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J Biol Chem, Vol. 274, Issue 10, 6031-6034, March 5, 1999
From the In the biosynthesis of fatty acids, the
In fatty acid biosynthesis, the chain elongation step consists of
the condensation of acyl groups, derived from acyl-ACP or acyl-CoA with
malonyl-ACP. These reactions are catalyzed by a group of enzymes, the
We recently determined the crystal structure of KASII from
Escherichia coli at 2.4-Å resolution (7). The subunit
consists of two mixed five-stranded Condensing enzymes have been identified with properties subject to
exploitation in the areas of plant oil modification (8), polyketide
engineering, and ultimately design of anti-cancer (9, 10) and
anti-tuberculosis (11, 12) agents. One of the molecular targets of
isoniazid, which is widely used in the treatment of tuberculosis, is
KAS (12). Cerulenin (Fig. 1A), a mycotoxin produced by the
fungus Cephalosporium caerulens, acts as a potent inhibitor
of KAS by covalent modification of the active cysteine thiol (13, 14).
Condensing enzymes from many pathways and sources have all been shown
to be inactivated by this antibiotic (6, 13-16) with the exception of
the synthase from C. caerulens (17) and KASIII (16), the
isozyme responsible for the initial condensation of malonyl-ACP with
acetyl-CoA in plant and bacterial fatty acid biosynthesis. Inhibition
of the KAS domain of fatty acid synthase by cerulenin is selectively
cytotoxic to certain cancer cells (9, 10).
We have now determined the structure of E. coli KASII
complexed with cerulenin with the objectives of defining the active site pocket, providing a basis for understanding differences in substrate specificities and sensitivity toward cerulenin between different condensing enzymes and laying a foundation for efforts at
structure based drug design with this target.
The KASII-cerulenin complex was prepared as described previously
(18). Crystals of the complex were grown by the hanging drop method.
Droplets consisting of equal amounts of protein solution (6 mg
ml Structure Determination of the Cerulenin Complex--
The complex
of KASII from E. coli with cerulenin crystallized in space
group P3121 isomorphously with the native enzyme (7), and
the crystal structure was determined to 2.65-Å resolution by
difference Fourier methods. The final protein model after refinement (R-factor = 0.213 and Rfree = 0.270 with good stereochemistry) contains 411 out of the 412 residues
of the subunit; no electron density for the N-terminal residue was
found. The overall real-space correlation coefficient (24) is 0.92, and
there is well defined electron density for the polypeptide chain except
for some side chains on the molecular surface. The inhibitor molecule
is well defined by the electron density (Fig.
1, B and C).
However, there is weaker than average electron density for the amide
group and no electron density for the last carbon atom of the
hydrocarbon tail, indicating considerable flexibility for the terminal
methyl group.
Cerulenin Binding Site--
The overall structure of the KAS dimer
is unchanged upon binding of cerulenin; the root mean square deviations
for the 411 C
From the initial Fo
In the KASII-cerulenin complex, the inhibitor amide carbonyl oxygen is
within hydrogen bond distance to the N
The structure of the cerulenin complex can be considered to mimic the
intermediate formed upon reaction of KAS with the acyl-ACP. In such a
complex the hydrophobic cavity would harbor the hydrocarbon tail of the
acyl intermediate. The acyl hydrophobic tails will not be restricted by
two double bonds (as in the case of cerulenin), and this will allow
longer acyl chains to be buried in this pocket. Inspection of the
active site cavity suggets that it would not be possible to harbor a
linear acyl chain longer than 14 carbon atoms without structural
changes. Such conformational changes must occur since KASII is able to
elongate 16:1 to 18:1 (29).
Cerulenin Resistance--
There are few exceptions to the
inhibition of KAS by cerulenin, one is the fatty acid synthase of the
fungus synthesizing this antibiotic, C. caerulens (17),
another is KASIII (16), the variant of KAS that catalyzes the initial
condensation of malonyl-acyl carrier protein with acetyl-CoA in plant
and bacterial fatty acid biosynthesis. Unfortunately there is no amino
acid sequence available of KAS from C. caerulens, therefore
the structural basis for the lack of inhibition can not yet be
analyzed. The sequence of a 12-residue peptide supposed to span the
active site cysteine (30) showed no homology to other KAS enzymes,
suggesting that the enzyme from C. caerulens might be very different.
Amino acid sequences for KASIII show very low degrees of sequence
identities to KASI, KASII, and to the fungal and animal fatty acid
synthases, which makes an alignment of the sequences very unreliable
and prevents a proper analysis. However, considering the size of its
substrates, a reasonable explanation for the lack of inhibition of
KASIII is that it has a smaller active site cavity available that can
not accommodate the hydrophobic tail of cerulenin.
