From the Department of Molecular Biology and
Biochemistry, the Department of Physiology and Biophysics, and the
Program in Macromolecular Structure, University of California, Irvine,
California 92697-3900, the § Institut fur Technische
Biochemie, Universität Stuttgart, Allmandring 31, D-70569
Stuttgart, Germany, and the ¶ Institute of Microbiology and
Genetics, University of Göttingen, Grisebachstrasse 8, 37077 Göttingen, Germany
Received for publication, July 2, 2002, and in revised form, September 4, 2002
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ABSTRACT |
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The second structure of a thermophile cytochrome
P450, CYP175A1 from the thermophilic bacterium Thermus
thermophilus HB27, has been solved to 1.8-Å resolution. The
overall P450 structure remains conserved despite the low sequence
identity between the various P450s. The CYP175A1 structure lacks the
large aromatic network found in the only other thermostable P450,
CYP119, thought to contribute to thermal stability. The primary
difference between CYP175A1 and its mesophile counterparts is the
investment of charged residues into salt-link networks at the expense
of single charge-charge interactions. Additional factors involved in
the thermal stability increase are a decrease in the overall size,
especially shortening of loops and connecting regions, and a
decrease in the number of labile residues such as Asn, Gln, and Cys.
Cytochromes P450
(P450s)1 catalyze the
monooxygenation of a vast array of organic compounds in the
following reaction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
P450s are ubiquitous enzymes essential for steroid biosynthesis,
catabolism of drugs, utilization of carbon compounds as an energy
source in bacteria, and in the production of various macrolide
antibiotics. P450s are found throughout the biosphere and various
genome projects have revealed a remarkable and unexpectedly large
number of P450s in a variety of organisms. For example, humans have
~180, Caenorhabditis elegans 81, Drosophila 90 (83 functional 7 pseudogenes) (1), and Arabidopsis 273 (drnelson.utmem.edu/CytochromeP450.html). Several bacteria also have
P450s, but normally bacteria produce only one or at most a few P450s.
Another surprising occurrence of a P450 was demonstrated in 1996 when
the first P450 from a hyperthermophile, CYP119, was discovered (2).
CYP119 was identified in the acidothermophilic archaeon
Sulfolobus solfataricus. This was not only the first thermostable P450 to be discovered but also the first demonstration of
an archaeon P450. Subsequent cloning, expression, and purification of
CYP119 led to the finding that CYP119 exhibits a melting temperature of
90 °C compared with
55 °C for mesophilic bacterial P450cam (3). This was soon followed by our determination of the CYP119 crystal
structure (4), followed by an independent structure determination by
Park et al. (5). The enhanced thermal stability of CYP119
was attributed to an unusually large clustering of aromatic side chains
not found in all other known P450 structures (3-6). The ever
increasing data base of known structures of proteins from both
mesophiles and their thermophilic counterparts reveals that aromatic
clustering does, indeed, correlate with enhanced thermal stability, but
that this is only one of several possible mechanisms for enhancing
thermal stability (7). Whether or not aromatic clustering is a
universal feature of thermostable P450s will require additional crystal
structures. Fortunately, the second P450 from a thermophile was
recently discovered, CYP175A1 from Thermus thermophilus.
The present report describes cloning, expression, purification,
crystallization, and structure determination of CYP175A1, a cytochrome
P450 from the thermophilic bacterium T. thermophilus HB27.
The 44-kDa protein is soluble and displays an absorption spectra in the
reduced, oxidized, and carbonyl adduct analogous to those of other P450
enzymes. We also demonstrate that this enzyme exhibits thermostability
with a melting temperature 30 °C higher than that of P450cam. To
understand whether CYP175A1 and CYP119 share common structural features
related to thermal stability, the two crystal structures are compared.
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MATERIALS AND METHODS |
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Gene Extraction, Expression, and Purification of P450 (CYP175A1) from T. thermophilus HB27(Tt)-- Plasmid DNA containing CYP175A1 was a kind gift from Dr. Hans-Joachim Fritz (University of Göttingen, Göttingen, Germany). Polymerase chain reaction (PCR) was used to engineer EcoRI and PstI restriction sites into the CYP175A1 gene. The oligonucleotide primers used for amplification of the new CYP175A1 gene from the original plasmid were the following: primer A, 5'-CCGGAATTCATGAAGCGCCTTTCCCTGAGG; primer B, 5-CCAATGCATTGGTTCTGCAGTCAGGCCCGCACCTCCTCCCTAGG.
