From the Department of Biochemistry,
Vanderbilt University School of Medicine, Nashville, Tennessee
37232-0146, the ¶ Argonne National Laboratory, Structural
Biology Center, Argonne, Illinois 60439, the
Department of
Microbiology and BioTechnology Institute, University of Minnesota,
Minneapolis, Minnesota 55455, and the ** Wolfson
Laboratory of P-450 Biodiversity, Institute of Biological Sciences,
University of Wales Aberystwyth, Aberystwyth,
Wales SY23 3DA, United Kingdom
Received for publication, December 2, 2002
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ABSTRACT |
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Evolutionary links between cytochrome
P450 monooxygenases, a superfamily of extraordinarily divergent
heme-thiolate proteins catalyzing a wide array of NADPH/NADH- and
O2-dependent reactions, are becoming
better understood because of availability of an increasing number of
fully sequenced genomes. Among other reactions, P450s catalyze the
site-specific oxidation of the precursors to macrolide antibiotics in
the genus Streptomyces introducing regiochemical diversity
into the macrolide ring system, thereby significantly increasing
antibiotic activity. Developing effective uses for Streptomyces enzymes in biosynthetic processes and
bioremediation requires identification and engineering of additional
monooxygenases with activities toward a diverse array of small
molecules. To elucidate the molecular basis for substrate
specificity of oxidative enzymes toward macrolide
antibiotics, the x-ray structure of CYP154C1 from Streptomyces
coelicolor A3(2) was determined (Protein Data Bank code 1GWI).
Relocation of certain common P450 secondary structure elements, along
with a novel structural feature involving an additional
The post-genomic era opens new opportunities for structural
insight into the evolution of a single protein family within and between species. Cytochrome P450
(CYP)1 monooxygenases are a
superfamily of heme-thiolate enzymes that are involved in a wide array
of NADPH/NADH- and O2-dependent reactions (1).
There are currently more than 2000 family members, including a large
number of putative P450 open reading frames found in fully sequenced prokaryotic and eukaryotic genomes
(drnelson.utmem.edu/CytochromeP450.html). Extensive studies have
firmly established their role in the biosynthesis of sterols, fatty
acids, and prostaglandins in animals, antibiotics, and other
biologically active molecules in bacteria, fungi, and plants as well as
in the metabolism of xenobiotic drugs and toxic chemicals (2).
Accordingly, the extraordinary diversity in amino acid sequence enables
wide variation in the substrates utilized and the patterns of oxidation
catalyzed by these enzymes.
A particularly rich source of CYPs is from Streptomyces
spp., a group of developmentally complex, Gram-positive bacteria that are known for production of a broad array of biologically active secondary metabolites. Streptomyces coelicolor A3(2) has
been investigated extensively as a model system for the study of
morphological and physiological development of Streptomyces
and for investigation of the genetic control of antibiotic production
(3). Over the past decade, an increasing number of
Streptomyces spp. have been investigated because of their
production of pharmaceutically important compounds including
anti-cancer agents, immunosuppressants, and antibiotics. In addition,
Streptomyces are being recognized as a source of versatile
biocatalysts for the detoxification of hazardous chemicals (4-9) in
bioremediation processes.
In an effort to analyze fully the complement of CYPs in the
industrially important Streptomyces, we decided to focus on
the 8.7 Mb S. coelicolor A3(2) genome whose sequence was
recently completed by the Sanger Centre (10)
(www.sanger.ac.uk/Projects/S_coelicolor/). Although 18 CYP open reading
frames are found dispersed throughout the S. coelicolor
A3(2) chromosome (10, 11), the functions of the corresponding gene
products and their role in various metabolic functions remain largely undefined.
Most of the currently identified antibiotics are produced by complex
biosynthetic systems comprised of clustered gene sets located
contiguously on the Streptomyces chromosome (12-18). The clustering of secondary metabolite genes has been an aid in the isolation of P450 monooxygenases involved in antibiotic biosynthesis. Cytochrome P450 monooxygenases are particularly common in polyketide biosynthetic gene clusters, and they catalyze site-specific tailoring reactions leading to the macrolide antibiotics, including methymycin, neomethymycin, and pikromycin (12, 19-22), novamethymycin (23), oleandomycin (24), amphotericin (17), and erythromycin (25, 26).
