From the Department of Molecular Biology and
§ The Skaggs Institute for Chemical Biology, The Scripps
Research Institute, La Jolla, California, 92037 and ¶ Center for
Novel Agricultural Products, Department of Biology (Area 8),
University of York, P. O. Box 373, York YO10 5YW, United Kingdom
Received for publication, October 2, 2002
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ABSTRACT |
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The crystal structures of an acetyl esterase,
HerE, and its complex with an inhibitor dimethylarsinic acid have been
determined at 1.30- and 1.45-Å resolution, respectively. Although the
natural substrate for the enzyme is unknown, HerE hydrolyzes the acetyl groups from heroin to yield morphine and from phenyl acetate to yield
phenol. Recently, the activity of the enzyme toward heroin has been
exploited to develop a heroin biosensor, which affords higher
sensitivity than other currently available detection methods. The
crystal structure reveals a single domain with the canonical Heroin (3,6-diacetylmorphine) is a highly addictive, synthetic
derivative of the alkaloid morphine (Fig.
1A). The instability of the
3-acetyl suggests that it can undergo rapid spontaneous hydrolysis to
give 6-acetylmorphine. HerE is known to hydrolyze the 6-acetyl of
6-acetylmorphine to yield morphine (kcat = 12.6 ± 0.6 s/
hydrolase fold with an acyl binding pocket that snugly accommodates the
acetyl substituent of the substrate and three backbone amides that form
a tripartite oxyanion hole. In addition, a covalent adduct was observed
between the active site serine and dimethylarsinic acid, which inhibits
the enzyme. This crystal structure provides the first example of an
As-containing compound in a serine esterase active site and the first
example of covalent modification of serine by arsenic. Thus, the HerE
complex reveals the structural basis for the broad scope inhibition of
serine hydrolases by As(V)-containing organic compounds.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
1,
Km = 0.5 ± 0.06 mM) and was
originally identified from the Rhodococcus sp. strain H1 by
selective growth using heroin as the sole carbon source (1).
Identification of this esterase has facilitated development of a
coupled enzyme biosensor for heroin detection (Fig. 1A) with
higher sensitivity than other currently available technology (2). In
brief, hydrolysis of heroin by HerE generates morphine, which is the
substrate for NADPH-dependent morphine dehydrogenase. The
activity of morphine dehydrogenase is directly monitored by bacterial
luciferase, which outputs a bioluminescent signal at 490 nm (Fig.
1A) (2). The higher activity (kcat
Km
1) of HerE toward smaller substrates
such as phenyl acetate (Fig. 1B)
(kcat = 3.0 ± 0.05 s
1, Km = 70 ± 8 nM at pH = 6.4) provides a benchmark for engineering
the esterase binding pocket to accept bulkier substrates more
efficiently. Hence, the crystal structure of this acetyl esterase
should provide substantial insights for structure-based design of
second generation heroin biosensors with improved sensitivity.
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Fig. 1.
Substrates and mechanism for arsenic
inhibition. A, schematic for heroin biosensor. The
3-acetyl ester of heroin undergoes rapid spontaneous hydrolysis,
whereas the 6-acetyl ester is hydrolyzed by acetyl esterase
(HerE) to give morphine. Morphine dehydrogenase
(MorA) oxidizes the alcohol to the ketone in an
NADPH-dependent reaction. The reduction of
NADP+ to NADPH is monitored directly by bacterial
luciferase, which outputs a signal at 490 nm. B, structures
of substrate phenyl acetate and inhibitor dimethylarsinic acid.
C, hydrolysis of the acyl enzyme intermediate. Hydrolysis of
both heroin and phenyl acetate yields the common acyl enzyme
intermediate. Hence, the complex with dimethylarsinic acid resembles
the transition state for hydrolysis of the acyl enzyme for both
substrates investigated.
In addition, our structure with the inhibitor dimethylarsinic acid (Fig. 1B) demonstrates for the first time the covalent modification of serine by arsenic. Arsenic lies directly below phosphorous in the periodic table and shares some similar properties, but in general, arsenic is more reactive. As(V)-containing organic compounds comprise a known class of broad scope serine esterase/protease inhibitors (3, 4). Usually, arsonic R-As(V)O3H2 and arsinic R2-As(V)O2H compounds have Ki values in the µM-mM range (3, 4), but surprisingly, the mechanistic basis for inhibition of esterase/proteases by As(V) compounds has never been defined from a crystal structure. The HerE esterase is the ideal model system for studying inhibition by dimethylarsinic acid, since the methyl groups correspond to the acetyl moiety that is hydrolyzed from heroin and phenyl acetate (Fig. 1C).