The new restriction sites, EcoRI and PstI, are underlined. The reaction mixture consisted of template DNA (100 ng), 2.5 units of Pfu DNA polymerase (Stratagene), 5 µl reaction buffer, 5 µl of Me2SO, 0.4 µM amounts of each oligonucleotide, 400 µM dNTPs, and H2O up to 50 µl. PCR cycling parameters were: 95 °C for 1 min; 30 cycles of 95 °C for 1 min, 53 °C for 1 min 30 s, and 68 °C for 1 min 30 s; and 68 °C for 4 min. The CYP175A1 gene sequence was confirmed by DNA sequencing.
After restriction enzyme digest of the gel-purified PCR product, the
CYP175A1 gene was cloned into the EcoRI/PstI
sites of the pKK233-3 plasmid (Amersham Biosciences). BL21
(DE3) Codon Plus Escherichia coli (Stratagene) were
transformed with a pKK233-3 plasmid containing the gene for CYP175A1
from Tt. Single colonies were selected for overnight growth
in 5-ml cultures, which were used to inoculate cultures (1 liter in
each 2.8-liter flask). At an A600 nm of 1.0, 500 µg/liter isopropyl -D-thiopyranogalactoside (United States Biochemical Corp.) was used to induce the expression of
CYP175A1. The conditions for cell growth were 2× YT (16 g of tryptone,
10 g of yeast extract, and 5 g of NaCl per liter), 0.1 g/liter ampicillin (Sigma), and 50 mg/liter chloramphenicol
(Sigma) at 37 °C, with shaking at 220 rpm for the starter cultures
and slow stirring (80-100 rpm) at 30 °C after induction.
Forty-eight hours after induction, the cells were harvested and the
cell paste was stored at
70 °C.
Cells were lysed in a French pressure cell in 50 mM
potassium Pi buffer, pH 7.5, 0.1% Emulgen (Japan), and 1 mM phenylmethylsulfonyl fluoride (Sigma). Cell debris was
removed by centrifugation, and the cell-free extract was brought to
35% ammonium sulfate concentration by adding solid ammonium sulfate.
Precipitated proteins were removed by centrifugation, and the
supernatant was brought to a concentration of 50% ammonium sulfate.
The resulting precipitate was centrifuged, resuspended in minimal
volume, and dialyzed against 50 mM potassium Pi
buffer, pH 7.5, overnight (3 times buffer exchange). The dialyzed supernatant was passed over a DE52 column. Contaminant proteins stuck
on the column, whereas CYP175A1 passed through because of its limited
affinity for DE52 resin. The flow-through containing CYP175A1 had an
absorbance ratio
(A418 nm/A280 nm) of
1.3. The protein was concentrated further and diluted in 50 mM Tris, pH 7.5. The protein was then loaded on to a
hydroxyapatite (Bio-Rad) column (2.5 × 20.0 cm). The protein was
washed with two to three column volumes of 50 mM Tris, pH
7.5, followed by two to three column volumes of 50 mM
potassium Pi buffer, pH 7.4. The protein was eluted with a
linear gradient of potassium Pi buffer, pH 7.4, from 50 to
300 mM over 10 column volumes at a flow rate of 0.5 ml/min.
Fractions with an absorbance ratio
(A418 nm/A280 nm) above
1.55 were pooled, concentrated, and exchanged into 50 mM Tris, pH 7.4, on a PD10 column (Amersham Biosciences). The protein was
then concentrated to 40 mg/ml and stored at
20 °C. A visible extinction coefficient of
418 nm = 104 mM
1 was determined by extracting and
measuring the total amount of heme present in a pure sample using the
pyridine hemochromagen assay (8) and compared with the spectra obtained
for the intact protein. Purity was measured by the appearance of a
single band on a Coomassie Blue-stained SDS-PAGE gel and had an
absorbance ratio
(A418 nm/A280 nm) of
1.60.