Additionally, CYPs are involved in the formation of the anticancer
agent epothilone (27, 28), immunosuppressant rapamycin (29, 30), the
growth promoter tylosin (14), and the antiparasitic agent avermectin
(15). These reactions typically occur during the late stages of
biosynthesis after formation of the core ring system by a polyketide
synthase. The hydroxyl or epoxide substituents provide an important
layer of structural variability into the final natural product
structures and often significantly influence biological activity (12,
22). P450 monooxygenases are also involved in one of the initial steps
in formation of the coumarin group of antibiotics (31), and of the
peptidyl nucleoside antibiotic nikkomycin (32), as well as in oxidative
tailoring of the vancomycin-like glycopeptides balhimycin (16) and
complestatin (33). Ultimately, the power to manipulate macrolide
metabolic systems using combinatorial biosynthetic technology (34-36)
will be extended by identification and/or engineering of additional
monooxygenases with versatile activities to provide novel biologically
active natural products.
Our current study was motivated by the intriguing amino acid sequence
relationship between a number of S. coelicolor A3(2) CYPs
that show significant similarity with P450 monooxygenases from other
microorganisms involved in regio-specific oxidation of macrolide
antibiotics. Among eight crystal structures reported for cytochrome
P450 over the last 17 years, only one has been reported for a macrolide
hydroxylase (P450 EryF from Saccharopolyspora erythraea)
(37). EryF is involved in hydroxylation of C-6 of the 14-membered ring
macrolactone 6-deoxyerythronolide B in the erythromycin biosynthetic
pathway. To elucidate structural, functional, and evolutionary aspects
of monooxygenases that tailor macrolide and xenobiotic molecules,
crystallographic analysis of the cytochrome P450 complement of S. coelicolor A3(2) (10, 11) was initiated. We report here the first
structural and functional analysis of the monooxygenase CYP154C1 from
this organism determined to 1.92 Å resolution.
CYP154C1 Purification--
The DNA sequence corresponding to
CYP154C1 with four histidine codons inserted at the 3' end was
generated by PCR and cloned into the Escherichia coli
expression vector pET17b as described elsewhere (11). The protein was
expressed in HMS174(DE3) cells (Novagen) and purified to homogeneity by
nickel-nitrilotriacetic acid (Qiagen) and Q-Sepharose (Amersham
Biosciences) chromatography.
CYP154C1 Crystallization and Data Collection--
Crystals grew
in hanging drops from 0.4 mM CYP154C1 in 10 mM
Tris-HCl, pH 7.5, 450 mM
NaCl, 0.5 mM EDTA mixed with
an equal volume of 1.6 M MgSO4,
100 mM MES, pH 6.5, 10 mM 2-methylimidazole at 22 °C. Native
diffraction data and multiple anomalous
dispersion data at three wavelengths (Tables
I-III)
were collected at 100 K at the laboratory source on R-AXIS IV mounted
on an RU-200 x-ray generator (Rigaku, Tokyo) and at the 19ID beamline
of the Structural Biology Center (Advanced Photon Source, Argonne
National Laboratory), respectively. Cryoprotectant contained 20% (v/v)
glycerol plus mother liquor. The crystals belong in space group
P212121, with unit cell
dimensions a = 62.728, b = 131.973, c = 134.886, CYP154C1 Phasing and Refinement--
The images were integrated,
and the intensities were merged using HKL2000 (38). The positions of
two iron sites were determined with SnB (39). The phases were
calculated and improved by CNS (40) yielding an interpretable
electron density map at 2.3 Å resolution. Crystallographic refinement
was carried out with CNS (40) using native data to a resolution of 1.92 Å. The final atomic model (Tables I-III) with an R factor
of 20.8% (22.8%) was obtained after iterations of refinement (CNS
(40)), evaluation (PROCHECK (41)), and manual building (O (42)). Two
protein molecules are present in the asymmetric unit. The quality of
the final structure (Tables I-III) was assessed with the program
PROCHECK (41). For the first molecule, 90.1% of residues were found in the most-favored regions of the Ramachandran plot, and 9.9% were found
in the allowed regions. For the second molecule, 91.7% of residues
were found in the most-favored regions of the Ramachandran plot, and
9.3% were found in the allowed regions.