The most common form of inorganic arsenic is
As(V)O, which
are less cytotoxic than the inorganic form but may have a longer term
carcinogenic impact in chronic exposure (5). These As(III) arsenicals
are generally regarded as the more reactive and most toxic species (5),
and crystal structures in the Protein Data Bank confirm that As(III)
compounds form covalent adducts with cysteine (6). Our HerE crystal
structure now demonstrates that dimethylarsinic acid, one of the key
metabolites associated with chronic arsenic toxicity, could also form a
covalent adduct with a reactive serine in a catalytic triad at
sufficiently high concentrations.
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EXPERIMENTAL PROCEDURES |
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Cloning, Expression, and Crystallization--
The HerE enzyme
was subcloned into pET-28a(+) (Novagen) (7) and transformed into
BL21-Gold (DE3) cells (Stratagene). Soluble native enzyme (60 mg
liter1 Luria broth) was obtained by overnight
induction with 1 mM
isopropyl-1-thio-
-D-galactopyranoside at ~23 °C and
was ~99% pure after 2 consecutive rounds of nickel nitrilotriacetic
acid-agarose chromatography (Qiagen). The amino-terminal His tag was
removed by overnight thrombin digestion at 4 °C, and the enzyme was
further purified by ion exchange chromatography with a MonoQ column
(Amersham Biosciences). Subsequently, HerE was dialyzed into 20 mM Tris, pH 7.5, 25 mM NaCl and concentrated to
15 mg ml
1 for crystallization trials.
Selenomethionine derivative enzyme was made and purified similarly from
the methionine auxotroph strain B834-(DE3) grown in minimal media
substituted with 0.3 mM selenomethionine (Sigma). The
selenomethionine enzyme was concentrated to 6-7 mg
ml
1 for crystallization in the same protein
buffer. The arsenic adduct, selenium-derivative crystals, were grown in
1.8 M ammonium sulfate, 0.1 M NaCl, 0.1 M cacodylate, pH 6.5, and native crystals were grown in 1.7 M ammonium sulfate, 0.1 M NaCl, 0.1 M BES1 buffer, pH
6.4.
Gel Filtration, Biochemical Activity, and Arsenic
Inhibition--
The apparent native molecular weight of the esterase
was determined using gel filtration chromatography on a Superdex 200 10/30 HR column calibrated with low and high molecular weight protein
standards (Amersham Biosciences). A calibration curve was constructed
from a plot of log molecular weight of the standard versus
Kav. Purified esterase (50 µg) was applied to
the column at a linear flow rate of 0.25 ml
min1, and the elution volume was determined
by monitoring absorption at 280 nm.
Enzyme concentrations were determined using the Coomassie Plus protein
assay reagent kit (Pierce) with bovine serum albumin as the standard.
Steady state kinetic measurements for 6-acetylmorphine were measured at
294 nm in the presence of 10 µg of protein in 100 mM
sodium phosphate buffer, pH 8.0, at 25 °C with a Hewlett-Packard 8452A single beam diode array spectrophotometer. A molar extinction coefficient of = 1050 M
1
cm
1 at 294 nm was used to determine the
concentration of product formed during the reaction.
Km and kcat values were
determined by varying the concentration of 6-acetylmorphine and fitting
the data to the Michaelis-Menten equation using the Grafit 4 software package (Erithacus Software Ltd, Staines, UK). Inhibition measurements for dimethylarsinic acid were performed using phenyl acetate as a model
substrate in conditions designed to mimic the crystallization conditions as closely as possible. The molar extinction coefficient E270 for phenol was determined from a standard curve to be
856.8 M
1
cm
1. The reaction was initiated by adding 150 µl of a 2× substrate solution to 150 µl of a 2× enzyme solution
in a 96-well UV plate (Costar). Steady state kinetics were monitored at
270 nm in the presence of 0.25 µg ml
1
protein in 50 mM BES buffer, pH 6.4 (uninhibited reaction),
and 100 and 50 mM cacodylate buffer, pH 6.4 (inhibited
reaction), at room temperature with a SpectraMax Plus 384 UV plate
reader (Molecular Devices). Km,
KM app, and kcat
values were determined by varying the concentration of phenyl acetate
in serial dilutions (50 µM-100 nM) and
fitting the data to the Michaelis-Menten equation using Kaleidagraph V
3.5 (Abelbeck Software).