Melting Temperatures--
The thermal melting temperatures were
measured by recording the absorbance of the heme Soret peak at a linear
temperature rate of 1 °C/min on a Cary 3E UV-visible
spectrophotometer equipped with a temperature controller. All P450s
were irreversibly denatured. P450BM-3 consists of the heme domain only
(residues 1-455). The absorbance increase at the Soret peak was
followed for CYP119 and CYP175A1, whereas the absorbance decrease was
followed for all other P450s. All measurements were carried out in the
absence of substrates in 50 mM Tris, pH 7.4, except
CYP175A1, which was carried out in 50 mM potassium
Pi buffer, pH 7.4. Protein concentrations ranged from 6 to
12 µM and corresponded to an absorbance from 1.0 to 0.8 at the Soret peak. The Soret peak was at 418 nm for P450BM-3, P450eryF,
P450cam, and CYP175A1, whereas the CYP119 Soret peak was at 414 nm. The
percentage denatured was calculated by the following equation depending
on whether the appearance or disappearance of the Soret peak was
measured (percentage of absorbance): appearance of the Soret peak = 1 (peak absorbance 100% natured
observed
absorbance); disappearance of the Soret peak = (peak
absorbance
observed absorbance).
Crystallization and Heavy Atom Derivative--
Crystals
belonging to the monoclinic space group P21 with unit cell
dimensions a = 64.54 Å, b = 90.70 Å,
c = 74.52 Å, = 99.67o were grown
by sitting-drop vapor diffusion at room temperature over a reservoir of
0.4 M sodium/potassium tartrate, 100 mM sodium citrate/phosphate, pH 4.2, and 1 mM 1-phenylimidazole or
4-phenylimidazole. Three µl of 20 mg/ml CYP175A1 was mixed with an
equal volume of reservoir solution and used to set up sitting drops on
siliconized glass cover slips. To prepare the heavy atom derivative,
crystals were transferred into artificial precipitant solution
containing saturating amounts of platinum diamino dichloride (Pfaltz
and Bauer) and allowed to soak for 36 h prior to data collection. Cryoprotectant soaking consisted of a four-step transfer to artificial precipitant solution (0.2 M
Na+/K+
tartrate, 50 mM Na+ citrate/phosphate, pH 4.2, 1 mM 1-phenyl- or 4-phenylimidazole) with increasing
concentrations of ethylene glycol from 5 to 30%. For crystals of the
platinum derivative, saturating amounts of heavy atom were also added
to the cryoprotectant solutions. A Matthews coefficient (9) calculation
indicated that there were most likely two molecules per
asymmetric unit.
Data Collection-- All data were collected using crystals flash-frozen in a stream of liquid nitrogen. Initial data sets were collected in-house on an R-Axis IV image plate equipped with a Rigaku rotating copper anode x-ray generator and Osmic focusing optics. Collection of in-house native data was optimized to maximize the anomalous dispersion signal from the heme iron by collection of a highly redundant data set resulting in greater than 1 million observations of 61,926 unique reflections to 1.95 Å (Table I). A high resolution data set (50-1.80 Å) used for the final refinement was collected at the Advanced Light Source, beamline 5.0.2 with an Area Detector Systems Co. Quantum-4 CCD detector. Optimization of data collection was guided by the STRATEGY function of MOSFLM (10). All data collected were reduced using DENZO and SCALEPACK (11).
