From the Department of Biochemistry, University of
Leicester, The Adrian Building, University Road, Leicester LE1 7RH,
United Kingdom, the
Structural Biochemistry Group, Institute of
Cellular and Molecular Biology, Michael Swann Building, University
of Edinburgh, The King's Buildings, West Mains Road, Edinburgh EH9
3JR, United Kingdom, the
Department of
Pure and Applied Chemistry, University of Strathclyde, The Royal
College, 204 George Street, Glasgow G1 1XL, United Kingdom, and the
** Department of Chemistry, University of Edinburgh, The
King's Buildings, West Mains Road, Edinburgh EH9 3JJ, United
Kingdom
Received for publication, September 27, 2002, and in revised form, October 31, 2002
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ABSTRACT |
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The first structure of a P450 to an atomic
resolution of 1.06 Å has been solved for CYP121 from
Mycobacterium tuberculosis. A comparison with P450
EryF (CYP107A1) reveals a remarkable overall similarity in fold
with major differences residing in active site structural elements. The
high resolution obtained allows visualization of several unusual
aspects. The heme cofactor is bound in two distinct conformations while
being notably kinked in one pyrrole group due to close interaction with
the proline residue (Pro346) immediately following the heme
iron-ligating cysteine (Cys345). The active site is
remarkably rigid in comparison with the remainder of the structure,
notwithstanding the large cavity volume of 1350 Å3. The
region immediately surrounding the distal water ligand is remarkable in
several aspects. Unlike other bacterial P450s, the I helix shows no
deformation, similar to mammalian CYP2C5. In addition, the positively
charged Arg386 is located immediately above the heme plane,
dominating the local structure. Putative proton relay pathways from
protein surface to heme (converging at Ser279) are
identified. Most interestingly, the electron density indicates weak
binding of a dioxygen molecule to the P450. This structure provides a
basis for rational design of putative antimycobacterial agents.
The cytochromes P450
(P450s)1 are a superfamily of
heme-containing mono-oxygenases (1). They are famed for their roles in drug metabolism/detoxification and steroid synthesis in mammals (e.g. Refs. 2 and 3) but are also found in virtually all other life forms. The vast majority of P450s bind and cleave molecular oxygen in a two-step reaction involving delivery of two electrons from
a redox partner. In eukaryotic P450s, this is typically the diflavin
enzyme NADPH cytochrome P450 reductase (4). However, in prokaryotes the
reducing equivalents are usually supplied via two enzymes: NAD(P)H
flavodoxin/ferredoxin reductase and either a ferredoxin or a flavodoxin
mediator of electrons to the P450 (5, 6). Many bacterial P450s are of
particular biotechnological and environmental relevance, given their
role in degradation of recalcitrant organic molecules for use as energy
sources (e.g. Refs. 7 and 8). Bacterial P450s have also
proven to be experimentally tractable systems for elucidation of P450
structure and mechanism, due largely to the fact that they are soluble
enzymes, as opposed to their eukaryotic membrane-associated
counterparts (5). A breakthrough was made recently with the atomic
structure of the first mammalian P450 (a form of P450 2C5). In this
case, extensive protein engineering was required to remove the
N-terminal membrane anchor peptide, to promote release from the
cellular membrane and to prevent aggregation of the resultant
solubilized P450 (9).
Whereas the genome sequence of E. coli is devoid of
P450-encoding genes (10), the genomes of the industrially important bacterium Streptomyces coelicolor and the pathogen
Mycobacterium tuberculosis (Mtb) indicate that there are 18 and 20 P450s encoded, respectively (11, 12). The genome sequence of Mtb
revealed several interesting features, most notably the preponderance
of genes involved in lipid metabolism; ~250 enzymes involved in lipid metabolism have been identified in Mtb (cf. ~50 for the
similarly sized Escherichia coli genome). The importance of
lipid metabolism in Mtb may explain the high P450 content, since
typical cellular functions for P450s in eukaryotes and other bacteria
are as mono-oxygenases for lipophilic fatty acids, polyketides,
steroids, and xenobiotics (e.g. see Ref. 2). Sterols are
rare in bacteria, but the P450 product of the Mtb Rv0764c gene (CYP51)
is highly related to eukaryotic sterol demethylases and has been shown
to catalyze demethylation of plant and eukaryotic sterols (13). The
crystal structure of the Mtb CYP51 was solved in complex with the azole
antifungal drug fluconazole (14). This structure is of biotechnological importance, given the importance of the eukaryotic CYP51s in physiology and also in view of the fact that the yeast and fungal CYP51 enzymes (lanosterol demethylases) are validated targets for the azole antifungal class of drugs of which fluconazole is a member (15). In
recent studies, the potency of these drugs as antibiotics against growth of Mtb's relative Mycobacterium smegmatis and the
related actinomycete S. coelicolor has been shown
(16).
