrate
37). In the case of 3, the iron is too far from the site of hydroxy- the stringent requirement for the DKP ring for cyclodipeptide
lation for such a reaction, and this may explain the low effi- binding to CYP121. Fortuitously, the DKP ring is an attractive
ciency. It was suggested recently that cis-trans isomerization of scaffold for medicinal chemistry and is commonly found in com-
the Val78–Pro79 peptide bond in CYP121 could trigger reposi- pound libraries (51). Our results suggest that a DKP ring bearing
tioning of the substrate, bringing it close to the heme iron (38). two aryl side chains, as in 8, may serve for anchoring various other
If so, it is clear that this mechanism is not sufficient to allow structures to the CYP121 binding cavity. This strategy could be
hydroxylation of 3. Finally, the poor transformation of 3 is con- used to identify novel chemical functions for binding to CYP121,
sistent with a CYP121 mechanism initiating with either hydro- circumventing the high specificity of binding of CYP121.
gen abstraction or electron transfer; the observed distances
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One exception to the poor transformation of the compounds
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