Phase Combination-- Phases were derived from molecular replacement, a platinum heavy atom derivative, and anomalous scattering from the heme iron. Molecular replacement was carried out using a combination of AMoRe (12) and BEAST (13) using data from 20 to 5 Å and a polyalanine search model of P450BM-3 (PDB accession no. 1FAG) with residues 1-15, 223-232, 293-303, and 337-350 deleted from the model. The rotation solution found in BEAST was used in AMoRe to find the translation solution for the first molecule. This provided a slightly higher signal to noise ratio than using the rotation solution generated in AMoRe alone, because the rotation solution generated by finer rotational step size in BEAST was closer to the final refined orientation. AMoRe was used to find both the rotation and translation solution for the second molecule. The log likelihood gain score for the first rotation in BEAST was 20.07 with a mean of 7.50. The rigid body fitting routine in AMoRe was applied to the final solutions and had a Patterson correlation coefficient score of 0.451 and a crystallographic R-factor of 52.7%. SOLVE (14) was used to interpret both the isomorphous difference Patterson from the platinum derivative and the native iron anomalous difference Patterson. SOLVE was able to locate two platinum sites and two iron sites in the asymmetric unit, in agreement with the predicted two molecules per asymmetric unit. The final Z score for the solution was 14.08 with an overall figure of merit of 0.41. Data from 12 to 2.8 Å were used for phase combination calculations using SIGMAA (15). The combined phases were further improved by density modification using DM (16), including solvent flattening, histogram matching, and 2-fold noncrystallographic symmetry averaging to provide an interpretable electron density map.
Model Building and Refinement--
Electron density map fitting
beginning with the truncated P450BM-3 model was carried out with the
graphic modeling package O (17). Several sections of the electron
density map, especially the core near the heme group, clearly showed
the identity of CYP175A1 side chains. Regions of the molecule that
lacked clear backbone electron density were deleted prior to refinement
in CNS (version 1.1) (18). Several rounds of model building, followed
by refinements with simulated annealing (at 3000 K) and minimization,
continued until the R-factor was ~30%, and then isotropic
B-factor refinement was introduced. In addition, simulated
annealing omit maps were generated in CNS to assist in model building.
When the R-factor reached ~25%, water molecules were
automatically picked at the 3.5 level in an
Fo
Fc difference map
using CNS and manually added in for the last few rounds of refinements.
The final stage of refinement was done with the program REFMAC5 (19).
The final model consists of all the residues in molecule A except the
last four at the C terminus, whereas molecule B is missing residues 11-15 as well as the last four residues. The final R-factor
is 18.2% (R-free 22.8%), and none of the main-chain
torsion angles were in the disallowed regions of a Ramachandran plot.
Relevant statistics are found in Table
I.
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RESULTS AND DISCUSSION |
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Thermal Stability--
Melting curves for the two thermophilic
P450s, CYP175A1 and CYP119, and three mesophilic P450s are shown in
Fig. 1. The three mesophilic P450s
exhibit Tm values ranging from 47 to 61 °C,
whereas CYP175A1 and CYP119 have Tm values of 88 and 91 °C, respectively. These values compare favorably for those
obtained using the more rigorous technique of differential scanning
calorimetry. In these experiments, the Tm for
CYP119 was found to be 91 °C, whereas that for P450cam was 54 °C
(3, 20).
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Structure Solution-- Surprisingly, the highest sequence identity to CYP175A1 is P450BM-3 at 26%. Based on this sequence identity, we chose P450BM-3 as the search model for molecular replacement. The solution to the phase problem for CYP175A1 by molecular replacement represents the only case where a known P450 structure could be used to solve both the rotation and translation functions. Previously, in the CYP119 structure determination (4), the rotation solution for only one of the two molecules in the asymmetric unit was obtained with a modified version of BRUTE (13, 21). The rotation parameters for the second molecule could not be found using routine molecular replacement programs and were generated by application of the self-rotation peak. The present structure determination demonstrates the ability to use preexisting P450 structures as molecular replacement models despite low sequence identity found throughout the P450 superfamily. Although the molecular replacement phases generated an interpretable electron density map, the map was significantly improved by incorporating phases derived from the platinum derivative and iron sites.
Overall Structure--
CYP175A1 exhibits the typical prism-like
P450-fold (Fig. 2) composed of 17 -helices and 11
-strands, corresponding to four
segments. At
the core of the molecule is a four-helix bundle composed of helices D,
E, I, and L and two
-helices (J and K) (22). The heme prosthetic
group is embedded between the distal I and proximal L helices, with
Cys-336 serving as the fifth axial thiolate ligand to the heme iron.
