From the RIKEN Institute, 2-1 Hirosawa, Wako,
Saitama 351-0198, the ** Department of Chemistry, Graduate
School of Science, Kyoto University, Kitashirakawa, Sakyo-ku,
Kyoto 606-8502, and
RIKEN Harima
Institute/SPring-8, 1-1-1 Koto, Mikazuki-cho, Sayo-gun,
Hyogo 679-5148, Japan
Received for publication, June 3, 2002, and in revised form, October 23, 2002
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ABSTRACT |
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The (R)-specific enoyl
coenzyme A hydratase ((R)-hydratase) from Aeromonas
caviae catalyzes the addition of a water molecule to
trans-2-enoyl coenzyme A (CoA), with a chain-length of 4-6 carbons, to produce the corresponding
(R)-3-hydroxyacyl-CoA. It forms a dimer of identical
subunits with a molecular weight of about 14,000 and is involved in
polyhydroxyalkanoate (PHA) biosynthesis. The crystal structure of the
enzyme has been determined at 1.5-Å resolution. The structure of the
monomer consists of a five-stranded antiparallel Metabolism of various fatty acids via The A. caviae (R)-hydratase is essentially
involved in the biosynthesis of an energy storage material of PHA,
functioning as a monomer-supplying enzyme (16, 17). It is encoded as a phaJ gene in the PHA biosynthesis operon, including the
phaC and phaP genes coding for PHA synthase (16)
and granule-associated protein (21), respectively. The hydratase acts
on the The reaction mechanism of the (R)-hydratase has not been
well characterized, in contrast to the rat crotonase. Recently, a highly conserved amino acid sequence, referred to as the hydratase 2 motif, has been identified by comparing the hydratase 2 domains of
peroxisomal MFE-2s and several fungal and bacterial proteins (15). On
the basis of the sequence comparison, pKa measurement, and mutagenesis, it has been proposed that in the human
hydratase 2, Glu366 and Asp510 play a critical
role in the catalytic reaction through an acid-base mechanism that is
similar to that for the crotonase (15). However, this cannot be applied
to the case of the A. caviae enzyme. Although Asp510 of the human enzyme, one of the hydratase 2 motif
residues, corresponds to Asp31 of the A. caviae
enzyme, there is no counterpart of Glu366 in the A. caviae enzyme, because of the lack of a region corresponding to
the N-terminal half (residues from 319 to 483) of the human enzyme
where Glu366 is located. Therefore, if the catalytic
reaction of the A. caviae hydratase also proceeds via an
acid-base mechanism, which residue partners with Asp31 to form the
catalytic dyad?
A preference for the chain length of the CoA thioester substrate is
another remarkable difference between bacterial and peroxisomal enzymes. Because no structure of either bacterial or peroxisomal enzymes has been reported, it is not clear whether or not the difference in substrate specificity is linked to the large difference in the polypeptide chain length of these proteins.
To elucidate the mechanisms of the catalytic reaction and recognition
of the chain length of substrates, a three-dimensional structure of the
enzyme is indispensable. Here we report the first crystal structure of
(R)-hydratase. The structure of A. caviae (R)-hydratase has a so-called "hot dog" fold as a main
frame, with an overhanging segment that contains conserved residues
including the hydratase 2 motif residues. The catalytic residues
Asp31 and His36 are on the overhanging segment
and are located at the dimer interface. The substrate-binding site is
tunnel-shaped, which accounts for the preference for a substrate with
chain length of four to six. The structure will provide invaluable
information for the application of this enzyme to the production of
PHAs that have attracted considerable attention because of their
potential as renewable and biodegradable plastics (23-27). It will
also provide a plausible model for the human enzyme, the deficiency of
which causes a peroxisomal disorder (28-30).