The biochemical functions of the remaining P450 enzymes in Mtb are not
immediately obvious. This is primarily because of limited similarity to
other members of the P450 superfamily with known catalytic properties.
Most show highest similarity to other P450 enzymes in Mtb, perhaps
suggesting co-evolution as oxygenases of related, complex lipids in the
bacterium (17). We decided to focus efforts on the product of gene
Rv2276, a P450 enzyme classified as CYP121 (1). This P450
shows amino acid sequence similarity to a number of polyketide
mono-oxygenase P450s, including the structurally characterized P450
EryF (CYP107A1) from Saccharopolyspora erythraea
(18), suggesting a potential role in polyketide metabolism in Mtb. In
addition, the known high affinity of P450 EryF for the azole
antifungal class of drugs suggests that CYP121 could be a potential
drug target in the pathogen, particularly since the role of CYP51, its
validity as a drug target, and whether sterol metabolism is really a
feature of the pathogen remain to be established. Indeed, the fact that
a Our initial expression and biophysical studies have established that
the Mtb P450 CYP121 has properties typical for members of this class
(20). Whereas the physiological role remains unclear, the enzyme binds
bulky azole antifungal drugs with high affinity, and the binding
constants for these drugs are perturbed by the presence of erythromycin
and other large polyketides, suggesting that CYP121 may metabolizes
polyketides or bulky polycyclics in vivo. However, what is
most remarkable is that in comparison with Mtb CYP51, the binding of a
variety of azole antifungal drugs to Mtb CYP121 is tighter and that the
order of their Kd values correlates well with the
minimal inhibitory concentration values for these drugs for
M. smegmatis and S. coelicolor (16). Since
the drugs retain their high potency against a S. coelicolor CYP51 knockout strain, other P450 enzyme(s) must be the true
targets in this bacterium; thus, the fact that potency of azole drugs versus mycobacteria correlates with CYP121
Kd values must make this a prime drug target
candidate (20). We have previously reported crystallization of CYP121
(21), and in this paper we report the crystal structure of CYP121 to
1.06 Å. This unprecedented level of resolution for a P450 system
allows, for the first time, a truly atomic description of the oxygen
scission site.
Expression and Purification of CYP121--
The Rv2276
gene encoding CYP121 was cloned by PCR from a Mtb chromosomal DNA
library (cosmid MTCY339, obtained from Prof. Stewart Cole at the
Institut Pasteur, Paris, France) and cloned into expression plasmid
pET11a to produce clone PKM2b, as described previously (20). The CYP121
protein was produced in E. coli strain HMS174 (DE3) with
growth of the culture after
isopropyl-1-thio- Crystallization and Structural Elucidation of
CYP121--
CYP121 crystals belonging to space group P6522
(unit cell dimensions a = 77.81 Å, c = 263.82 Å) were obtained as described previously (21). A complete
native data set up to 1.06 Å could be measured on a single
flash-cooled crystal on ID14-4 at the European Synchrotron Radiation
Facility. Different sections of the crystal were exposed to
avoid excessive radiation damage due to the intense beam. Whereas these
crystals clearly diffract further, both the long cell axis of 263 Å and the rapid decay of diffraction quality hampered the collection of
higher resolution reflections. Data were processed and scaled with the
HKL package programs DENZO and SCALEPACK (22). Despite the availability
of several P450 structures, the crystal structure could not be solved
using molecular replacement and was solved using a Multiple Isomorphous
Replacement with Anomalous Scattering (MIRAS) approach instead.