The propionate side chains of the heme are tethered to the protein
through interactions with Trp-87, Arg-273, and Arg-334. The I helix
spans the length of the entire molecule and contains the highly
conserved Thr (Thr-225) residue, which is hydrogen-bonded to the
peptide backbone. This region has been postulated to be involved in a
proton shuttle network considered to be important for delivering
solvent protons required for the activation of O2 during
the catalytic cycle (23, 24). At 389 residues, CYP175A1 is comparable
in size to CYP119 (378 residues), but considerably smaller than other
known soluble bacterial P450s such as P450BM-3 (heme domain) at 455 residues.
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Crystals used in this study were prepared in the presence of
1-phenylimidazole. As a result, we expected to find this ligand in the
active site pocket coordinated to the heme iron. As shown in Fig.
3, there is clearly defined electron
density for the inhibitor with continuous density connecting the
inhibitor and heme iron atom consistent with an imidazole N atom
coordinating to the heme iron. Nevertheless, attempts to accurately
model inhibitor to the electron density failed to give good fits. In
sharp contrast, the similar inhibitor, 4-phenylimidazole, fit the
electron density very well in CYP119. It appears that the inhibitor may
either be partially occupied, thus sharing space with solvent, or the inhibitor is partially disordered. Spectral titration data do indicate
that the inhibitor does not bind as well to CYP175A1 as to CYP119. The
apparent Kd is 0.3 µM for CYP119
and
83.7 µM for CYP175A1 in the same buffer used for
crystallization. In both cases there are clearly defined spectral
shifts characteristic of forming an N-Fe bond. Although the inhibitor
binds more weakly to CYP175A1, it remains surprising that the inhibitor
structure was not more well ordered and/or fully occupied given the
large excess of 1-phenylimidazole (1 mM) used for
crystallization.
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Comparison with P450BM-3--
Because the closest homologue to
CYP175A1 is P450BM-3, a bacterial fatty acid monooxygenase, we will
next compare these two structures. The r.m.s. backbone deviation
between CYP175A1 and palmitoleic acid-bound P450BM-3 (PDB accession no.
1FAG) (25) is 1.64 Å for 304 C atoms. Table
II provides a comparison of secondary
structural elements, and Fig. 2 provides a structural picture of the
two P450s. Most of the differences between CYP175A1 and P450BM-3 are
located in the loops connecting helices. For example, P450BM-3 has a
seven-residue insertion in the loop connecting helices E and F
(residues 159-172 in P450BM-3, residues 146-152 in CYP175A1). Another
difference includes the
-5 region in P450BM-3 (residues 239-250)
compared with the loop found in CYP175A1 (residues 202-205), a
difference of eight residues. This region forms a tight loop between
the H and I helices, and resembles what was previously found in
P450nor, which does not have a redox partner (26), and CYP119. In
contrast, all other soluble bacterial P450 structures show this ill
defined
-hairpin similar to P450BM-3. Interestingly, the crystal
structure of a complex formed between the heme domain of P450BM-3 and
FMN domain has shown that this hairpin region makes direct contact with
the FMN domain (27).
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Although the redox partner for CYP175A1 remains unknown, it generally
is thought that electrostatic interactions are important in redox
partner recognition. Therefore, it is instructive to examine the
electrostatic surface potential of CYP175A1 compared with P450cam and
P450BM-3, where there is a fairly clear picture on the location of the
docking site. The electrostatic surface potential of CYP175A1, P40cam,
and P450BM-3 are shown in Fig. 4. The
arrows indicate the approximate location of the surface above the proximal Cys ligand. This region is known to be
the docking site for the P450BM-3 redox partner (27) and is generally considered the docking site in P450cam (28). The excess positive charge
in this region provides favorable electrostatic interactions with
negative charges on the redox partner. CYP175A1 also has a large
positive electrostatic potential in this region, suggesting that this,
too, may be a recognition site for redox partners. The larger positive
potential on CYP175A1 on the proximal surface CYP175A1 compared with
the other P450s is caused in part by an extra arginine, Arg-319, in
CYP175A1 with no homologue in P450cam or P450BM-3. In addition, Arg-319
occupies approximately the same position in three dimensions as Glu-286
in P450cam; hence, CYP175A1 has one less negative charge in this
region. This might provide an especially strong electrostatic docking
site for the CYP175A1 redox partner.