Data Collection, Structure Solution, and Refinement--
A
single wavelength anomalous diffraction (SAD) data set was collected at
Stanford Synchrotron Radiation Laboratory (SSRL) beamline 11-1 from a
selenomethionine derivative crystal at a high energy remote wavelength,
and a native data set was collected at SSRL beamline 9-2 (Table I).
The space group is P3221 with a = b = 71.4 Å, c = 105.7 Å (native), and
a = b = 71.6 Å, c = 106.2 Å (complex), with one molecule per asymmetric unit and a Matthews coefficient of 2.1 Å3
Da1 (solvent content of ~42%). The data
were integrated and scaled with HKL2000 (8), and two selenium sites and
one arsenic site were found using the program SOLVE (9), which was
sufficient to phase the data at 2.0 Å (figure of merit = 0.36, all reflections). Density modification was performed using RESOLVE
(figure of merit = 0.61, all reflections) (10), and the
selenomethionine derivative phases were extended to 1.45 Å using
Arp/Warp (11). Arp/Warp then successfully traced and built ~80% of
the structure (11). The remaining model was built manually in O (12),
and the refinement was started in CNS (13) and finished using SHELXL
(14) with final Rcryst = 15.7% and
Rfree = 22.3% for all data. The native structure was refined using the SAD structure as a starting model with
the final Rcryst = 15.3%, and
Rfree = 21.3%.
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RESULTS |
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General Overview of the Structure--
The crystal structure of
HerE was determined by SAD from a high energy remote wavelength (Table
I). The native enzyme and cacodylate
complex were determined at 1.30 and 1.45 Å, and refinement converged
to a final Rcryst = 15.3%,
Rfree = 21.3% and Rcryst = 15.7%, Rfree = 22.3%, respectively. The
enzyme consists of 323 residues with a molecular weight of ~35,000
(GenBankTM accession number U70619). In each structure,
residues 1-317 were modeled in the electron density. The globular
structure has approximate dimensions of ~35 Å × ~45 Å × ~50
Å, and the overall secondary structure is a mixture of -sheet
(15%) and
-helix (36%) (Fig. 2,
A and B). The enzyme is related to the
hormone-sensitive lipase subfamily of enzymes, sharing ~30% sequence
identity with related structures, brefeldin A esterase (15), esterase
2 (16), and Archaecoglobus fulgidus esterase (17). In
brief, HerE has a single domain with the canonical
/
hydrolase
fold, which typifies esterases with a Ser/Cys-Asp/Glu-His catalytic
triad. The fold consists of repeating
-
-
motifs that form a
central, predominantly parallel (except
2) eight-stranded
-sheet
(strand order
1,
2,
4,
3,
5,
6,
7,
8)
surrounded on either side by
-helices (Fig. 2, A and
B). The core
-sheet and the
-
-
motifs are all right-handed, except for the unusual left-handed junction between strand
8 and helix F', which is necessary to properly position the
catalytic histidine residue in the active site (18). In the hydrolase
family of enzymes, insertions are commonly observed in the canonical
structure at the amino and carboxyl termini and at the junction between
strands
6 and
7. Such insertions are believed to modulate the
substrate specificity of the enzyme (18, 19). HerE has two
predominantly helical, ~50-amino acid insertions, one at the amino
terminus (H1, H2) and the other (H3, H4) between strands
6 and
7
(Fig. 2, A and B).
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Crystal Packing Supports a Dimer Model--
Analysis of the
packing within the crystal shows that HerE forms a dimer, where each
monomer is related to the other by a crystallographic 2-fold (Fig.