Data were collected on station 14-1 at Synchrotron Radiation
Source for two independent heavy metal derivatives. Initial
heavy atom sites were found using the program RSPS (23) and used for
detection of minor sites by difference Fourier analysis. Heavy atom
sites were refined using the program MLPHARE (24), and the weak
anomalous signal of the iron was included in the final phase
calculations. Data collection and phasing statistics are given in Table
I. The program ARP-WARP (25) was used to
build an initial model in the solvent flattened maps. This initial
model was manually adjusted using TURBO-FRODO (26) and refined using
REFMAC5 (27). The structure of the uncomplexed enzyme was refined with
anisotropic B-factors for all atoms including a riding
hydrogen atom model. With successive rounds of refinement and model
building, the entire CYP121 sequence was fitted to the electron density
map with the exception of the N-terminal three residues. Multiple
conformations were observed for 33 of the 395 residues defined. The
final R-factor was 0.132 (R-free = 0.153)
for the model consisting of the protein, 800 water molecules, and 3 sulfates per asymmetric unit. The enzyme was complexed with hydrophilic
compounds by soaking in the mother liquor (2.3 M ammonium
sulfate, pH 6.5) supplemented with a large excess of the ligand. In
order to obtain data on complexes with hydrophobic compounds, the
crystals were placed in 30% polyethylene glycol 8000, 0.2 M ammonium sulfate, pH 6.5, supplemented with the compound
of interest. Only the hydrophilic iodopyrazole proved to bind in the
active site, and data were collected for complexed crystals to 1.8 Å.
Final refinement statistics for the two crystal structures are given in
Table II.
General Structural Features--
Globally, the CYP121 structure
resembles a triagonal prism, as observed for all other P450 structures
to date. As with other P450s, there are two distinct structural
"domains," with the heme sandwiched between a smaller Overall Enzyme Fold--
Despite the elucidation of several P450
crystal structures, new cytochrome P450 models often reveal distinct
structural features that are unpredictable from primary sequence
alignments. Given the relative low homology (25.3% identity over 396 amino acids) with P450 EryF (CYP107A1; a macrolide
monooxygenase) and the unsuccessful molecular replacement, the high
three-dimensional similarity of the substrate-free CYP121 with that of
substrate/inhibitor-bound CYP107A1 comes as a surprise (28, 29).
Notwithstanding this high similarity in fold (Z score 42.3, root mean
square deviation 2.6 Å for 379 C-
Upon comparison of CYP121 with CYP107A1 from N to C terminus, the first
region that is drastically different is that immediately following the
conserved B helix (Fig. 3). Starting at
residue Met62, the polypeptide chain proceeds first with a
310 helix and then with an extended loop forming the dome
of the active site relative to the heme plane. This loop proceeds into
a second 310 helix that is immediately followed by helices
B' and C. The corresponding polypeptide region in CYP107A1 is longer
and bears little or no resemblance, having no regular 310
helical structure, whereas helix B' is separated from C by several
residues. The C helix in CYP121 is immediately adjacent to helix B' and
makes a different angle with other conserved structural elements. It is
separated from the short D helix by a flexible region that precedes
Glu110. From this position onward, both polypeptide chains
again assume very similar positions. After helix D and E, the helices F
and G are slightly reoriented with respect to the remainder of the structure, and the FG loop that forms a part of the active site dome is
bent inward with respect to the CYP107A1 structure. Whereas the CYP121
helix H still occupies a distinct conformation, as does the beginning
of helix I, the polypeptide chain trace is once again highly similar to
the main part of helix I and remains so for the rest of the structure.
Two minor exceptions are the loop following the K helix and the
C-terminal region, which are both slightly reoriented and form part of
the dome of the active site. These reorganizations in the secondary
elements and loops lining the active site cavity result in a slightly
increased volume for the CYP121 active site of 1337 Å compared with
1115 Å for the substrate-bound CYP107A1. Whereas in CYP121 the main
part of the active site is more compressed due to the closer packing of
loops forming the dome of the active site, a significant part of the
cavity extends between helices F and G (Fig. 1), accounting for the
increased volume in CYP121. As has been observed for the majority of
P450 crystal structures (with the notable exceptions of Mtb CYP51 and
Bacillus megaterium P450 BM3) (14, 30), the active
site is apparently inaccessible from the surface in this substrate-free from of the enzyme. This obviously raises the question of how the putative substrate reaches the active site. As has been
proposed for other P450s, we envisage the CYP121 structure as
oscillating between open and closed conformations whereby the "breathing motions" of the protein, specifically movements of the
secondary elements and loops lining the active site, allow for
transient exposure of gaps allowing substrate access. It is especially
interesting that the active site seems to protrude in between helices F
and G, allowing small movements in the relative positions of these
helices to open an active site channel (Fig. 1). Similar motions in
helices F/G and the interconnecting loop region have been demonstrated
to occur for the CYP119 monooxygenase from Sulfolobus
solfataricus (31).