Mutations in the gene coding for fatty acid synthase that result in
cerulenin resistance in S. cerevisiae have been observed (31). One of these mutations is a Gly We thank the staff at MAX-lab, Lund
University for help at the beamline.
*
This work was supported by a grant from the Swedish Natural
Science Research Council and by the Foundation for Strategic Research.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 structure factors (codes 1b3n and
r1bnsf) have been deposited in the Protein Data Bank,
Brookhaven National Laboratory, Upton, NY.
¶
To whom correspondence should be addressed. Tel.:
46-8-7287695; Fax: 46-8-327626; E-mail: ylva{at}alfa.mbb.ki.se.
The abbreviations used are:
ACP, acyl carrier
protein;
KAS,
COMMUNICATION
Structure of the Complex between the Antibiotic Cerulenin and Its
Target, 
-Ketoacyl-Acyl Carrier Protein Synthase*
,,¶
Department of Medical Biochemistry and
Biophysics, Doktorsringen 9A1, Karolinska Institutet, S-171 77 Stockholm, Sweden and the § Oils Division, Calgene, Inc.,
Davis, California 95616
ABSTRACT
Top
Abstract
Introduction
References
-ketoacyl-acyl carrier protein (ACP) synthases catalyze chain
elongation by the addition of two-carbon units derived from malonyl-ACP
to an acyl group bound to either ACP or CoA. The enzyme is a possible
drug target for treatment of certain cancers and for tuberculosis. The
crystal structure of the complex of the enzyme from Escherichia
coli, and the fungal mycotoxin cerulenin reveals that the
inhibitor is bound in a hydrophobic pocket formed at the dimer
interface. Cerulenin is covalently attached to the active site cysteine
through its C2 carbon atom. The fit of the inhibitor to the active site is not optimal, and there is thus room for improvement through structure based design.
INTRODUCTION
Top
Abstract
Introduction
References
-Ketoacyl synthases, condensing enzymes, comprise a
structurally and functionally related family that play critical roles in the biosynthesis of a variety of natural products, including fatty
acids (1, 2) and the polyketide precursors leading to antibiotics,
toxins, and other secondary metabolites (3, 4). They catalyze
carbon-carbon bond forming reactions by condensing a variety of acyl
chain precursors with an elongating carbon source, usually malonyl or
methylmalonyl moieties, that are covalently attached through a
thioester linkage to an acyl carrier protein (ACP).1 Condensing enzymes
can be part of multienzyme complexes, domains of large, multifunctional
polypeptide chains as the mammalian fatty acid synthase, or single
enzymes as the -ketoacyl synthases in plants and most bacteria
(1-5).
-keto-ACP synthases (KAS, EC 2.3.1.41). Several species of
-keto-ACP synthases in plants and bacteria have been identified,
distinct in amino acid sequence, chain length specificity for their
substrates, and sensitivity to cerulenin, an inhibitor of condensing
enzymes (6).
-sheets surrounded by
-helices. The two sheets are packed to each other in such a way that
the fold can be described as consisting of five layers,
----. The enzyme is a homodimer, and the subunits are
related by a crystallographic 2-fold axis. The proposed nucleophile in
the reaction, Cys-163 is located at the bottom of a mainly hydrophobic
pocket at the dimer interface. The structure allowed us to suggest the
specific function of conserved residues in the catalytic reaction. The conservation of key residues in the whole family of condensing enzymes
suggests that they all exhibit a similar three-dimensional molecular architecture.
EXPERIMENTAL PROCEDURES
1 protein, 0.3 M NaCl, 25 mM
Tris, pH 8.0, 5 mM imidazole, and 10% v/v glycerol) and
reservoir solution were equilibrated against 26% w/v polyethylene
glycol 8000 and 0.1% v/v 2-mercaptoethanol in water. Data from two
crystals were collected at 298 K at the synchrotron in MAX-lab,
beamline I711, in Lund. The data was processed with DENZO (19) and
programs from the Collaborative Computating Project 4 Suite (20) and
the two data sets were scaled together in SCALA (21) (Table
I). The crystals are very
radiation-sensitive, but cannot be frozen in a cryostream. Due to
non-isomorphism, data of only two crystals could be merged. The
crystals of the complex have space group P3121 with similar
cell dimensions as the native enzyme (Table I). The coordinates of the
native enzyme (7) were used to calculate initial electron density maps
with SIGMAA (22). All data were used in the refinement; no sigma cutoff
was applied. After an initial cycle of positional refinement, the model
was rebuilt and a model of cerulenin was included. Further cycles of
refinement of the complex were carried out using the program REFMAC
(23) including a bulk solvent correction, interspersed with inspection
and correction of the model using O (24), OOPS (25), and PROCHECK (26)
(see Table I). Structure comparisons were performed using O (24) with
default parameters.
Statistics of data collection and structure refinement
RESULTS AND DISCUSSION
View larger version (34K):
[in a new window]
Fig. 1.