Protein Expression, Crystallization, and Data
Collection--
Recombinant (R)-hydratase was overexpressed
in Escherichia coli and purified according to the procedure
described by Fukui et al. (17). Because the sample was found
to be degraded perhaps due to contaminated proteinases, further
purification by gel filtration was performed with a Superdex 70pg
column (Amersham Biosciences). Crystals of the enzyme were obtained by
sitting-drop vapor-diffusion using a mother liquor containing 20%
polyethylene glycol 4000, 5% 2-propanol, and 20 mM HEPES
(pH 7.0), as described previously (31). The crystallization experiments
were set up at 25 °C. Large crystals were grown in 2-3 weeks to
maximum dimensions of 0.5 × 0.1 × 0.04 mm and were
stabilized in a solution containing 28% polyethylene glycol 4000, 5%
2-propanol, and 20 mM MES (pH 6.0). These crystals belong
to space group C2, with cell parameters a = 111.5 Å, b = 59.3 Å, c = 47.3 Å and
For the preparation of heavy atom derivatives, the
(R)-hydratase crystals were transferred to a synthetic
mother liquor containing 28% polyethylene glycol 4000, 5% 2-propanol,
and 100 mM MES (pH 6.0). Three isomorphous heavy atom
derivatives were prepared with 1 mM HgCl2, 3 mM K3IrCl6, or 3 mM
K2PtCl4. All sets of derivative data were
collected using CuK Structure Determination and Refinement--
Initial phase
calculation was conducted with the Native 1 and derivative data sets.
Two mercury sites were identified by calculating the
difference-Patterson function using PHASES (34). Two iridium sites and one platinum site were identified using the
difference-Fourier technique. Heavy atom parameters were refined at
3.5-Å resolution in PHASES, including the anomalous dispersion signals
of mercury. Following solvent-flattening density modification
and phase extension to 3.0-Å resolution, the electron density map was
clearly interpretable. A model was built interactively using XTALVIEW
(35) and TURBO-FRODO (36). Because the encoded first Met residue does
not exist in the mature enzyme due to a post-translational modification
(17), the sequence number was started with the encoded second residue Ser as the first. This model was positioned in a unit cell by molecular
replacement using REPLACE (37) calculated against the Native 2 data
set, and then refined with a simulated annealing protocol using X-PLOR
(38) and CNS (39). The final model includes residues 2-133 and 1-133
for chains A and B, respectively, one 2-propanol, and 347 water
molecules. Fig. 1 shows a representative (2mFo Mutagenesis, Activity Assay, and CD Spectra
Measurement--
Site-directed mutagenesis was performed by means of
the PCR method (41) and was confirmed by DNA sequencing. The PCR
primers containing mutational sites were designed in inverted
tail-to-tail directions and used for the amplification reaction with
the expression vector for the (R)-hydratase (pETNB3) as a
template. The oligonucleotide sequences of the primers used are as
follows: 5'-CTCTCGGAGGCCTTCAACCCCCTGC-3' for the D31A
mutation; 5'-CTCTCGGAGGACTTCAACCCCCTGGCCCTGGACCCGGCC-3' for
the H36A mutation; 5'-CGCGGCGAAGGCGGCTACCTCCGCC-3' for the D31A and
H36A mutations; and 5'-CTCTTCGCCGGGCTGCTGGGCCAGCAG-3' and
5'-GCTGGCGAGCAGCATGCCGTGGACTATGGG-3' for the S62A mutation (the changed
codons causing the mutations are indicated as boldface letters). The
products were self-ligated and transformed into E. coli BL21
(DE3). The expression, purification, and activity assay for the mutant
enzymes were carried out in the manner described previously (17) for
the wild-type enzyme. The structural conformations of the mutants were
evaluated by far-UV CD spectroscopy using a Jasco J720
spectropolarimeter in a range of 200-250 nm with a 1-mm path length
quartz cuvette at 20 °C.