P450 Heme Binding in Atomic Detail--
As in other P450 enzymes,
a cysteine (Cys345) provides the proximal axial ligand to
the heme iron, with a well defined water molecule as the distal axial
ligand. The Fe-S distance is 2.30 Å, whereas the Fe-O distance is 2.21 Å, both values being highly similar to those in other P450 structures obtained.
An unusual aspect of the CYP121 structure is the fact that the heme is
clearly bound in one of two distinct conformations, related by a 180°
rotation through an axis of symmetry across the
Ch
One of the four pyrrole rings of the heme is distorted out-of-plane by
the close interaction with the side chain of Pro346. The
pyrrole is kinked toward the distal face by an angle of ~30° (Fig.
4b). Pro346 immediately follows the cysteinate
heme ligand (Cys345), and this Cys-Pro motif is found in a
number of other cytochrome P450 enzymes, with several of the Mtb P450s
having this feature (Fig. 2). This structural perturbation to heme
planarity may therefore well be a feature common in other P450 enzymes
but previously unrecognized due to the absence of the Cys-Pro motif in
P450 structures solved to date. This structural element could indeed be
essential to the specific electronic properties of the heme in these
oxidases. Recently, we have demonstrated in the P450 BM3 system that a
phylogenetically conserved Phe residue (Phe393 located 7 amino acids before the cysteine ligand) that also stacks with both heme
plane and packs with the cysteinate ligand exerts a large effect on the
heme electronic properties (Fig. 2) (35). It is therefore highly likely
that this proline residue, being closer to both heme and cysteinate
ligand than even the phenylalanine (Phe338 in CYP121), has
a considerable impact on the heme catalytic and thermodynamic properties.
The Oxygen Scission Site to Atomic Detail--
The water ligand to
the heme in the resting state of the enzyme is hydrogen bonding to
Ser237. This residue replaces the more general threonine
residue found in cytochromes P450, which has been implicated in oxygen
binding and/or proton delivery (36, 37). In turn, Ser237
hydrogen-bonds to the carbonyl backbone of Ala233 and to
the side chain of Arg386 (Figs. 4b and
5). Whereas a distinctive deviation of
conventional
In the last stages of refinement, additional density became apparent
immediately adjacent to the heme water ligand. The shape of this
density is nearly spherical, and the center is located 1.2 Å from the
sixth ligand (Fig. 4b). Only two models seem to fit this
density equally well: either the sixth ligand occupies two distinct and
mutually exclusive conformations, or a diatomic species with ~1.2-Å
bond length (e.g. dioxygen) is bound to a proportion of the
heme groups in the crystal, effectively superposing the densities of
water ligation and dioxygen ligation. The presence of hydrogen peroxide
can be excluded on the basis that the bond length of the observed
species is significantly shorter than that of hydrogen peroxide (1.5 Å). The immediate environment of the additional density does not,
however, correlate with the first model (i.e. two distinct
water conformations), since there are no significantly stabilizing
structural elements present to explain the fact that the ligating water
molecule would preferentially bind outside, but close to, the ligation
sphere of the iron. Therefore, we favor the second model
(i.e. bound dioxygen), with an angle of nearly 120°
between dioxygen and heme plane highly similar to other dioxygen-heme
structures (e.g. see Ref. 40). However, it is unprecedented
that dioxygen binds to ferric heme, and the possibility exists that the
crystal might have partially reduced in the intense beam. However,
during data collection, several parts of the crystal were exposed each
for less than 10 s, minimizing the chance of significant reduction
due to repetitively exposing fresh, fully oxidized parts of the
crystal. Additionally, several data collections at medium resolution
involving significantly longer exposures and higher irradiation doses
in similar conditions did not reveal any additional density. Second,
the putative bond length for the dioxygen-ferric ion is 2.2 Å, which
is highly similar to the water-ferric ion distances but in significant
deviation for dioxygen-ferrous ion distances of 1.8 Å. Therefore, we
conclude that P450 enzymes might weakly bind dioxygen in the ferric
form, a consequence of the electron rich character conferred upon the heme by the cysteinate ligation.
The putative dioxygen species forms an ideal model for analyzing the
initial steps of the catalytic cycle, during which both electrons and
protons need to be consecutively passed onto the ligand to break the
dioxygen bond and create the highly reactive oxidizing intermediate.