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Another important region is the substrate binding environment, the F
and G helices, B' helix, and neighboring loops (Fig. 5). The F and G helices and the loop
connecting these helices are known to undergo a large open/close motion
between the substrate-free and -bound forms of P450BM-3 (25). In
addition, CYP119 undergoes a rather large structural change in the F/G
region, depending on the type of ligand bound in the active site (4).
When a small inhibitor, imidazole, is bound, the C-terminal end of the F helix in CYP119 unwinds, which increases the length of the F/G loop,
thus allowing the F/G loop to dip down into the active site and
interact with the inhibitor. These two examples illustrate the
importance of the F/G region in substrate recognition and binding. It
therefore is important to note that the major portions of the F-G
region and the B' helix, known to be important in substrate binding
(29), are in nearly identical conformations in CYP175A1 and P450BM-3
(Fig. 5). The B' helix forms a portion of the lid to the substrate
access channel in P450BM-3 and makes intimate contact with the G helix.
The average r.m.s. deviation for the B' helix is 1.23 Å, reflecting
the general overlap of this structural element. The F helix in CYP175A1
(residues 150-170) is nearly identical in both size and location to
the F helix in P450BM-3 (residues 171-190). The core region of the F
helix, residues 157-167 in CYP175A1, has an average r.m.s. deviation
of 1.66 Å, despite being shifted by approximately half a turn. The G
helix is approximately two turns shorter in CYP175A1 (residues 176-198
in CYP175A1 compared with residues 197-226 in P450BM-3). Although the
G helix is shorter, residues 176-195 in CYP175A1 align very well with
residues 198-217 in P450BM-3 with an average r.m.s. deviation of 1.14 Å.
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Based on the sequence identity and close structural homology in regions associated with the active site, it seemed possible that CYP175A1 may act as a fatty acid hydroxylase similar to P450BM-3. However, we did not observe the characteristic low to high spin shift that accompanies the binding of substrates to P450s using a range of even chain, commercially available fatty acids (C10-C20) either at room temperature or temperatures up to 70 °C. In addition, fatty acid hydroxylation could not be detected using H2O2 or bovine adrenodoxin/adrenodoxin reductase as redox partners.2
A superimposition of the P450BM-3 structure with fatty acid bound
(1FAG) on to CYP175A1 provides clues on why we failed to observe fatty
acid binding (Fig. 6). The main problem
is steric crowding owing to the close approach of Gln-67, Trp-269, and
Ile-270 to the fatty acid substrate. The corresponding residues in
P450BM-3 are, respectively, Ala-74, Pro-329, and Ala-330, which are all smaller side chains, thus providing additional space for the substrate to fit into the pocket. These close contacts involve the
polar half of the fatty acid substrate nearer to the substrate entry channel, whereas the non-polar half of the substrate closer to the heme
fits well into the CYP175A1 active site pocket. In addition, CYP175A1
also is missing Arg-47 and Tyr-51, both of which help to stabilize the
fatty acid carboxyl group in P450BM-3. The corresponding residues in
CYP175A1 are, respectively, Phe-41 and Leu-45 in CYP175A1. Those
observations suggest that CYP175A1 may bind substrates considerable shorter than palmitoleic acid (16 carbons).
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Comparison with CYP119-- The only other known thermostable P450 is CYP119 from the acidothermophilic organism S. solfataricus (2) with a Tm of 91 °C (3). At 368 amino acids, CYP119 is slightly smaller than CYP175A1. The r.m.s. backbone deviation is ~1.7-1.8 Å. The overall (sequence and structure) similarity between the two thermophilic P450s is less than that between CYP175A1 and P450BM-3. Most of the increase in length of CYP175A1, relative to CYP119, resides at the N-terminal region, which typically has the lowest sequence identity between P450s. CYP175A1 has an extra A' helix, similar in conformation to a short 310 helix found in P450BM-3, which accounts for the most of the increase in length between the two thermophilic P450s. The CYP119 A helix starts at approximately residue 22 in CYP175A1. The largest structural differences between the two P450s reside in regions surrounding the active site: the F-G region, including the helices and all connecting loops, and the B' helix. As noted earlier, the F/G region was found to be highly flexible between two crystal forms of CYP119, where the C terminus of the F helix unwraps by one full turn to dip down into the active site, depending on what ligand is bound at the active site. CYP119 is unusual because it contains a long loop preceding the B' helix that enables it to adopt a position away from the active site toward the molecular surface. The B' helix in CYP175A1 is in a similar conformation to P450BM-3 and most likely forms the lid of the substrate access channel.