The crystal structure of the (R)-hydratase was solved
by isomorphous replacement with anomalous scattering using the
mercury, iridium, and platinum derivatives. The final model was
refined to 1.5-Å resolution with crystallographic and free (5% of the total reflections) R-factors of 20.3 and 23.1%,
respectively (Table I). The asymmetric
unit contained two polypeptide chains, referred to as A and B, which
together formed a functional dimeric molecule. Because the first
encoded residue, methionine, is post-translationally deleted (17) and
not present in the mature protein, the encoded second residue serine
represents residue 1 in our numbering system. Chain B consists of all
of the 133 amino acid residues, whereas chain A contains 132 residues,
because Ser1 was not well defined in the electron density
map. Both chains are effectively identical with a root-mean-square
(r.m.s.) deviation between the 130 C-sheet and a
central
-helix, folded into a so-called "hot dog" fold, with an
overhanging segment. This overhang contains the conserved residues
including the hydratase 2 motif residues. In dimeric form, two
-sheets are associated to form an extended 10-stranded
-sheet,
and the overhangs obscure the putative active sites at the subunit
interface. The active site is located deep within the substrate-binding
tunnel, where Asp31 and His36 form a catalytic
dyad. These residues are catalytically important as confirmed by
site-directed mutagenesis and are possibly responsible for the
activation of a water molecule and the protonation of a substrate
molecule, respectively. Residues such as Leu65 and
Val130 are situated at the bottom of the substrate-binding
tunnel, defining the preference of the enzyme for the chain length of
the substrate. These results provide target residues for protein
engineering, which will enhance the significance of this enzyme in the
production of novel PHA polymers. In addition, this study provides the
first structural information of the (R)-hydratase
family and may facilitate further functional studies for members of the family.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-oxidation includes a
hydration step in which 2-enoyl coenzyme A
(CoA)1 intermediates are
converted to 3-hydroxyacyl-CoAs. Two stereoisomers of the product are
used in different metabolic pathways, and, accordingly, enzymes with
different stereospecificities are involved in each pathway. The enzyme
responsible for the production of the S-isomer is the
classical enoyl-CoA hydratase (crotonase) and is involved in
degradation of fatty acids in mitochondria, peroxisomes, and bacterial
cells. The rat mitochondrial enzyme has been studied extensively. The
rat enzyme has a specific activity for CoA thioesters of straight chain
fatty acids with a broad range of chain lengths
(C4-C16) (1). It is a hexamer of identical subunits composed of 261 residues in the mature form (2). Extensive studies (3-5), including crystallographic analyses (6-8), have revealed that two amino acid residues, Glu144 and
Glu164, are important for the catalytic reaction that
proceeds through an acid-base mechanism. However, the enzyme
responsible for the production of the R-isomer,
(R)-specific enoyl-CoA hydratase, hydratase 2, or
D-hydratase (hereafter referred to as
(R)-hydratase), has been identified recently in mammals,
yeast, and some polyhydroxyalkanoate (PHA)-producing bacteria. In
mammals, this enzyme occurs as a domain of the peroxisomal
multifunctional enzyme type 2 (MFE-2) and is involved in the
degradation of very long chain and 2-methyl-branched fatty acids and
the biosynthesis of bile acids (9-13). The yeast enzyme is also
localized in the peroxisome as a part of MFE-2 but is involved in the
degradation of straight chain fatty acids (14). These eucaryotic
enzymes are similar in size, composed of about 300 amino acid residues,
and are highly homologous (15). Bacterial (R)-hydratases
have been found to date in Aeromonas caviae (16, 17),
Pseudomonas aeruginosa (18), Rhodospirillum rubrum (19), and Methylobacterium rhodesianum (20), all
of which are known to accumulate PHA granules in their cells. These enzymes have been characterized at the molecular level except for the
case of M. rhodesianum. However, the A. caviae
enzyme is the only bacterial one whose physiological role has been
identified to date.
-oxidation intermediates trans-2-enoyl-CoAs to
produce (R)-3-hydroxyacyl-CoAs, which are subsequently
polymerized to form PHA by PHA synthase. The hydratase, and the
synthase as well, preferably act on the substrates with a chain length
of 4-6 carbon atoms, accounting for the fact that the produced PHA is
a copolymer of (R)-3-hydroxybutyric (C4) and
(R)-3-hydroxyhexanoic (C6) acids (22). Unlike
the eucaryotic enzymes, this bacterial (R)-hydratase is a
monofunctional enzyme forming a homodimer of identical subunits with a
molecular weight of 13,954. The mature polypeptide chain contains 133 amino acid residues (17). Interestingly, despite the lack of a
physiological relationship with the eucaryotic enzyme, the A. caviae enzyme has significant sequence homology with the
C-terminal region of the eucaryotic enzyme (e.g. a 38.4%
identity with the yeast enzyme for a limited region of 73 amino acid
residues (16)), indicating that they are derived from a common ancestor.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
= 112.9°, and contain two subunits per asymmetric unit. They
diffracted CuK
radiation up to a resolution of 2.0 Å.
The intensity data (Native 1) were processed to a resolution of 2.5 Å with an Rmerge of 7.8% using PROCESS (Rigaku).