Upon initial reduction of the heme, the dioxygen molecule can either
accept a proton from proximal water molecules or from
Ser237. However, we assume that the active site will be
devoid of water molecules in the vicinity of the heme plane when
complexed with substrate. Ser237, in turn, can easily
receive a proton from Arg386. This residue contacts a water
molecule buried in the interior of the protein. At this point, there
are several other residues contacting this water molecule that could
provide a proton. Most notable is Ser279, which occupies
two distinct conformations in the CYP121 structure. Each of these
conformations is in contact with a different series of residues and
buried water molecules that eventually lead up to the surface of the
molecule. Thus, it appears plausible that there may be a bifurcated
proton delivery pathway from the P450s surface, with pathways
converging at Ser279. This would have the potential to
transport one proton at a time through each individual path (Fig.
6).
Structure of the Substrate Binding Site--
The active site
cavity of 1350 Å3 is filled with water molecules and is
remarkably rigid. Whereas a significant proportion of both
surface-exposed and interior residues exhibit multiple conformations,
all of the residues involved in lining the active site adopt a single
conformation. The dome of the active site chamber is 12 Å above the
heme plane, being most open above pyrrole ring D (main conformation).
The heme is 62 Å2 solvent-exposed with access to pyrroles
B and C limited by residues from the I helix. The majority of residues
lining the active site are hydrophobic with the notable exceptions of
Arg386 and Gln385. Whereas several antifungal
agents bind tightly to CYP121 in solution (20), we have been unable to
date to obtain crystallographic complexes with these compounds by
either soaking native crystals or conducting co-crystallization trials.
This is highly likely due to the extreme insolubility of the polycyclic
azoles in the ammonium sulfate mother liquor but could also be due to
restricted access of the azoles to the heme due to obstruction by
Arg386. The single azole compound that was proven to bind
into the active site cavity by soaking the crystals was iodopyrazole.
This clearly demonstrates the fact that, even in the tight crystal
packing, breathing motions must allow transient access from the
outside into the otherwise inaccessible active site cavity.
Surprisingly, the pyrazole compound does not ligate to the heme iron
but binds instead in the small channel region between helices F and G. The pyrazole plane stacks at ~4 Å with Trp182 on one
side and Phe168 on the other side (Fig.
7, a and b). The
bulky iodide atom points toward the active site cavity and is buried
between the hydrophobic side chains of Phe168,
Thr229, and Ala233. A similar unexpected
observation was made upon complexation with 4-phenylimidazole. In this
case, the compound bound to the surface of the protein instead of
acting as the sixth heme ligand. In spectral titrations, 4-phenyl
imidazole was shown to bind to the heme iron, giving a typical (and
complete) shift of the Soret maximum to ~423 nm. This clearly
demonstrates that CYP121 can adopt conformations in solution distinct
from that observed in the crystal and that these solution conformations
are compatible with ligand binding to the iron.
CYP121 is similar in overall fold to other structurally
characterized P450s. It contains unique structural elements involved in
lining the active site. The heme binding and oxygen scission sites are
particularly interesting, since these reveal several new features that
could be general to P450s. First, the heme cofactor is bound in two
distinct, mutually exclusive conformations. Furthermore, it is
contorted due to close interaction with the
Cys345-Pro346 motif, a common structural
element in Mtb P450s and present also in other prokaryotic and
eukaryotic forms. A series of hydrogen-bonded amino acid and water
molecules define two potential proton delivery pathways, and these
apparently converge at Ser278, which has distinct
conformations in the CYP121 structure. Most interestingly, the oxygen
scission site is markedly different from other bacterial P450s, and,
while resembling the eukaryotic CYP2C5 structure, it contains a unique
arginine residue (Arg386). The atomic resolution obtained
has allowed visualization of a species (probably dioxygen) weakly bound
to the ferric heme iron.
Future studies on CYP121 will involve establishing its substrate
selectivity through screening for activity against mycobacterial lipid
extracts (and possibly infected tissue samples) and use of rational
mutagenesis to define roles of key amino acids identified from this
structural study in determining the biophysical and catalytic
properties of the P450. A further priority is the solution of an
azole-ligated CYP121 structure, since this may provide critical information required to facilitate de novo design of novel
and more highly specific azole drugs that are potent
anti-mycobacterials but that lack the cross-reactivity with human
isoforms. Through such an approach, anti-P450 drugs may prove useful
new agents in the war against multidrug-resistant strains of M. tuberculosis, which the World Health Organization describes as
having the potential to cause a "global catastrophe."