Spectral Substrate and Inhibitor Binding
Assays--
YC-17-induced (43) and narbomycin-induced (44) spectral
shifts were monitored at 23.5 °C by using a Shimadzu UV-2401
spectrophotometer. The sample contained 1 ml of 5 µM
CYP154C1 in 10 mM Tris-HCl, pH 7.5, 10% glycerol. YC-17
and narbomycin were dissolved in ethanol at a stock concentration of 25 mM. Spectral binding assay was carried out by difference
spectra with the addition of substrate dissolved in ethanol to the
sample cuvette and ethanol to the reference cuvette by 2-µl aliquots.
The assays were performed by using eight YC-17 or narbomycin
concentrations ranging from 50 to 390 µM. The data were
linearized in the form of the S0/ Catalytic Activity--
The CYP154C1 and PikC conversion of YC17
or narbomycin was compared using a modification of the assay developed
for PikC (19). The assay mixture contained 1 µM enzyme,
3.5 mM spinach ferredoxin, 0.1 unit of spinach
ferredoxin-NADP+ reductase, 1 mM NADPH, and
~500 µM YC17 or narbomycin in a total volume of 1 ml of
conversion buffer (100 mM NaH2PO4,
pH 7.3, 1 mM EDTA, 0.2 mM dithiothreitol, and
10% glycerol). The reaction was carried out at 37 °C for 20 min and
terminated by extraction with ethyl acetate (3 × 1 ml). The
extracts were combined, dried, and resuspended in 100 µl of ethyl
acetate. The extracts were compared with purified compounds using
silica TLC developed with chloroform:methanol:NH4OH
(9:1:0.1) and stained with vanillin.
Sequence Homology with Other
Monooxygenases--
CYP154C1(Q9L142; accession numbers are
according to SWISS-PROT/TrEMBL Protein Knowledgebase,
tw.expasy.org/sprot/) is one of the 18 cytochromes P450 revealed in the
S. coelicolor A3(2) genome (10), and one of the two assigned
to the CYP154 family (11). The closest homolog of CYP154C1 identified
in the data base using program BLAST (45) is a cytochrome P450 CYP154B1 from Streptomyces fradiae with 44% sequence identity
(Q9XCC6) localized within the tylosin biosynthetic gene cluster (13). However, because macrolactone ring hydroxylations at C-20 and C-23 that
occur during tylosin biosynthesis are catalyzed by the products of
different genes, one of which encodes another cytochrome P450, CYP105L1
(Q9ZHQ1) (14), the role of the CYP154C1 homolog in S. fradiae remains unknown. CYPs with lower identity include the
second member of CYP154 family in S. coelicolor A3(2),
CYP154A1 (Q9KZR7) (with 42% identity), as well as CYP107B1 (B42606) and EryF, or CYP107A1 (Q00441), from S. erythraea (both with 37% identity and 54 and 51% homology, respectively). There are also a
number of P450s from different Streptomyces species having about 36% identity, including CYP107C1 from S. thermotolerans (Q60005), CYP107L1 (PikC) from Streptomyces
venezuelae (O87605), PTED from Streptomyces
avermitilis (Q93H80), and CYP107D1 (OLEP) from Streptomyces
antibioticus (Q59819). This last group of related P450s is known
to be involved in modification of macrolide antibiotics such as
carbomycin (46), methymycin, neomethymycin, pikromycin (19, 21, 22),
novamethymycin (23), avermectin (15), and oleandomycin (24).
Two S. erythraea enzymes, CYP107B1 and EryF (46% identical
to each other) show the same level of identity to CYP154C1 from S. coelicolor A3(2) (Fig. 1).
Having 46% identity, the enzymes have different enzymatic activity.
Specifically, EryF catalyzes C-6 hydroxylation of the
6-deoxyerythronolide B (Scheme 1) in the
biosynthesis of erythromycin (47). CYP107B1 shows no detectable activity toward 6-deoxyerythronolide B (48), and its function remains
unknown. Interestingly, CYP107B1 has 51% identity and 65% homology
with the PikC from S. venezuelae (Fig. 1A).
