From the
Endogenous fatty acids are synthesized in all organisms in a
pathway catalyzed by the fatty acid synthase complex. In bacteria,
where the fatty acids are used primarily for incorporation into
components of cell membranes, fatty acid synthase is made up of several
independent cytoplasmic enzymes, each catalyzing one specific reaction.
The initiation of the elongation step, which extends the length of the
growing acyl chain by two carbons, requires the transfer of the malonyl
moiety from malonyl-CoA onto the acyl carrier protein. We report here
the crystal structure (refined at 1.5-Å resolution to an R factor of 0.19) of the malonyl-CoA specific transferase from
Escherichia coli. The protein has an
Endogenous synthesis of fatty acids is essential to all living
organisms. The reactions involved in the process are complex but can be
visualized in a simplified form as an iterative process of linear
decarboxylative condensations of several (normally seven) molecules of
malonate onto an acetyl primer. Subsequent to condensation, each such
elongation step requires NADPH-dependent modifications including
ketoreduction, dehydration, and enoyl reduction
(1, 3) .
Individual reactions are catalyzed by the components of the fatty acid
synthase (FAS)
The initiation of
each elongation step in the fatty acid synthesis cycle requires the
transfer of a malonyl moiety from the respective CoA thioester to the
-SH group of the phosphopantetheine arm of the acyl carrier
protein (ACP), the central component of any FAS. Depending on the FAS
type this reaction may either be catalyzed by a general transferase
that equilibrates between short chain CoA and ACP thioesters (FAS I) or
a specialized enzyme that is involved only in the elongation step (FAS
II). The transferase domain of the rat liver FAS I has been recently
characterized both at the protein and DNA levels
(7, 8) .
In Escherichia coli FAS II includes a specific malonyl-CoA:ACP
transacylase (MCAT), a 32-kDa single chain enzyme that has been
purified to homogeneity and characterized at the protein level (9, 10).
More recently the fabD gene coding for this protein has been
cloned and overexpressed
(11) . We now report the crystal
structure of native MCAT, solved by multiple isomorphous replacement
and refined at 1.5-Å resolution. Apart from the two-dimensional
NMR study of the E. coli ACP
(12) , this report
constitutes the first structural study of a component of an FAS
complex.
The enzyme was overexpressed, purified, and crystallized as
described previously
(13) ; only the hexagonal crystal form was
used in the present study (space group P6
Early characterization of the E. coli MCAT at the
protein level
(9, 10) indicated that the enzyme is
serine-dependent, with the catalytic residue located at the center of a
GHSLG pentapeptide that compares well with the frequently invoked
GXSXG consensus sequence
(25) of various
acylhydrolases. All hitherto determined structures of these enzymes
exhibit the
The presence of the Ser-His dyad
stabilized by an H-bond between the imidazole of His-201 and a main
chain carbonyl group was also a surprise. An analogous stereochemistry
has been recently found in an unrelated esterase from Streptomyces
scabies(29) . A reassessment of the mechanism of serine
hydrolases based on the comparison of MCAT with other enzymes appears
elsewhere.
It can be assumed that in MCAT
the acylation step and subsequent transfer of the acyl moiety proceed
via tetrahedral intermediates in a fashion similar to the hydrolytic
reaction pathway of serine proteinases. There is, however, a major
difference between MCAT and the hitherto characterized hydrolases: the
acyl-enzyme complex of MCAT is stable in aqueous solution, and
deacylation can only take place in the presence of specific thiol
acceptors.
Finally, we address the critical issue of substrate specificity. In
FAS I the transferase domain is capable of equilibrating various short
chain acyl-CoA thioesters with respective acyl-ACP complexes. This
includes acetyl-CoA and malonyl-CoA, both of which are involved in the
fatty acid synthesis process
(1, 3) , as well as
methylmalonyl-CoA in the biosynthesis of some methyl branched fatty
acids, and propionyl-CoA in the priming reaction leading to the
synthesis of fatty acids with an odd number of carbon atoms. The
synthetic route is in this case determined by the available pool of CoA
thioesters
(35) . The bacterial MCAT, on the other hand, is
highly specific toward malonyl-CoA and does not participate in the
priming transfer of the acetyl moiety onto ACP
(3) . In the
polyketide synthase, the analogues of MCAT most commonly use
methylmalonyl-CoA
(36) . Thus, these pathways are determined by
the transferase specificity.