Structural Basis for Thermal Stability-- The enhanced thermal stability of proteins from extreme thermophiles has been attributed to a variety of global factors. Early on it was thought that amino acid composition was correlated with an increase in thermal stability (30). Later, general traffic rules of substitutions were loosely established (31-33). For example, an increase in the Arg content at the expense of Lys can aid in thermal stability because of an increase in hydrogen bonding possibilities, a decrease in chemical reactivity of the guanidino group, and resonance stabilization relative to Lys (34). Interestingly, CYP175A1 does show an increase in Arg content at 12.1% and a decrease in Lys (2.8%) relative to some of the mesophilic P450s such as P450BM-3 (Arg 4.4%, Lys 7.9%), whereas CYP119 shows a normal Arg and Lys content (7.6 and 7.1%, respectively). Another general traffic rule is the decrease in the number of polar-uncharged residues (Asn and Gln) and an increase in charged residues (Lys, His, Arg, Glu, and Asp) (34, 35). CYP175A1 shows a decrease in the polar uncharged residues (2.6%), whereas CYP119 shows only a slightly lower than normal content (6.3%) when compared with a mesophile such as P450BM-3 (9.0%). CYP175A1 also has a very large number of hydrophobic residues such as Ala and Leu (11.5 and 18.0%, respectively), but a normal hydrophobic content of 33.9%. Ala residues were first thought to increase thermal stability (31) because Ala is the best helical forming residue. However, in recent reviews based on genome sequencing projects researchers have noticed a slight decrease in the number of Ala residues (34). Another factor noted was an increase in the number of charged residues in thermophiles (35). However, the percentages of charged residues in both CYP119 and CYP175A1 and the mesophilic P450s remain fairly constant at ~30%.
As the data base grows, a common recurring theme in thermophilic proteins is the potential importance of salt bridges (36), especially salt bridge networks involving more than 2 charged residues. It therefore is not too surprising that both thermophilic P450s have a greater number of such networks compared with their mesophilic counterparts. As shown in Table III, CYP175A1 has eight salt bridge networks whereas CYP119 has 10. By comparison the bacterial P450s listed in Table III have from four to six. P450cam has six salt bridge networks, but one of these contains His and hence is expected to be weaker at neutral pH. In addition to the larger number of salt bridge networks in CYP175A1, these networks are more evenly distributed over the surface than those in P450cam (Fig. 7). Additionally, what is substantially more significant is the total fraction of salt bridges that are in networks. In the thermophilic P450s , ~60% of the total number of salt bridges are in networks, whereas, in the bacterial mesophilic P450s, the maximum is 36.8% (P450cam, Table III). Mutagenesis work on CYP119 showed that the disruption of a salt-link network between Glu-114, Arg-363, and Glu-342 lowered the thermal melting transition by ~3.8 °C (20).
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It is generally thought that salt bridges should not be stabilizing because the energetic cost of desolvation cannot be fully compensated by electrostatic interactions between side chains and/or between the salt bridge side chains and the rest of the protein. An obvious difference with proteins is that the charged side chains are fixed to a polypeptide backbone. Thus, the folding of the polypeptide chain, driven by hydrophobic factors, "forces" charged groups close to one another on the surface. Nevertheless, such "forced" interactions might well oppose folding because there remains the problem of balancing unfavorable desolvation with favorable electrostatic interactions. One property of salt bridges that, perhaps, has not been fully appreciated is the importance of geometry (37) and electrostatic interactions between the salt bridge residues and the remainder of the protein (38). A survey of 222 salt bridges (37) using electrostatic calculations indicates that good geometry can substantially increase the stability of salt bridges. In this case, the balance between desolvation versus electrostatic interactions tips in favor of electrostatics. In addition, the electrostatic interactions between salt bridge groups and the rest of the protein are significantly greater in thermophilic proteins than in their mesophilic counterparts (37, 38). This suggests that salt bridges alone do not fully account for enhanced thermal stability but that the precise positioning of the charged residues within the electrostatic context of the rest of the protein is critical.