Higher resolution data (Native 2; up to 1.5-Å resolution) from a
flash-cooled crystal were collected using synchrotron radiation at
SPring-8, Harima, Japan (32), and were processed with an
Rmerge of 5.5% using MOSFLM (33). On
flash-cooling, the cell parameters of the crystal changed slightly to
a = 110.0 Å, b = 57.8 Å,
c = 47.0 Å, and
= 112.7°. The statistics of
the data collection are summarized in Table I.
radiation at room temperature and
processed with PROCESS (Table I).
DFc) electron density
map of the protein. The stereochemistry was verified using PROCHECK
(40). The atomic coordinates have been deposited in the Protein Data
Bank (accession code 1IQ6).
View larger version (72K):
[in a new window]
Fig. 1.
A representative
A-weighted
(2mFo
DFc)
electron density map at 1.5-Å resolution (contoured at
1
).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
atoms of each monomer (residues
4-133) of 0.59 Å. However, small conformational differences occur in
the region from Asn33 to Arg49 and its adjacent
region from Leu65 to Ile75 (r.m.s. deviation of
1.28 Å for 28 C
atoms). As a result, the segment from
Pro39 to Ala42 forms a 310-helix in
chain B, whereas it is an
-helix in chain A. This helix in chain B
has more contact with another molecule in the crystal than that in
chain A. Thus, it is not clear whether the differences for these
regions are merely due to the different environment in the crystal
lattice or are linked to the enzymatic function of the molecule. On the
other hand, the conformational difference for the N-terminal residues
before Ser4 is very large, probably due to the intrinsic
flexibility, and was excluded from the calculation of the deviation.
Apparently this N-terminal region is not functionally important. The
Ramachandran plot shows that almost all of the non-glycine residues are
in allowed regions, with the exception of Glu48A ((
,
) = (67.6°,
31.1°)) which is in a disallowed region. This unusual conformation for Glu48A is, however, validated with
the omit map calculated from the model without this residue. Note that
Glu48E has very similar values for the dihedral angles
((
,
) = (68.1°,
28.5°)), which lie in the allowed
region for the left-handed helical conformation.
Data statistics
The Monomer Structure--
The (R)-hydratase monomer
consists of a five-stranded antiparallel -sheet and four helices
(Fig. 2, a and b).
The order of strands in the
-sheet is 1-3-4-5-2, and the length of
each strand ranges between 7 and 11 residues. All the helices (H1-H4)
are connected between the first strand, S1, and the second strand, S2.
Strand S1 is followed by the first
-helix H1 having three turns. Two
contiguous short helices with one turn for each follow helix H1. In
chain A, both helices are in the
-helical conformation, whereas
in chain B, helices H2 and H3 are in the
- and
310-helical conformations, respectively, as described
above. Helix H3 is then followed by a long extended loop of about 10 residues, and finally the central helix H4, with four turns, comes to
be connected to strand S2. Helix H4 has a very interesting feature. It
is a complex of three helices that are fused continuously along a
common axis. The first and the last portions,
Gly54-Leu57 and
Phe61-Gln68, form
-helices, whereas the
middle portion, Leu57-Phe61, forms a
310-helix. Helix H4 is wrapped around by the largely wound
-sheet like a hot dog, where the
-sheet and the central helix H4 are a "bun" and a "sausage," respectively.
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The hot dog fold has been originally observed for the
-hydroxydecanoyl thiol ester dehydrase from E. coli (42)
and later for the 4-hydroxybenzoyl-CoA thioesterase from
Pseudomonas sp. CBS-3 as well (43). Two hot dog repeats in a
single peptide, called the "double hot dog" fold, have also been
reported for the medium chain length acyl-CoA thioesterase II from
E. coli (44). Although they have a fold similar to the core
structure, (R)-hydratase has a feature that distinguishes it
from other hot dog fold enzymes. The most notable difference is an
overhanging segment situated between the first strand S1 and the
central helix H4. This region in other hot dog fold enzymes is a
relatively short loop of around 15 residues, whereas in
(R)-hydratase, it is a large fragment of about 35 residues.
This overhang in (R)-hydratase is rather compact and has
little interaction with the hot dog main body, other than the central
helix H4. The B-factor values for the main chain atoms in
the region from Asn33 to Arg49 (from helix H2
to the loop after helix H3; see Fig. 2b) are relatively high
compared with those in the hot dog main frame (the average B-factors for the C
atoms of chains A and B are 18.01 and
17.03 Å2, respectively, whereas those in the region from
Asn33 to Arg49 of both chains are 23.65 and
23.24 Å2, respectively), indicating the intrinsic lability
of this region. This is consistent with the observed conformational
differences between chains A and B as described above. The residues in
this overhang are highly conserved among the (R)-hydratases,
indicating the importance of this overhang. In fact, the catalytically
important residues Asp31 and His36 are involved
in this segment (see below). Thus, this overhang is both structurally
and functionally characteristic to the (R)-hydratase.