The structure of the native E. coli MCAT fails to provide an obvious rationale for its specificity. It
is tempting to speculate the Arg-117 might play a role in the binding
of the free carboxyl group as is the case, for example, in citrate
synthase
(37) . This arginine, however, is invariant among all
known MCAT homologues (), including two sequenced FAS I
transferase domains that shuttle the acetyl group with equal ease. We
note that in these two cases there are non-conservative mutations
within the substrate binding site, replacing polar Ser-200 and Gln-63
(MCAT notation) with hydrophobic phenylalanines
(Fig. 1b). Mutational studies, under way in our
laboratories, will no doubt shed more light on the structure-function
relationships in this interesting family of enzymes.
Native
data were collected using
The atomiccoordinates of MCAT (accession code 1MLA) have been deposited
in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
We acknowledge Drs. Masao Fujinaga, Peter Sheffield,
and Stuart Smith for discussions and helpful suggestions in the course
of the preparation of the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
/
type
architecture, but its fold is unique. The active site inferred from the
location of the catalytic Ser-92 contains a typical nucleophilic elbow
as observed in
/
hydrolases. Serine 92 is hydrogen bonded to
His-201 in a fashion similar to various serine hydrolases. However,
instead of a carboxyl acid typically found in catalytic triads, the
main chain carbonyl of Gln-250 serves as a hydrogen bond acceptor in an
interaction with His-201. Two other residues, Arg-117 and Glu-11, are
also located in the active site, although their function is not clear.
(
)
complex. Type I FAS, found in
animals and man, is a single polypeptide chain, approximately 2500
amino acids in length, and is made up of eight distinct domains
including seven with discrete catalytic functions
(1) . The
molecule functions as a homodimer
(2) . In contrast, bacteria and
plants contain type II FAS, which is composed of structurally
independent proteins
(3) . Their exact number is
species-dependent, and there may be several isoforms of some
components. The fatty acid biosynthesis pathway is not unique in the
way it utilizes short chain acyl-CoA thioesters as building blocks for
larger organic molecules. It has been shown that analogous pathways are
involved in the generation of polyketides, where multifunctional and/or
multienzyme polyketide synthases catalyze the syntheses of such diverse
biologically active molecules as immunosuppressants, anti-tumor drugs,
and antibiotics
(4, 5, 6) .
, a = 68.2 Å, c = 118.6 Å). A
preliminary search for heavy atom derivatives was carried out using the
X1000 Siemens area detector mounted on a Siemens rotating anode source
(
= 1.54178 Å). Three heavy atom derivatives
(mercury, platinum, and gold) were identified. To improve the quality
of x-ray data and to enhance the anomalous scattering effects, the
final experiments were conducted using synchrotron radiation (beamlines
BW7B and X31, EMBL Outstation, Hamburg, Germany) using MAResearch
imaging plate systems (for further details see ). Scaling
between the data sets, difference Patterson maps and other calculations
were carried out using the CCP4 suite of programs
(14) . The
major site in the mercury derivative was identified in both isomorphous
and anomalous difference Patterson maps; SIROAS (single isomorphous
replacement optimized anomalous scattering)
(15) phases were
calculated for data between 15 and 2.4 Å using
MLPHARE
(16) . These were used to calculate Fourier difference
maps and identify both the secondary mercury sites and the heavy atoms
sites in the other two derivatives. Final phase calculation (MIROAS
(multiple isomorphous replacement optimized anomalous scattering))
using all three derivatives resulted in a figure of merit of 0.64.
Density modification involving solvent flattening and histogram
matching using SQUASH
(17) led to an average shift in phases of
20.2°. To resolve space group ambiguity phases were calculated in
both P6
and P6
; the latter yielded an easily
interpretable map showing elements of secondary structure folded in
agreement with accepted principles of protein chemistry. Model building
was done using O
(18) on a SGI Indigo Extreme. The initial
conventional crystallographic R factor (
F
- F
/
F
), for the model that included
all amino acids from residue 4 to 306, was 0.47 for all the data
between 8.0- and 2.0-Å resolution and decreased to 0.24 after one
cycle of crystallographic refinement with simulated annealing using
X-PLOR
(19) . The resulting electron density map allowed for
manual reconstruction of some loops, addition of residues 3 and 307, as
well as water molecules identified using an automated procedure of
ARP
(20) . Further crystallographic refinement using the
energy-restrained algorithm of X-PLOR resulted in an R factor
of 0.17 (all data in the 8.0-2.0-Å range). Final details of
the 1.5-Å refinement currently in progress (R factor of
0.19 for all the data) will be described elsewhere.