Cerulenin structure and fit to electron
density maps. A, free cerulenin in the open and ring
closed form and as observed bound to the enzyme. B,
stereoview of the initial Fo 
Fc map at the cerulenin binding site at a contour
level of 2
. The figure was generated using O (24). The final,
refined model of the complex is superimposed. C, stereoview
of the final 2Fo Fc,
-weighted map at the binding site at 1. The figure was generated
using O (24). The final, refined model of the complex is
superimposed.
atoms of the subunit is 0.23 Å between the two
structures. These differences are mainly localized in the active site,
in particular in the loop comprising residues 398-401. The main
differences in structure between the native enzyme and the cerulenin
complex are in the conformation of the side chains of Phe-400 (which
was anticipated already from the native structure) and of Ile-108, which have completely new rotamer conformations, and in the positions of the side chains of Cys-163, His-340, and Leu-342, which also have
moved substantially. These conformational changes provide access for
cerulenin to the active site cysteine and open a hydrophobic pocket for
the hydrophobic tail of the inhibitor.
Fc
electron density map these structural changes could be readily seen as
well as the binding site for the inhibitor (Fig. 1B).
Cerulenin is bound covalently through its C2 carbon atom to the Cys-163
S
atom. Its hydrocarbon tail fits in a hydrophobic pocket formed at
the dimer interface (Figs. 2 and
3). The structure of the adduct of cerulenin and cysteine, isolated by tryptic digestion of the
cerulenin-fatty acid synthase complex, has been determined by NMR and
mass spectroscopy (14). This study revealed that the inhibitor reacts
at its C2-epoxide carbon with the SH group of cysteine and that
cerulenin formed a hydroxylactam ring (Fig. 1A). The
electron density observed in the KASII-cerulenin complex is not
consistent with this structure. It was not possible to model bound
cerulenin in the closed ring form but the open form of the inhibitor
could readily be fitted to the electron density map (Fig. 1,
B and C). We thus conclude, that the
hydroxylactam ring, which is formed preferably in protic solvents (14),
is not present in the hydrophobic environment of the protein.
View larger version (83K):
[in a new window]
Fig. 2.
Schematic diagram of the dimer of KASII with
cerulenin bound at the interface as a CPK model. The figure
was generated using the programs Molscript (32) and Raster3D
(33).
View larger version (57K):
[in a new window]
Fig. 3.
The cerulenin binding groove of KASII.
The figure was prepared using Voidoo (34), Bobscript (35), and Raster3D
(33).
atoms of the side chains of
His-340 and His-303, while the amide NH2 group does not
make any close interactions (Fig. 3). It is, however, not possible from
the structure to exclude the opposite conformation and interactions for
the amide group. The hydroxyl group at C3 forms a hydrogen bond to the
main chain NH of Phe-400. The carbonyl oxygen at C4 does not form any
polar interactions, in fact, it is located in a very hydrophobic pocket
formed by side chains Phe-400, Phe-202, and Val-134 from the other
subunit in the dimer. The binding site for the hydrophobic part of the
inhibitor is also lined with hydrophobic residues: Ala-162, Gly-107,
Leu-342, Phe-202, Leu-111, Ile-108, Ala-193, Gly-198; and from the
second subunit in the dimer, Ile-138, Val-134, and Phe-133. The two
double bonds with trans configuration give the hydrophobic
tail a shape that fits to the hydrophobic groove once residue Ile-108
has changed rotamer. In comparison, binding of tetrahydrocerulenin
would cost entropy, and as expected it shows more than 2 orders of
magnitude less inhibitory activity (27). The influence of the length of the hydrocarbon chain, maintaining the double bond positions, has been
studied using fatty acid synthase from Saccharomyces cerevisiae (28). Cerulenin (12 carbons) had the highest inhibitory activity, with slightly decreasing binding strength upon increase in
chain length. However, when increasing the length from 16 to 18 carbon
atoms, the inhibition decreased by 2 orders of magnitude. The size of
the hydrophobic pocket in KASII, which binds the hydrocarbon tail of
cerulenin, suggests that there is space for a longer hydrophobic tail
only if the side chains of Leu-111 and of Phe-133 in the second subunit
change their conformation. Thus, possible differences in the
sensitivity of condensing enzymes toward cerulenin might be controlled
by the size of this cavity.
Ser replacement. This glycine
residue corresponds to Gly-107 in KASII, adjacent to Ile-108, which
changes rotamer upon binding of cerulenin. Exchange of this glycine for
serine introduces steric hindrance to binding of cerulenin and
influences the polarity in the hydrophobic cavity.
ACKNOWLEDGEMENT
FOOTNOTES
ABBREVIATIONS
-keto-ACP synthase.
REFERENCES
Top
Abstract
Introduction
References
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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