3). In the dimer interface, strands 8
from each monomer align antiparallel to each other to form four
backbone hydrogen bonds so that the
-sheet from one monomer is
continuous with the
-sheet of the other. In addition, helices
E
and
F' from each monomer form coiled-coil motifs,
E-
E and
F'-
F' at the interface. In total, the dimer interface buries 1890 Å2 (20) and forms 144 van der Waals contacts, 11 hydrogen
bonds, and 8 salt bridges (21). In comparison, the
VH/VL interface of a Fab typically buries
1000-1400 Å2 (22), and hence, the HerE dimer is likely to
be relatively stable in solution. Determination of the native molecular
weight by high resolution gel filtration chromatography supports this model of a stable dimer with an apparent molecular mass of 73 ± 4 kDa. This mode of dimerization is similar to that seen in other members
of the hormone-sensitive lipase subfamily (15).
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Identification of Arsenic in the Active Site--
Crystals grown
in 50 mM cacodylate buffer (dimethylarsinic acid) yielded a
tetrahedral covalent adduct between the Ser-160 O and arsenic (Fig.
4, A and B).
Arsenic was unequivocally identified as the heavy atom from an x-ray
fluorescence scan, an anomalous difference map calculated with data
collected at the arsenic absorption peak (
= 1.0439 Å), and
comparison to native crystals grown in the absence of cacodylate
buffer. From this arsenic adduct, the prototypic catalytic triad was
identified as Ser-160, Asp-260, and His-290 at the carboxyl end of
strands
5,
7, and
8, respectively. Ser-160 (
= 55°,
=
121°) is located in the so-called nucleophilic elbow
within the conserved
GX1SX2G motif (GQSAG), a
signature sequence for the hydrolase family (18). In the arsenic
adduct, the divalent oxygen is in a tridentate oxyanion hole, where it could form hydrogen bonds with the backbone amides of Ala-161, Gly-88,
and Gly-89, located between strand
3 and helix
A (Fig. 4B). Two rotamers of the nucleophilic Ser-160 were refined
in both the native structure (Fig. 4A) and the As complex
(Fig. 4B), with arsenic bound to only one of the possible
conformers. The occupancy of arsenic, therefore, was refined and found
to be 0.61, which implies a mixture of bound and unbound species within
the crystal.
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The Acyl and Alcohol Specificity Pockets--
The
dimethylarsinic acid adduct is 91% buried (20) and forms 32 van
der Waals contacts (21) in the active site. The pro-S methyl
group corresponds to the methyl group of the heroin acetyl ester that
is hydrolyzed by HerE (Figs. 1 and 4B). This methyl sits
snugly inside the acyl binding pocket (Fig. 4B) and suggests that the enzyme may preferentially hydrolyze small acyl groups (three
carbons or less) from esters. The pro-S methyl abuts the -face of Trp-209, forming nine favorable van der Waals contacts. In
addition, Glu-190 is buried at the back of the acyl binding pocket,
forming hydrogen bonds with Ser-217 and a water molecule. The presence
of the charged residue is unexpected given the neutrality of the acyl
ester and differs from the acyl binding pocket of acetylcholinesterase,
which is entirely hydrophobic (23). The electronegative potential in
HerE may facilitate expulsion of the negatively charged product,
acetate, as part of an evolved negative design strategy to avoid
product inhibition. Alternatively, the negative charge may be important
for modulating the specificity of the enzyme for other as yet
unidentified substrates.
The putative alcohol binding pocket is fairly wide compared with
the 6-acetylmorphine substrate. Therefore, it is possible to model
morphine in several different orientations, which taken together
implicate hypothetical interactions with Val-21, Phe-23, Tyr-33,
Ile-92, Gln-159, Leu-208, and Leu-294. Based on this modeling alone, we
speculate possible hydrogen bonding between the morphine nitrogen and
the side chains of either Gln-159 or Tyr-33 but defer definitive
characterization until a complex with morphine becomes available.
Leu-208 ( = 72°,
=
53°) is located at the apex of a
-turn, which may be important in positioning Trp-209 in the
acyl binding pocket. The amino-terminal insertion contributes many of
the residues that line this putative alcohol binding pocket. This
insertion has an average isotropic B value of ~38 Å2
compared with only ~17 Å2 for the rest of the protein,
and portions of this domain are relatively disordered in the electron
density. These elevated B values suggest inherent flexibility in this
part of the molecule that may be of relevance for substrate recognition.