A secondary structure-based sequence alignment shows that
(R)-hydratase from A. caviae has some homology
with the -hydroxydecanoyl thiol ester dehydrase from E. coli (Fig. 3). A total of 12 identical residues are localized in S1, H4, S3, and S5 (the
(R)-hydratase notation). This suggests that
(R)-hydratase and dehydrase may have been
derived from a common ancestor protein. This is not surprising because
the functions of both enzymes are very similar; both catalyze the
addition/elimination of a water molecule to/from 2-enoyl/(R)-3-hydroxyacyl thioesters (CoA and acyl-carrier
protein thioesters for (R)-hydratase and dehydrase,
respectively). In the case of (R)-hydratase, the overhang,
including the functionally important residues, may have been inserted
in the course of molecular evolution.
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The Dimer Structure--
In the dimeric form (Fig. 2, c
and d), two monomers make extensive contacts, burying
1426.02 Å2 (about 20%) of the surface area of the
monomer, 68% of which is apolar (values from
www.biochem.ucl.ac.uk/bsm/PP/server/). Helices H1 and H4 and strand S2
are situated at the subunit interface. Strands S2s are associated in an
antiparallel direction so that the two -sheets form a 10-stranded
-sheet as a whole. The extended 10-stranded
-sheet wraps around
the two central helices, with the overhangs covering the gaps that
occur at the interface of the two subunits. Helices H1 and H4 are
associated with their counterparts across the molecular dyad, facing
side by side in an antiparallel direction. These paired helices are
then packed so that the axes of the helices run almost perpendicular to
each other. Thus, the two H4 helices are sandwiched between the two H1
helices and the 10-stranded
-sheet. This results in the helix H4
being almost entirely buried in the molecule, with the C terminus of
the helix partially exposed to the solvent. In fact, helix H4, which is
composed of 14 residues (Gly54-Gln67), is
dominantly hydrophobic with the exception of only three polar residues
(Ser59, Ser62, and Gln67).
Gln67, located at the C terminus of the helix, is exposed
to the protein surface, but Ser59 and Ser62 are
buried in the molecule. Ser59 is situated at the
310-helix portion of helix H4 facing Ser59'
(the prime indicates that the residue is from the adjacent
polypeptide chain) across the molecular dyad. But these two serine
residues are not hydrogen-bonded to each other; rather, they are
hydrogen-bonded to the carbonyl oxygen of Met55 (in chain
A) or Leu56 (in chain B) in the same helix, stabilizing the
310-helix structure. Ser62 hydrogen bonds to
O
2 of Asp31' and a water molecule and may be
functionally important (see below). On the other hand, helix H1 is
half-exposed to solvent. It contains 11 residues
(Ala19-Ser29), including two polar ones
(Glu21 and Ser29). On the exposed face of the
helix are lined alanine residues (Ala19, Ala20,
Ala23, Ala24, and Ala27) in
addition to Glu21. Ser29 is buried in the
protein, hydrogen-bonded to the carbonyl oxygen of Phe25
and the amide nitrogen of Asp31. The dimeric molecule has
an ellipsoidal shape, with dimensions of ~56 × 46 × 35 Å.
The Hydratase 2 Motif and Other Homologous Proteins-- Only a few examples (17-19) of bacterial (R)-hydratases have been described thus far. They are similar in polypeptide chain length (133-174 residues), substrate specificity (for short chain length enoyl-CoA), and amino acid sequence (about 45%). Eucaryotic enzymes (around 300 residues), on the other hand, are about twice as large as the bacterial enzymes and have a substrate specificity for very long chain length, 2-branched, and C27 bile acid intermediate 2-enoyl-CoAs. Qin et al. (15) recently identified the highly conserved amino acid sequence, referred to as the hydratase 2 motif ([YF]-X(1,2)-[LVIG]-[STGC]-G-D-X-N-P-[LIV]-H-X(5)-[AS]), by comparing the hydratase 2 domains of MFE-2s and several fungal and bacterial proteins. The sequence alignment shows that the bacterial (R)-hydratases also have this motif, with a deviation in which the glycine residue is replaced by Glu30 for the A. caviae enzyme (Fig. 3). The strictly conserved residues in the motif must be important for either catalysis or structural stability.