Previous studies have revealed that PikC has remarkable substrate
flexibility. It is capable of accepting 12- and 14-membered ring
macrolides as substrates and catalyzes conversion of the 12-membered
ring macrolide intermediate YC-17 to methymycin, neomethymycin (12, 19,
21), and novamethymycin (23). PikC also converts the 14-membered ring
macrolide narbomycin to pikromycin (12, 19, 21, 22) (Scheme 1).
The Biological Role of CYP154C1--
The endogenous function and
biological role of CYP154C1, as with all other S. coelicolor
A3(2) CYPs, remain unclear. CYP154C1 lies on the chromosome adjacent to
CYP157A1 in an operon that does not contain polyketide synthases or
nonribosomal peptide synthetase gene sequences. Adjacent sequences
include a sensor kinase, two open reading frames of unknown function,
and an ATP-binding protein. The expression of this operon and the
function of CYP154C1 may be related to secondary metabolism through a
regulatory cascade induced by an environmental stress or to
modification of xenobiotics.
Functional Analysis of CYP154C1: Conversion of YC-17 and Narbomycin
in Vitro--
Sequence similarity of CYP154C1 with monooxygenases
involved in tailoring of macrolide antibiotics (Fig. 1A) led
us to investigate its catalytic activity toward these important
compounds. Thus, the protein was tested against 12- and 14-membered
ring macrolide intermediates YC-17 and narbomycin, which are converted
to methymycin, neomethymycin, novamethymycin, and pikromycin by PikC in
S. venezuelae (12, 19-23) (Scheme 1). Interestingly,
CYP154C1 showed catalytic activity toward both substrates. The reaction
with the 14-membered ring macrolide narbomycin was notably faster and
resulted in a more complete conversion to pikromycin compared with
conversion of the 12-membered ring macrolide, YC-17. However,
transformation of YC-17 was accomplished with notable position
selectivity at the C12 position, leading predominantly to
neomethymycin. This substrate selectivity is in marked contrast to
PikC, which converts YC-17 with equal efficiency to methymycin and
neomethymycin (19). CYP154C1 binds YC-17 and narbomycin with similar
affinity (Kd = 405 and 403 µM,
respectively), producing type I binding spectra resulting from
expulsion of a water molecule from the iron coordination sphere
followed by transition of the heme iron to a pentacoordinate high spin
state (Fig. 2, A and
C). The similarities in binding characteristics of CYP154C1,
while at the same time showing significant substrate specificity toward
the two macrolactone ring systems, represent a significant opportunity
to explore structure-function relationships within and between other
members of the CYP enzyme class.
X-ray Structure Determination of CYP154C1--
The growing number
of cytochrome P450 structures in the Protein Data Bank (eight are
currently available) provides a pool of search models to aid in solving
new P450 structures by the molecular replacement technique. However,
amino acid sequence diversity of enzymes within the superfamily has
limited use of the molecular replacement approach for cytochrome P450
structure determination (49). Fortunately, the intrinsic heme-iron atom of cytochromes P450 can be used as an anomalous scatterer to facilitate protein structure determination using the multiple anomalous dispersion technique.
CYP154C1 has 37% sequence identity and 51% sequence similarity to the
macrolide monooxygenase EryF, the highest homology to date between
P450s with reported x-ray structure. However, this similarity is
insufficient to successfully use EryF atomic coordinates to find a
molecular replacement solution for CYP154C1, even when poorly conserved
regions are omitted from the search model. Thus, CYP154C1 is the first
cytochrome P450 whose structure has been determined solely by the
multiple anomalous dispersion technique using the endogenous heme-iron
as an anomalous scatterer. The final model (Tables I-III) consists of
two molecules in an asymmetric unit each bearing a heme group and
residues 8-407, plus three residues from the C-terminal His tag for
the first molecule, along with 520 water molecules and eight sulfate
ions. Nine residues in the first molecule and three residues in the
second molecule were truncated to Ala because of insufficient electron density.