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
CYP51 strain of S. coelicolor is nonlethal
(16, 19) and that the strain exhibits no significant alteration in
sensitivity to azole antifungal drugs (16) confirms that CYP51 is not
the target in this actinomycete.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
-D-galactopyranoside induction
performed at low temperature (18 °C) to promote production of
soluble enzyme. The cells were broken using a French press and
sonication, and CYP121 was purified to homogeneity by ammonium sulfate
fractionation (30-70% fraction retained), followed by successive
column chromatography steps on phenyl-Sepharose, Q-Sepharose, and
hydroxyapatite resins, as described (20).
Data collection and phasing statistics for CYP121
Final refinement statistics for the CYP121 crystal structures
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
sheet-rich module and a larger helix-rich domain (Fig.
1). The P450s have relatively few
definitive amino acid motifs, and evolutionarily distinct P450s may
exhibit as little as 15% amino acid identity. However, the cysteine
ligand to the heme iron and the region immediately surrounding this
amino acid (the "heme-binding" motif) at the C-terminal section of
the P450 are strongly conserved (Fig.
2).
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Fig. 1.
Overall fold of Mtb CYP121. The atomic
structure of CYP121 is composed mainly of helices. The heme (in
space fill) is sandwiched between the two major
domains of the structure: an
helix-rich domain
(dark gray) and the smaller
sheet-rich domain
(light gray). The large water-filled active site
cavity of the enzyme is shown as a transparent surface. An
arrow indicates the possible site of substrate entry between
helices F and G.
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Fig. 2.
The heme binding motif in M. tuberculosis CYP121 and other cytochromes P450. The
heme-binding motif region for Mtb CYP121 (10 amino acids either side of
the cysteinate ligand to the heme iron) is shown aligned
versus those from prokaryotic and eukaryotic P450s using the
ClustalW program (EBI). Four amino acids are conserved throughout,
including the cysteine (Cys345 in CYP121), and are
highlighted in white against a black background.
These identical residues are also indicated by an asterisk
at the base of the alignment, and other positions where the amino acid
class is retained in each P450 are indicated by dots.
Highlighted in gray are those individual amino acids present
in 40% or more of the P450s at the respective positions. P450 enzymes
are indicated by their formal classification "CYP" numbers (1) and
by their species of origin. Mtu, M. tuberculosis;
Ser, S. erythraea; Bsu, B. subtilis; Sso, S. solfataricus;
Atu, Agrobacterium tumefaciens; Bme,
B. megaterium.
atoms), several distinct
features can be observed in CYP121 for polypeptide stretches lining the
active site cavity.
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Fig. 3.
Structural comparison of CYP121 with CYP107A1
(P450 EryF) from S. erythraea.
Shown is a stereo view of the overlay of Mtb CYP121 (dark
gray) with S. erythraea CYP107A1
(light gray). Loops and secondary elements lining
the active site cavity are clearly more divergent than the remainder of
the structure.
-Fe-CH
atoms of the molecule. In short,
the heme is bound either in the "a" or "b" conformer, a
phenomenon well known for other heme proteins (e.g.
cytochrome b5) (e.g. see Ref. 32).
This mixture of conformers does not otherwise impact on the overall
fold of the enzyme. One of the conformers predominates, and the
relative occupancy is refined to a 70:30 mixture of the species (Fig.
4a). In cytochrome
b5, there is dynamic exchange of the heme, such
that the proportions of the a- and b-type conformers can be perturbed
considerably by temperature (e.g. see Ref. 33). The
situation is extremely unlikely to be the same in the cytochromes P450,
where the heme is bound deep in the protein (not superficially as in
b5) and requires protein denaturation to be
released. Whereas insertion of heme and modified hemes into P450 has
been demonstrated, this was achieved only very slowly by denaturation
of the P450 and refolding by gradual removal of denaturant after the
addition of the exogenous heme (e.g. see Ref. 34). The
situation is highly unlikely to occur in vivo, and we infer
that the heme-binding step in CYP121 (and other P450s) occurs at a
relatively early stage in the protein folding process, and the position
of the heme as bound commits the P450 to this conformation
throughout its lifetime in vivo.
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Fig. 4.