In the (R)-hydratase structure, the residues corresponding
to the hydratase 2 motif are located in the region from the C-terminal half of helix H1 to the short helix H3
(25FAALSEDFNPLHLDPAFA42). An inspection of the
structure of (R)-hydratase indicates that most of these
residues, except for Asp31 and His36 which are
important in catalysis (see below), have structural roles, stabilizing
the structure via hydrophobic and hydrogen bonding interactions. It is
interesting to note that Glu30 adopts a left-handed helical
conformation ((,
) = (71.8° and 16.3°) (chain A) and
(74.3° and 13.0°) (chain B)), which accounts for the high
preference for the glycine residue at this position in other hydratase
2 motif enzymes. Due to the conformation of Glu30, the side
chain of the following catalytic residue Asp31 can be
properly oriented to the catalytic site cavity. Note that the side
chain of Glu30 is hydrogen-bonded to Lys15',
which may contribute to stabilizing the energetically less favorable conformation. Another notable residue is Ser29. This
residue, situated at the C terminus of the helix H1, is buried in the
protein, hydrogen-bonded to the carbonyl oxygen of Phe25
and the amide nitrogen of Asp31. This interaction may also
stabilize the conformation of the peptide
Glu30-Asp31.
Other homologous proteins include the -subunits of
fatty-cid synthases and hypothetical proteins from various sources, and collectively, they are classified into a family in which a conserved domain referred to as the MaoC-dehydratase domain in the Pfam data base
(CD ID: pfam 01575) (45) is shared. Therefore, the (R)-hydratase structure described here provides the first
and representative example for this conserved domain.
Active Site and Implications for the Catalytic
Mechanism--
Attempts to prepare crystals of the enzyme with
substrates or competitive inhibitors have, thus far, been unsuccessful.
It is possible, however, to speculate on the location of the active site of the (R)-hydratase on the basis of the structural
similarity with the E. coli -hydroxydecanoyl thiol ester
dehydrase, for which the active site has been identified (42).
Two active sites related by the molecular dyad are formed at the
interface of the two monomers. Each active site is located deep within
the substrate-binding tunnel, which is contributed by both of the
monomers. Although the environment of the tunnel is dominantly
hydrophobic, three charged or polar residues, Asp31,
His36, and Ser62', are localized at the bottom
of the tunnel, making a complex hydrogen bond network with five water
molecules (Fig. 4a). An inspection of the active site structure suggests that Asp31
and His36 are the most promising candidates for the
catalytic residues.
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To examine the importance of these residues, as well as
Ser62, for catalysis, four mutants, D31A, H36N, H36A, and
S62A, were prepared and assayed. For the former three mutants, the
Vmax values were reduced by
~105-fold compared with the wild-type enzyme, from
6.2 × 103 units mg1 to 9.7 × 10
2, 5.7 × 10
2, and 9.4 × 10
2 units mg
1, respectively. The
Km values were largely unchanged (23, 11, and 16 µM for the three mutants, respectively, compared with 29 µM for the wild-type enzyme). These data indicate that Asp31 and His36 are essential for catalysis. It
should be noted that the CD spectra for these mutants showed profiles
similar to the wild-type enzyme (data not shown), indicating that the
observed reduced activities for these mutants are not due to
conformational changes of the proteins. The mutant S62A, on the other
hand, showed Vmax and Km
values of 9.6 × 10 units mg
1 and 27 µM, respectively, and thus retained significant activity, suggesting that Ser62 is less important for catalysis. An
observation of the hydrogen bond between Asp31 and
Ser62' may suggest that Ser62 plays a
significant role in maintaining the orientation of the side chain of
Asp31 to be favorable for catalysis and in increasing the
overall efficiency of the catalysis.