Overall CYP154C1 Crystal Structure--
Although P450
monooxygenases have been studied extensively by x-ray crystallography,
new superfamily members continue to reveal structural features as yet
unpredictable from primary sequence. Despite relatively high homology
to EryF, the three-dimensional appearance of the substrate-free
CYP154C1 is quite distinct from substrate/inhibitor bound EryF.
CYP154C1 appears to be separated along the interface between the
The open conformation in CYP154C1 is achieved by relocation of certain
secondary structure elements constituting the common P450 fold, as well
as being due to a novel
An important issue regarding the open conformation of CYP154C1 is
whether its presence is due to the empty active site pocket. If so,
CYP154C1 might oscillate between open/closed conformations triggered by
substrate binding/product release. Conformational changes leading to
closure of the active site entrance caused by substrate binding have
been demonstrated previously for P450BM3 (51, 52). S. coelicolor A3(2) CYP154C1 is the second P450 (after mycobacterial
CYP51 (53)) whose active site is accessible from the surface in
substrate-free form. Obtaining structures for P450s in substrate-free
and substrate-bound forms are expected to address the complex
conformational changes that occur when substrate binds, and following
hydroxylation the product is released from the enzyme. These dynamic
affects could account for the open structure of substrate-free CYP154C1
and the closed structure of substrate-bound EryF.
Analysis of the Putative CYP154C1 Substrate-binding Site--
The
putative CYP154C1 substrate-binding site in the absence of substrate is
shaped as a cleft open from the distal surface, with both walls built
by hydrophobic residues (Fig. 4). The
back side of the cleft is lined by polar residues and has negative potential because of Asp398, the only charged residue
residing in the substrate-binding site. The left cleft wall is
predominantly built by the residues from the BC loop, whereas
the right wall is comprised of residues from the FG region and the I
helix (Fig. 4).
Sequence alignment of CYP154C1 with other highly related monooxygenases
shows variation in the BC loop region (enclosed in box in
Fig. 1A). Significantly, this region is two residues shorter in CYP154C1 than in EryF and is substantially shorter (11 residues) in
PikC that has broad substrate specificity having ability to hydroxylate
both 12- and 14-membered ring macrolide antibiotics. It is worth noting
that sequence alignment of CYP154C1 with CYP105C1 from
Streptomyces griseus also shows significant
shortening of the BC loop (20 residues shorter in CYP105C1 and five
residues shorter in three other family members) (Fig. 1B).
It seems likely that the shortened BC loop would allow more space above
the heme plane and confer more freedom to accommodate substrates of
variable size. This is consistent with the biological function of
monooxygenases with broad substrate specificity, including PikC and
xenobiotic functionalizing enzymes, which may be inherently more flexible.
A water molecule bound to the iron in the sixth coordination site
(W1 in Fig. 5) was modeled
into electron density in the active site of each molecule in an
asymmetric unit. Although iron-bound water has a relatively high
temperature factor, 40.1 Å2 with the occupancy of 1.0 in
both molecules, omitting this water from the model results in
appearance of extra electron density in a Fo The I Helix Motif in CYP154C1--
Water in the sixth coordination
site (W1) is hydrogen-bonded to the carbonyl oxygen of
Ala242 (with the distances 2.65 and 2.78 Å in both
molecules), which in turn is hydrogen bonded to the side chain of
Thr246 occupying the n + 4 position along the
same face of the I helix (Fig. 5). A similar hydrogen bond is formed
between Gly243 and Thr247. A motif occurring in
the CYP154C1 I helix as
Ala242-Gly243-His244-Glu245-Thr246-Thr247
is partially conserved among cytochromes P450, although the growing number of P450 enzymes in the data base show variations of amino acids
in every single position. For S. coelicolor A3(2) CYPs, this
motif is summarized as
Ala(Gly)242-Gly(Ala)243-Xaa244-Glu(Asp,Gln,Ala,Val)245-Thr(Ala,Pro,Val)246-Thr(Met,Leu,Ile)247
(residue numbers are according to CYP154C1). To a large degree the
motif conservation seems to be determined by the position of the I
helix relative to the heme plane. The I helix is positioned so that
residues 242 and 246 face the heme plane to the exclusion of bulky side
chains because of space constraints (Fig. 5). Because of the small size
of these side chains, the I helix approaches near the heme plane and
enables a carbonyl oxygen of residue 242 (usually Ala, sometimes Gly)