Geometry of the Arsenic Adduct--
Our structure demonstrates
that As(V)-containing organic arsenicals react specifically with
serine, forming an adduct that resembles the tetrahedral transition
state for deacylation (Fig. 4B). Although the electron
density shows the arsenic is coordinated by four ligands (two methyl
groups, the Ser-163 O, and divalent oxygen), the bond angles are
distorted from the expected tetrahedral geometry seen in small molecule
crystal structures of cacodylate. This deviation from ideal geometry
may indicate considerable strain or chemical reactivity within the
enzyme inhibitor complex.
In the refined complex, the bond lengths of As to Ser-O is 1.98 Å,
from As to divalent oxygen is 1.59 Å, and from As to the methyl groups
is 1.82 Å (pro-S) and 1.88 Å (pro-R). These
bond lengths are within the range of possible values for arsenate
compounds surveyed in the small molecule Cambridge Structural Database
(24). Importantly, these refined values show that the oxygen is
divalent, and As is in the plus five oxidation state. The bond angles
of the arsenic adduct reveal additional strain from classic tetrahedral geometry seen in covalent adducts with phosphonate transition state
analogs (25, 26). In fact, the bond angles suggest the configuration of
the arsenic ligands in some respects is closer to trigonal bipyramidal
than tetrahedral geometry. For example, the Ser-160 O
is apparently
in an apical position as the bond angles are 90.2°
(O
-As-pro-S CH3), 108.2°
(O
-As-pro-R CH3), and 95.3°
(O
-As==O). In addition, the arsenic ligands CH3
(pro-R and -S) and divalent oxygen are nearly
coplanar and are separated by 115.3° (pro-S
CH3-As==O), 116.5° (pro-R
CH3-As==O), and 122.2° (CH3-As-CH3) (Fig. 4B). These values
differ from the ideal tetrahedral bond angles seen in the small
molecule crystal structure of cacodylate. Considerable strain should be
expected given that the natural transition state of the enzymatic
reaction involves carbon, which has a much smaller atomic radius than
arsenic (0.91 versus 1.33 Å).
Comparison of Native and Complex Structures--
The native and
complex structures closely superimpose (root mean square deviation = 0.2 Å for all C atoms). In both crystal structures, the His-290
N
2 atom is ~3.3 Å from the Ser-160 O
, which approaches the
upper limit of separation for these two residues in a catalytic triad
(Fig. 4, A and B). His-290 is precisely oriented in this position by close interaction with Asp-260, Leu-208, and Trp-209. In the arsenic complex, the His-290 N
2 is ~3 Å from the
pro-R methyl and, in the native structure it is ~3.4 Å from a water molecule (Fig. 4, A and B). This
water molecule forms hydrogen bonds with His-290 N
2 and Ser-160 O
and would nearly superimpose on the pro-R methyl of the
arsenic adduct (Fig. 4A). Hence, this water molecule would
be ideally positioned for activation and subsequent nucleophilic attack
of the acyl intermediate. In both structures, alternate conformations
were visible for the active site Ser-160. However, the alternative
rotamer in the native structure differs from that in the arsenic adduct
structure. Multiple conformations for the active site serine may be of
importance mechanistically during initial substrate binding or product
release. The observation of three serine rotamers is unusual in a
catalytic triad (27) and is possibly attributable to the relatively
distant interaction with His-290.
Structural Homology with Other Esterases--
Three crystal
structures are known from the hormone-sensitive lipase subfamily of
hydrolases, brefeldin A esterase (15), esterase 2 (16), and
Archaecoglobus fulgidus esterase (17). The DALI Z score (28)
for HerE and brefeldin A esterase was 37, which is exceptionally high
(a Z-score > 12 is significant homology). Superposition
illustrates that all of these homologous enzymes share similar helical
insertions at the amino terminus and between strand 6 and
7, and
all of these enzymes exhibit a similar mode of dimerization.
Mechanistically, this subfamily employs three backbone amides in the
oxyanion hole, similar to scattered reports for several other lipases
and acetylcholinesterase, which belong to related esterase subfamilies
(16, 29). This tripartite oxyanion hole differs from the classic
bipartite oxyanion hole seen in most proteases and esterases (19). The
third hydrogen bond in acetylcholinesterase has been invoked to
explain, in part, the remarkable diffusion-limiting rate acceleration
for this particular enzyme (29).