The Quality of the Model
The final model
contains residues from 3 to 307 and 170 water molecules. The
stereochemistry was assessed using PROCHECK
(21) ; root mean
square deviations from ideal bond lengths and angles were 0.012 Å
and 2.58°, respectively. Of all amino acids, 95.4% are in the most
favored regions of the Ramachandran plot. The only outlier is the
active site serine 92, which is in a strained conformation
(
= 50°,
= -99°),
characteristic of the nucleophile in
/
hydrolases
(22, 23) . There is one cis-peptide
that precedes Pro-52. The mean isotropic atomic displacement
(B) factor for all atoms is 19.6 Å
.
Overview of the Structure
The tertiary fold of the
enzyme is best described in terms of two subdomains
(Fig. 1a). The larger subdomain is made up of two
non-contiguous segments including residues 3-123 and
206-307. It contains a short four-stranded parallel -sheet
and 12 helices from 4 to 17 residues in length. The smaller subdomain,
residues 124-205, contains a four-stranded anti-parallel
-sheet capped by two
-helices. The fold is, in general terms,
of the
/
type, but (to our knowledge) is unique among known
proteins.
Figure 1:
A, schematic representation of the
three-dimensional structure of E. coli MCAT.
-Strands and
-helices were defined according to the DSSP
algorithm (24); helices: H1, 20-25; H2, 28-40; H3,
44-50; H5, 53-56; H6, 59-79, H7, 94-101; H8,
107-123; H9, 140-150; H10, 173-185; H11,
206-217; H12, 240-250; H13, 257-266; H14,
280-288; H15, 300-306;
-strands: B1, 5-8; B2,
87-90; B3, 130-136; B4, 156-163; B5, 166-172;
B6, 190-193; B7, 271-274; B8, 293-296. The major
subdomain is made up of two non-contiguous parts: one containing
helices H1-H8 and strands B1-B2 (yellow), and the
other made up of H11-H15 and strands B7-B8
(orange). The smaller subdomain (green) is formed by
a contiguous stretch including stands B3-B6 and helices
H9-H10. Three amino acids forming the modified triad at the
active site are shown in violet. B, stereochemistry
at the active site of MCAT. The green color represents
invariant residues among MCAT homologues; Ser-200 and Gln-63, both of
which are mutated to Phe in FAS I, are shown in pink. Water
molecules are shown in red. C, comparison of MCAT
(violet) with R. miehei lipase complexed with
n-hexylphosphonate ethyl ester (paleblue).
H-bonds are indicated by dashedlines. The two water
molecules in the active site of MCAT believed to occupy potential
oxyanion binding sites are shown in orange. The yellow
phosphorus-bonded oxygen shown in yellow indicates the
location of the oxyanion hole in lipase. The figure was generated after
C
atoms of residues 90-94 of MCAT were superimposed on the
corresponding atoms in residues 142-146 in the inhibited lipase.
The figure was generated using RIBBONS (38).
The Active Site
The active site of MCAT is located
in a gorge between the two subdomains (Fig. 1a). The
nucleophile, Ser-92, is located in a sharp turn between a -strand
and an
-helix within the major subdomain. There is an H-bond
between the side chain hydroxyl of Ser-92 and N
-2 of His-201,
reminiscent of similar interactions in serine hydrolases containing
so-called catalytic triads (Fig. 1b). However, the
N
-1 of His-201 acts as a donor in an H-bond with a main chain
carbonyl oxygen of Gln-250 and not a carboxyl acid typically observed
in triads. The hydroxyl of Ser-92 is also within hydrogen bonding
distance (3.07 Å) of N
-2 of Arg-117, a completely buried
residue as assessed by DSSP (24). Two well resolved water molecules are
found in the proximity of Ser-92 (Fig. 1, b and
c). Water 321 (B = 14 Å
)
accepts H-bonds from the main chain amides of Leu-93 (3.05 Å) and
Gln-11 (2.87 Å) and donates an H-bond to water 335 (2.77
Å). The latter (B = 20 Å
)
accepts a second H-bond from N
-1 of Arg-117 (3.07 Å) and
donates to the hydroxyl of the Ser-92 (3.08 Å) as well as
O
-1 of Gln-11 (3.13 Å). Residues Ser-92, His-201, Gln-11,
and Arg-117 constitute 4 out of 8 invariant residues in the hitherto
sequenced homologues of MCAT ().