To gain further insights into the energetics of salt bridge networks, we employed the method of Hendsch and Tidor (39) to compute the electrostatic free energy of each salt bridge network in CYP175A1 using DELPHI (40, 41). We found that none of the salt bridges in CYP175A1 are stabilizing (data not shown). One problem with such an analysis, however, is that these comparisons are based on room temperature calculations where the dielectric constant of water is 80. As the temperature increases, the dielectric constant of water decreases and, hence, the energetic cost of desolvation will decrease. The problem, however, is far more complex than a simple change in dielectric constant. Elcock (42) carried out a computational analysis of charge interactions at elevated temperatures and found that the desolvation penalty for charged side chains is substantially decreased at thermophile temperatures. This analysis was extended to a comparison of proteins from thermophiles and mesophiles, and it was found that the effect the electrostatic component of hydration free energy results in greater stability for the thermophiles at elevated temperatures (42). Therefore, the more salt bridges, the more stable the protein is at elevated temperatures. Based on this view, it appears that the primary factors controlling thermostability in CYP175A1 and also CYP119 are salt bridge networks and shorter loops between major elements of secondary structure.
An additional structural feature possibly relevant to enhanced
stability unique to CYP119 is the presence of a large aromatic network
extending ~39 Å (C--C-
) down one side of the molecule that
was correctly predicted in the homology models (3, 6) and further
confirmed in the crystal structure (4). Mutagenesis work by Maves
et al. (20) showed that disruption of this aromatic network
by an F24S mutation lowered the thermal melting transition by
~10.2 °C. Enhanced thermal stability owing to aromatic
interactions also has been found in other thermophiles (34).
Surprisingly, there was no large aromatic networks present in CYP175A1
despite the fact that the aromatic content was nearly the same at 10% in CYP175A1 and 9.8% in CYP119. The largest network found was ~13 Å and was composed of residues 117, 118, 121, 234, 353, 354, and 357.
In summary, it appears that the two thermophilic P450s share some
common structural features expected to contribute to enhanced thermal
stability. Both are smaller than mesophilic P450s, which is especially
notable in CYP175A1. CYP175A1 closely resembles P450BM-3 without the
loops. In addition, both contain a significant increase in salt bridge
networks compared with mesophilic P450s. Where the two thermostable
P450s sharply differ is the extensive aromatic network found in CYP119,
which is not present in CYP175A1 or any other P450 for which a
structure is available. Although the function of both CYP119 and
CYP175A1 remain unknown, there have been recent advances in using an
engineered CYP119 to catalyze fatty acid hydroxylation (43). Thus, it
may be possible to engineer thermostable P450s to catalyze reactions of
interest using both directed evolution (44) and structure-based design.
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ACKNOWLEDGEMENTS |
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We thank Drs. Rebecca Wade (EMBL, Heidelberg, Germany), Bruce Tidor (Massachusetts Institute of Technology, Cambridge, MA), and Ray Luo (University of California, Irvine, CA) for advice and help with electrostatic calculations.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM32688 (to T. L. P.) and by a grant from BASF AG, Ludwigshafen, Germany (to R. D. S.).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.
To whom correspondence should be addressed. Tel.:
949-824-7020; Fax: 949-824-3280; E-mail: poulos@uci.edu.
Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M206568200
2 F. Blasco, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: P450, cytochrome P450; CYP175A1, cytochrome P450 from T. thermophilus strain HB27; CYP119, cytochrome P450 from S. solfataricus; P450eryF, cytochrome P450 from Saccaropolyspora erthrea; P450cam, cytochrome P450 from Pseudomonas putida; P450BM-3, cytochrome P450 from Bacillus megaterium; P450nor, cytochrome P450 from Fusarium oxysporum; PDB, Protein Data Bank (www.rcsb.org/pdb/); r.m.s., root mean square.
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