to hydrogen bond a water molecule bound to the heme iron. This
interaction violates the hydrogen bonding pattern and the geometry of
the I helix. To compensate for missing helical hydrogen bonds, the
hydroxyl of Thr246 (or Ser in many P450s) hydrogen bonds
with the carbonyl oxygen of Ala242, whereas the hydroxyl of
Thr247 hydrogen bonds to the carbonyl oxygen of
Gly243. This unconventional
Gly in position 243 is the most highly conserved within the I helix
motif, and we believe is related to the molecular dynamics of P450
molecules. Gly confers flexibility to the I helix, which might allow
the I helix water binding cleft (Fig. 5) to adjust its size expelling
or accepting a water molecule (55) and also allow the I helix N
terminus to perform swinging motions in response to substrate or
inhibitor binding in the active site (52, 53, 56). Conservation of
residue functionalities in positions 245, partially 246, and perhaps
247 is likely due to mechanistic reasons for electron and/or proton
transfer and for oxygen activation (57-59). Apparently, structural
reasons dominate conservation of this motif because positions 242, 243, and 246 are never occupied by residues with bulky side chains, whereas
functionalities of residues in 245, 246, and 247 alternate. Whether
these variations correlate with reaction mechanism is unknown because
most P450s have not been studied in sufficient detail to address this question.
The CYP154C1 structure illustrates that functional diversity within the
cytochrome P450 family arises from the structural diversity of a few
externally positioned elements: the F and G helices with the FG loop in
between and the highly variable B' helix, whereas the oxygen scission
site remains largely intact. Relocation of the structural elements
allows an almost endless number of configurations for P450
substrate-binding sites, particularly in the absence of substrate, and
might be a serious obstacle in attempts to predict new cytochrome P450
structures based on homology modeling approaches.
Surface Electrostatic Potential of CYP154C1--
Electrostatic
potential distribution shows the presence of a cationic patch on the
proximal surface of CYP154C1 (Fig. 6), the region that has been implicated in redox partner interactions in
P450s (60). A similar cationic patch is present on the surface of EryF,
although it has a larger dipole moment (694 versus 305 Debye in CYP154C1) pointed in a different direction (Fig. 6). The dipole vector of CYP154C1 points directly in the middle of the
patch on a proximal surface almost perpendicular to the heme plane,
whereas the dipole vector of EryF is strongly inclined toward the C and
C' helices. A key landmark of the proximal surfaces of both CYP154C1
and EryF is a protruding Arg344 (Arg339 in
EryF) coming from the region known as the meander. Together with other
positively charged residues that form similar patterns on the proximal
surfaces of both CYP154C1 and EryF (Fig. 6), the prominent Arg might
play a role in anchoring the electron donor on the cationic patch.
Biological Implications--
This effort represents the first
structural analysis of S. coelicolor CYP enzymes and is
expected to provide information on hydroxylation patterns of endogenous
as well as exogenous substrates. Cytochrome P450 monooxygenases from
Streptomyces catalyze site-specific oxidations of the
precursors to many macrolide antibiotics and degrade a wide range of
xenobiotic compounds. Use of Streptomyces in the production
of natural products of pharmaceutical importance as well as in
bioremediation technologies provides an important incentive to identify
native enzymes and engineer novel monooxygenases with activities toward
alternative substrates. Together with the opportunity to manipulate
entire biosynthetic gene clusters, it allows design of biosynthetic
pathways giving rise to novel polyketides and other natural product
structures. CYP154C1 is the first three-dimensional structure for P450
monooxygenase with activity toward polyketides of diverse structures
and will contribute to understanding the molecular basis for the
specificity of oxidative tailoring in macrolide antibiotic
biosynthesis, as well as in development of approaches that lead to
fully elaborated and biologically active macrolide structures.
-strand transforming the five-stranded
-sheet into a six-stranded
variant, creates an open cleft-shaped substrate-binding site between
the two P450 domains. High sequence similarity to macrolide
monooxygenases from other microbial species translates into catalytic
activity of CYP154C1 toward both 12- and 14-membered ring macrolactones
in vitro.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
=
=
= 90°.