Asp31 and His36 are conserved in other bacterial and eucaryotic (R)-hydratases (Fig. 3), which also indicate the importance of these residues for enzyme function. In the case of the human enzyme, on the other hand, it has been proposed that Glu366 and Asp510 play critical roles in the hydratase reaction, acting in a similar manner to the two catalytic glutamic acid residues of the rat crotonase (15). Whereas Asp510 of the human enzyme corresponds to Asp31 of the A. caviae enzyme, there is no counterpart of Glu366 in the A. caviae enzyme due to the lack of a corresponding region to the N-terminal half of the human enzyme, where the Glu366 residue is located. Unfortunately, the investigators in Ref. 15 failed to examine the importance of His515 in the human enzyme, which is equivalent to His36 of the A. caviae enzyme, because the H515A mutant could not be correctly folded. Thus, the possibility that His515 of the human enzyme has a catalytic role cannot be ruled out. For the A. caviae enzyme, the catalytic importance of His36 has been demonstrated, as described above.
On the basis of the crystal structure analysis and mutagenesis
experiments, and with the aid of a docking model of crotonyl-CoA to the
substrate-binding tunnel (Fig. 4b), we propose that the Asp31 and His36 residues play critical roles in
the catalytic reaction of the enzyme as follows. Asp31 may
activate a water molecule by abstracting a proton from a water
molecule. The activated water molecule would then attack the carbon
atom C3 of crotonyl-CoA, and cooperatively His36 may donate
a proton to the C2 carbon atom of the substrate, thus completing one
cycle of the catalytic reaction. Note that the docking model also
suggests the importance of Gly54. The amide group of
Gly54 may hydrogen-bond to the carbonyl group of the
thioester bond of the substrate, thus keeping the substrate in an
orientation in which the activated water molecule is restricted to
attacking the C3 atom of the substrate from the re-face,
which is related to the C2-C3 double bond, so as to form the
R-isomer of the product. The amide group of
Gly54 may also function as the oxyanion hole, which, in
addition to the dipole of the central -helix H4, stabilizes the
reaction intermediate. This interaction between the substrate and the
protein requires a glycine residue at position 54 exclusively,
otherwise steric hindrance between the carbonyl group of the substrate
and the side chain of the residue would have disruptive influences.
The proposed reaction mechanism is very similar to that of
-hydroxydecanoyl thiol ester dehydrase from E. coli,
which utilizes His70 and Asp84' as catalytic
residues (42). However, there is a large difference between the two
proteins with respect to the location of the two active site residues.
In the case of the dehydrase, the active site residues Asp and His are
located on different subunits, whereas in the case of
(R)-hydratase, they are on the same subunit. The C
atoms
of the histidine and aspartic acid residues from both proteins are
located spatially in different positions. Interestingly, even the
location of the C
atoms of the catalytic residues are different, the
architecture of the active site is very similar, that is, the
catalytically significant N
1, O
1, and amide nitrogen atoms of the
His, Asp, and Gly residues, respectively, can be well superimposed.
Note that Ser62' in the A. caviae enzyme, which
is located on the central helix, is topologically equivalent to
Asp84' of the E. coli dehydrase and that the
O
1 atom of the Ser residue and the O
2 atom of the Asp residue
come close in the same superposition.
Substrate-binding Site-- The substrate-binding site is a tunnel-shaped pocket 15 Å long and 6 Å wide. The mouth of the tunnel is formed by Phe47 and residues from the region immediately after strand S2 (Phe83-Pro86) and the N-terminal region of strand S2' (Ile75'- Leu77') (Fig. 4a). Of these residues, Phe47, Phe83, Pro86, Ile75', and Tyr76' are conserved in bacterial (R)-hydratases (Fig. 3), indicating that they are important for interactions with the substrate or the formation of the mouth of the tunnel. The docking model (Fig. 4b) indicates that the 3'-phosphate ADP moiety of the substrate may be bound at the surface of the enzyme and that the pantetheine and acyl chain moieties are bound inside the tunnel. Arg103, Arg106, and Lys131 are located around the mouth which may be involved in the binding of the phosphate groups of the 3'-phosphate ADP moiety. The tunnel can be divided into two parts at the catalytic site. They correspond to the regions for binding of the pantetheine and acyl chain moieties. The pantetheine-binding site is contributed by the two subunits, whereas the acyl chain-binding site is formed by a single subunit. The acyl chain-binding site is surrounded by the side chain atoms of Ser62, Leu65, Pro70, Ser74, Tyr76, and Val130, in addition to the main chain atoms of these residues and Gly66 and Ile75. These residues define the size of the acyl chain-binding pocket, which is sufficient to accommodate a substrate with a carbon chain length of four to six, but which is insufficient to accommodate a longer one. To accommodate substrates with much larger chains, the tunnel may need to be burrowed further into the hydrophobic core of the protein. Thus these residues can be targets for site-directed mutagenesis to modify the preference for the chain length of the substrate, which will generate monomers with various structures and enable novel PHAs to be designed.