A, different heme conformations in
CYP121. The main conformation of the heme B group of CYP121
is shown, surrounded with 1.5 (blue) and 4 (purple) contoured 2FoFc electron density. The
additional FoFc density (contoured at 3
in green) and
the lack of strong 2FoFc density of the vinyl atoms of the main
conformation clearly indicate a second conformer. The two different
conformers are related by the indicated 180° rotation about the heme
plane. b, stereo view of the active site of CYP121. Residues
surrounding the heme are shown in addition to the putative oxygen
ligand. The corresponding 2FoFc electron density is contoured at 1.5
in blue. The heme is notably kinked due to the
close interaction with Pro346. The FoFc density is
contoured at 3
in green and reveals a second atom close
to the position of the sixth water ligand.
-helix hydrogen bonding patterns in the region of the
water ligand-contacting residues can be seen in all P450s of known
structure, with the exception of CYP2C5 (9), the I helix in CYP121 is
devoid of any significant alteration of hydrogen bonding patterns and
resembles the eukaryotic CYP2C5 in this region. The presence of the
bulky and positively charged residue Arg386 immediately
adjacent to the oxygen scission site is unique to CYP121 and makes it
one of the key residues in this enzyme (Fig. 4b). The side
chain of Arg386 is wedged in between Ile236 and
Phe280, whereas hydrogen bonding to Ser237,
Gln385, and two water molecules in the active site cavity.
The close proximity of the arginine and serine residues may define a
binding site for a negatively charged functional group, possibly a
carboxylate, as observed previously for e.g. P450 BM3
(CYP102A1) with its fatty acid substrates (38, 39).
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Fig. 5.
Hydrogen bonding pattern in the CYP121 I
helix. Residues of the CYP121 I helix close to the heme group are
shown in gray ball and
stick mode. All bacterial cytochromes P450 show a severe
disruption of the hydrogen bonding pattern between Ala233
and Ser237 (CYP121 numbering). Remarkably, this distortion
is not seen in CYP121 in the vicinity of the relevant amino acid
(Ser237), similar to the mammalian CYP2C5 structure
(9).
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Fig. 6.
Putative proton relays in CYP121. Shown
is a view of two putative proton pathways leading from the solvent into
the active site. Both pathways are mainly made by buried water
molecules in addition to a single amino acid (Thr244 and
Glu310, respectively) in the two pathways. These pathways
merge at Ser279 that occupies two distinct conformations,
each contacting a water molecule that is hydrogen-bonded to
Arg386. The latter residue is proposed to donate protons
either directly or via Ser237 (not shown) to the oxygen
intermediates.
View larger version (43K):
[in a new window]
Fig. 7.
The iodopyrazole complex of CYP121.
A, key residues of the CYP121 active site in complex with
iodopyrazole. Residues are depicted in ball and
stick mode, color-coded according to
property as follows. Blue, basic; purple,
aromatic; yellow, hydrophobic; green,
hydrophilic. The iodopyrazole (I-Pyr) ligand is shown in
pink, and the heme group is shown in red. Part of
the I helix is shown as a blue ribbon.
B, overall view of the active site in complex with
iodopyrazole. The active site cavity is depicted as a gray
transparent surface with key residues shown as
sticks. Secondary structure elements are
color-coded according to the legend to Fig. 1.
CONCLUSIONS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to Prof. Stewart Cole (Institut Pasteur, Paris) for provision of cosmid DNA and for helpful scientific discussions. We gratefully acknowledge beamtime received at Synchrotron Radiation Source (Daresbury, UK), European Synchrotron Radiation Facility (Grenoble, France), and EMBL X11 beamline at the DORIS storage ring, Deutsches Electronen-Synchrotron (Hamburg, Germany).
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FOOTNOTES |
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* This work was supported by the Biotechnology and Biological Sciences Research Council, Engineering and Physical Sciences Research Council, and European Union (Project "X-TB"). Access at EMBL Hamburg was supported by the European Community-Access to Research Infrastructure Action of the Improving Human Potential Program to the EMBL Hamburg Outstation (Contract HPRI-CT-1999-00017).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1N40 (RCSB017490) and 1N4G (RCSB017506)) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Member of the TB Structural Genomics Consortium.
¶ To whom correspondence should be addressed. Tel.: 44-116-252-3484; Fax: 44-116-252-3473; E-mail: dl37@le.ac.uk.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M209928200
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ABBREVIATIONS |
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The abbreviation used is: P450, cytochrome P450.
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REFERENCES |
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