There are two molecules/asymmetric unit with solvent content in the
crystal 64%.
Crystallographic data and statistics
Multiple anomalous dispersion data phasing statistics
Refinement statistics
A versus S0 plot, where
S0 is a total concentration of substrate in the
reaction mixture. The difference in absorbance between 384 nm (peak)
and 418 nm (trough) for each spectrum was taken as the
A
of the reaction. Kd was estimated from the intercept
of the linear plot on the S0 axis.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Sequence alignment between CYP154C1 from
S. coelicolor A3(2) and homologous P450
monooxygenases. A, sequence alignment between CYP154C1
from S. coelicolor A3(2), EryF (CYP107A1) from S. erythraea, and PikC from S. venezuelae. B,
fragment of sequence alignment between CYP154C1 from S. coelicolor A3(2) and CYP105 proteins from S. griseus
accession numbers for CYP105A1, B1, C1, and D1 in the SWISS-PROT data
base are P18326, P18327, P23296, and P26911, respectively. The
black shading shows regions of conservation, whereas
gray shading denotes similarity of amino acid residues in a
given position. Residue numbers for each protein correspond to their
sequences deposited in the data base. Secondary structure elements are
assigned based on crystal structures for CYP154C1 determined here and
for EryF (Protein Data Bank code 1OXA). The - and
310-helices are marked by open bars,
-strands
are marked by arrows, and T indicates turns in
the protein chain. The BC loop region is enclosed in a box.
The star indicates the conserved Cys.
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Scheme 1.
Reactions catalyzed by cytochromes P450
EryF and PikC.
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Fig. 2.
Substrate binding and catalytic activity of
CYP154C1. A, type I binding spectra resulting from
CYP154C1 titration with increasing concentrations of YC-17 or
narbomycin ranging from 50 to 390 µM. B,
linearization of the titration data in the form of
S0/ A versus
S0 plot. C, thin layer chromatography
analysis of products of the catalytic conversion of YC-17 or narbomycin
by PikC and CYP154C1. MM is for methymycin, NMM
is for neomethymycin, NBM is for narbomycin, and
PKM is for pikromycin.
-sheet and
-helical domains from the distal surface all the way
down to the heme (Fig. 3A; see
also Fig. 6). The two domains remain separated by a slit about 10 Å wide without additional stabilizing forces. Despite the distinct packing environment in the crystal, two molecules in an asymmetric unit
show minor deviations (root mean square deviation of 0.3 Å as
calculated using an algorithm implemented in SWISS PDB VIEWER (50)) in
the major part of the structure, whereas more significant deviations up
to 1.8 Å are observed in the FG region (blue in Fig.
3A). Repositioning of the FG loop results in slightly more open access to the active site in the one molecule compared with the
second.
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Fig. 3.
Superimposition of structures.
A, superimposition between two CYP154C1 molecules in an
asymmetric unit. B, superimposition between CYP154C1 and
EryF. Superimposition was performed by using SWISS PDB VIEWER
(50) according to an algorithm implemented in the program. The diagrams
here and in Figs. 4 and 5 were prepared using SETOR (61). CYP154C1 is
shown in gray with the FG region highlighted in
blue, the BC loop and the B' helix in violet, and
the -sheet 1 in magenta. EryF (Protein Data Bank code
1OXA) is in green with the BC loop highlighted in
orange. The EryF substrate 6-deoxyerythronolide B is denoted
by gray balls. Heme is in red.