/
hydrolase fold
(23) in which the
consensus pentapeptide forms a tight turn between a
-strand and an
-helix, a motif known as the nucleophilic elbow
(23) or the
-
Ser-
motif
(22) . Furthermore, in all
serine-dependent
/
hydrolases the active sites contain
Ser-His-Asp(Glu) catalytic triads analogous with that originally
described in chymotrypsin
(26) . It is noteworthy that the
sequence of the active site pentapeptide in MCAT is identical to the
one found in a well studied family of extracellular lipases from
filamentous fungi
(27) and also found in the mammalian
pancreatic lipases
(28) . Hence, we expected that MCAT would
exhibit a typical
/
hydrolase tertiary structure.
Surprisingly, MCAT has a unique fold, albeit the 18-residue-long
oligopeptide containing the active serine is structurally very close to
the nucleophilic elbow of
/
hydrolases.
(
)
This is the first observation of the nucleophilic elbow
outside the context of the
/
hydrolase fold and a dramatic
example of convergent evolution.
(
)
(
)
The comparison of the active site of
MCAT with that of the Rhizomucor miehei lipase complexed with
n-hexylphosphonate ethyl ester (entry 5TGL in the Protein Data
Bank, Brookhaven National Laboratory; Ref. 32), a transition state
analogue, provides some clues as to the possible structural roots of
this difference. There are two possible sites for the oxyanion hole in
MCAT occupied by solvent molecules water 335 and water 321,
respectively (see ``Results,'' Fig. 1c).
However, with the exception of the main chain NH of Leu-93 the
potential H-bond donors (NH of Gln-11; N
-1 of Arg-117, and
N
-2 of Gln-11
(
)
) are located 1.0-1.5
Å further away from the nucleophile than in other serine
hydrolases. It is this relative displacement of the putative oxyanion
hole from the nucleophile in MCAT that may be responsible for the
absence of hydrolytic activity. Assuming that a potentially hydrolytic
water molecule would approach the carbonyl carbon of the acyl-enzyme
adduct in a fashion similar to that seen in serine
proteinases
(33, 34) , there are no interfering steric
constraints in the MCAT structure. However, if the target carbon is
pulled away from His-201 into the oxyanion hole, the water molecule
cannot be suitably oriented and activated by an H-bond to N
-2 of
His-201. Since sulfur has a larger radius than oxygen and a C-S
bond is
0.4 Å longer than a C-O bond, -SH might
be more suitable as an acceptor, as indeed is the case in MCAT.
Table:
Data collection and phasing
= 0.87 Å while derivative
data were collected using
= 0.993 Å
(L
absorption edges for mercury, gold, and
platinum are 1.0091, 1.0400, and 1.0723 Å, respectively). Data
were processed and reduced with the DENZO-SCALEPACK programs (39). The
derivatives were prepared shortly before data collection by soaking
native crystals as follows: mercury, 0.6 mM thimerosal, 12 h;
platinum, 0.5 mM K
PtCl
for 4 days;
gold, 5 mM NaAuCN
for 1 day. In contrast to the
original mother liquor (45-50% saturated ammonium sulfate, 0.1
M Mes, pH 6.0, 1% (w/v)
-octyl glucoside) the soaking
solutions were detergent-free. R
is the measure
of the internal consistency of the data, defined as
I
-
I
/
I
, where
I
is the intensity of the ith
observation and
I
is the mean intensity of the
reflection; R
is the measure of the mean
relative isomorphous difference between the native protein
F
and the derivative
F
data defined as
F
-
F
/
F
;
phasing power is defined as
F
/
, where
F
is the mean heavy atom
contribution and
the mean lack of closure;
R
is defined as
/
F
- F
.
Table:
Partial sequence alignment of MCAT
homologues: active site residues
atoms of residues 86-103 of MCAT
superposed by least squares on the analogous fragment of the
Rhizomucor miehei lipase (residues 138-155, entry 3TGL
in Protein Data Bank, Brookhaven National Laboratory) is 0.73 Å.
-alanyl)-cysteamine, and
N-acetylcysteamine (31).
-2 of
Gln-11 can serve as an H-bond donor in the putative oxyanion hole only
after a rotation of the side chain 180° around
with respect to the structure described here.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.