Compared with the bacterial enzyme, the eucaryotic enzymes have a
larger molecular size of about 300 residues and prefer substrates with
a longer carbon chain. For example, the yeast enzyme can act on
2-decenoyl-CoA (14), and the human enzyme on 2-methylhexadecenoyl-CoA and 3,7
,12
-trihydroxy-5
-cholest-24-enoyl-CoA (11). It does not appear that the acyl chain-binding site housed in the hot dog
structure is able to provide sufficient room for these large substrates. Thus it is likely that the architecture of the acyl chain-binding site in the eucaryotic enzyme is different from that in
the bacterial one, that is, the N-terminal portion of the eucaryotic
enzyme may contribute to the large part of it. Furthermore, it should
be noted that residues corresponding to Ile75,
Tyr76, and Leu77 of the A. caviae
enzyme around the mouth are not conserved between the eucaryotic and
bacterial enzymes, in contrast to residues Phe47,
Phe83, and Pro86 which are highly conserved in
both groups. This suggests that the N-terminal region of strand S2
might not be involved in interactions with substrates. This further
indicates that in the eucaryotic enzyme, the C-terminal portions
corresponding to the bacterial enzyme do not associate to form a
homodimer with a 10-stranded
-sheet. If this is true, the N-terminal
portion might form another hot dog fold and associate with the
C-terminal hot dog moiety, that is the enzyme would have a double hot
dog fold, as is observed in the E. coli medium chain
acyl-CoA thioesterase II (44).
In conclusion, the data herein reveal that (R)-hydratase is
a member of the hot dog fold enzyme superfamily. Furthermore, the
functionally important overhanging segment, including the hydratase 2 motif residues, is inserted into the hot dog core structure. The
structure identifies the key catalytic residues Asp31 and
His36, which were further confirmed by mutagenesis. It is
proposed that Asp31 may function as an activator of a water
molecule which attacks a substrate molecule and His36 as a
proton donor to the substrate. The acyl chain-binding pocket is
circumvented by residues such as Leu65 and
Val130, which define the selectivity for the chain length
of substrates. The issue of whether the overhang, which has relatively
high B-factor values, changes its conformation on binding of
substrates is an intriguing one. Crystallographic studies of the
enzyme-substrate complex are currently in progress to address this
issue as well as the detailed catalytic reaction mechanism. In
addition, future studies will include the modification of the
specificity for the carbon chain length of substrates by targeting the
residues involved in the acyl chain-binding site, in an attempt to
obtain enzymes with a specificity useful for the production of PHA
polymers with desired structures. Finally, this study provides the
first insight into the structure of the (R)-hydratase family
with a signature of the hydratase 2 motif, which will facilitate
further functional studies of this class of enzymes.
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ACKNOWLEDGEMENTS |
---|
We thank S. Adachi and S.-Y. Park for assistance with the x-ray diffraction measurements at the BL44B2 station at SPring-8, Harima, Japan. We also thank H. Miyatake, H. Shimizu, and S. Kobayashi for technical assistance.
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FOOTNOTES |
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* This work was supported by CREST of the Japan Science and Technology Corporation, by the Special Postdoctoral Researchers' Program in RIKEN (to T. H.), and by Grants-in-aid for Scientific Research 12780431 from the Ministry of Culture, Education, Science and Sports of Japan (to T. H.).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 1IQ6) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Present address and to whom correspondence should be addressed: RIKEN Harima Institute/SPring-8, 1-1-1 Koto, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan. Tel.: 81-791-58-2912; Fax: 81-791-58-2913; E-mail: hisano@postman.riken.go.jp.
¶ Present address: Dept. of Innovative and Engineered Materials, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, 4259 Nagatsuda, Midori-ku, Yokohama 226-8502, Japan.
Present address: Dept. of Synthetic Chemistry and Biological
Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto 606-8501, Japan.
Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M205484200
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ABBREVIATIONS |
---|
The abbreviations used are: CoA, coenzyme A; PHA, polyhydroxyalkanoate; MFE-2, multifunctional enzyme type 2; MES, 2-morpholinoethanesulfonic acid; r.m.s., root-mean-square.
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