-strand unique to CYP154C1. As seen from of
the alignment (Fig. 1A) and from stereo views of the
structure (Fig. 3),
-sheet 1 in CYP154C1 consists of six strands
instead of the five observed in EryF and other P450s. The new strand
(indicated for CYP154C1 as
1-1 (Fig. 1A)) precedes the
invariant helix A. At residues Pro14-Phe15 the
polypeptide chain makes a 180° turn so that the A helix runs in a
direction anti-parallel to the
1-1 strand. The result of this new
arrangement is absence of the A' 310 helix present in EryF
or a loop preceding the A helix in some other P450s that normally
contribute to interactions between the
-helical and
-sheet
domains. In the
-helical domain, significant dislocation of the F, G
(blue in Fig. 3A), and B' (violet) helices
relative to their positions in substrate-bound EryF (green
in Fig. 3B) occurs. The F and G helices are translocated
away from the distal and toward the proximal surface by about one
-helical turn. Together with the shorter length of the G helix in
CYP154C1 and the FG loop being open compared with EryF, a significant
separation between the two protein domains is achieved. Additionally,
the B' helix in CYP154C1 is repositioned relative to its EryF
counterpart so that it becomes almost parallel to the G helix (Fig.
3B). These two helices complete the formation of an opening,
which we believe is a substrate-binding site in CYP154C1.
View larger version (51K):
[in a new window]
Fig. 4.
Putative substrate-binding site of
CYP154C1. For a better view of the cleft entrance, the CYP154C1
molecule in Fig. 3 was rotated toward the viewer approximately along
the horizontal axis in the plane of the drawing. Residues
building the left wall of the binding site cleft are from
the BC loop (violet), and residues building the right
wall are from the FG region (blue) and the I helix
(green). Residues lining the back side of the
cleft (gray) are from the last turn within -sheet 3 and
from the junction between the K helix and the strand
1-5.
Fc map (dark blue in Fig. 5). The
iron-oxygen distances in CYP154C1 are refined to 2.42 and 2.54 Å for
the two molecules, which is considerably longer than the 2.28 Å observed in substrate-free P450cam (54). High B-factors for water at
the sixth ligand position are likely a result of partial occupancy of
this site. The relatively long iron-oxygen distance also indicates that
this bond is weakened in CYP154C1 compared with P450cam.
View larger version (48K):
[in a new window]
Fig. 5.
Oxygen scission site of CYP154C1. The
fragment of the I helix shown represents the partially conserved I
helix motif positioned relative to the heme plane. Atoms are colored
according to elements: oxygen in red, nitrogen in
blue, sulfur in yellow, and carbon in
gray. Red was also used for the heme iron. Two
water molecules in the active site are represented by the red
spheres: W1 is for water in the sixth heme ligand position, and W2
is for water in the water-binding cleft of the I helix. Fragments of
the 2Fo Fc electron
density composite omit map are contoured at 1.0
(cyan)
and 2.5
(red). Fragment of the Fo
Fc electron density map generated with W1
omitted is contoured at 1.5
(dark blue).
-helix hydrogen bonding
pattern is observed in the I helix of most structurally defined CYPs
(53).
View larger version (76K):
[in a new window]
Fig. 6.
Surface electrostatic potential of CYP154C1
and EryF. The deepest shades of red and blue
correspond to potentials of 22.6 and 27.2 kcal, respectively, whereas
neutral points are white. Heme partially seen through the
cleft is green. Cyan three-dimensional vectors
show the direction and relative size of molecular dipoles for CYP154C1
and EryF.
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ACKNOWLEDGEMENTS |
---|
We thank Jarrod A. Smith and the Vanderbilt University Center for Structural Biology computing facilities for expert technical assistance, Dr. Stanley N. Cohen and Dr. Jianqiang Huang (Stanford University) for providing microarray information on expression of individual CYP genes, and Dr. Thomas Poulos (University of California, Irvine) for interest in the work, helpful discussions, and critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM37942 and ES00267 (to M. R. W.), P30 ES00267 (to L. M. P.), GM48562 (to D. H. S.), by Biotechnology and Biological Sciences Research Council and a Welcome Trust Grant (to S. L. K. and D. C. L.), and by NCI Cancer Biology Training Grant CA09138 (to B. J. B.).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 1GWI) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ To whom correspondence should be addressed: Dept. of Biochemistry, Vanderbilt University, 23rd South at Pierce, Nashville, TN 37232-0146. Tel.: 615-343-4644; Fax: 615-322-4349; E-mail: larissa.m.podust@vanderbilt.edu.
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M212210200
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ABBREVIATIONS |
---|
The abbreviations used are: CYP, cytochrome P450; MES, 4-morpholineethanesulfonic acid.
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