Substrate Specificity in the Active Site--
Because the 3-acetyl
ester appears to undergo rapid spontaneous hydrolysis, it is
impractical to measure Michaelis constants for this substrate.
Therefore, the specificity of HerE was determined for two different
substrates, 6-acetylmorphine (kcat = 12.6 ± 0.6 s1, Km = 500 ± 60 µM) at pH = 8 (data not shown) and phenyl acetate (kcat = 3.0 ± 0.05 s
1, Km = 70 ± 8 nM) at pH = 6.4 (Fig.
5). The Michaelis constants for phenyl
acetate were measured under mildly acidic conditions to directly
compare the uninhibited reaction with the inhibited reaction in the
cacodylate crystallization buffer, pH = 6.4. The catalytic
efficiency of HerE for 6-acetylmorphine (kcat Km
1 = 2.5 × 104
M s
1) is several orders of
magnitude less than for phenyl acetate (kcat
Km
1 = 4.3 × 107
M s
1) (Fig. 5). The large
difference in Km for the two substrates accounts for
the wide variation in efficiency, suggesting that the enzyme is unable
to bind heroin as well as phenyl acetate. The high bimolecular rate
constant kcat
Km
1 for phenyl acetate at a non-ideal
pH 6.4 implies the enzyme would approach the bimolecular diffusion
limit (109 - 1011 M
s
1) at a more optimal pH 8, which would be
similar to the high efficiency seen for acetylcholinesterase. Further
biochemistry within the hormone-sensitive lipase subfamily will
illuminate whether or not other members approach a comparable catalytic
efficiency as acetylcholinesterase. The structural homology to other
lipase enzymes further suggests that HerE may have acylglycerol
hydrolytic activity (e.g. 1,2,3-triacetylglycerol) (30) and,
hence, provides a basis for additional mutagenesis and biochemistry
experiments.
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Arsenic Inhibition in the Acetyl Esterase--
The
Ki of dimethylarsinic acid was determined under
steady state conditions using phenyl acetate as a model substrate. All
reactions were conducted at pH 6.4, which is similar to the pH of the
crystallization mother liquor and coincides with the pKa of cacodylate (Fig. 5). The apparent shift in
Km for phenyl acetate was determined for two
different inhibitor concentrations, and the Ki for
dimethylarsinic was determined to be ~12 ± 2 mM.
The inhibition is most likely competitive, although it was impractical
to confirm that Vmax converges to the same value
in the limit of high substrate concentrations. The specific interaction
between dimethylarsinic acid and serine in the active site, however,
supports this model of competitive inhibition. The relatively high
Ki for the inhibitor also suggests that the
inhibition is reversible. Based on this Ki value, one would expect that at equilibrium ~80% of the enzyme would be
bound in the 50 mM cacodylate crystallization buffer. This value agrees closely with the crystal structure where the refined occupancy of the arsenic adduct was ~60%.
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DISCUSSION |
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Several novel observations have emerged from the crystal structure of HerE. The HerE enzyme is the first structure reported of an esterase known to hydrolyze heroin. Hence, the structure provides a framework for improving the specificity and sensitivity of a recently reported heroin biosensor (2). The high bimolecular rate constant for phenyl acetate (approximately 1000-fold higher than 6-acetylmorphine) implies that with engineering of the active site, the specificity constant for heroin could be substantially improved. Specifically, the structure highlights residues in the acyl and alcohol binding pockets that could be targeted by random and rational mutagenesis to create a binding pocket with higher shape complementarity to morphine. From a more biochemical perspective, truncation of the flexible amino-terminal domain could assess its contribution to activity. These capping domains have been proposed to act as flexible lids in other esterase and lipase structures that regulate access into and out of the active site. In addition, the negatively charged Glu-190 in the acyl binding pockets could be mutated to Gln or Leu to create a binding pocket with higher electrostatic complementarity to heroin.
We also employed unconventional structure determination methodology by using SAD with a high energy remote wavelength rather than the optimal peak wavelength (because of constraints at the beam line). Surprisingly, the anomalous contributions to the structure factors were sufficient for phasing even though the data were not collected at the optimal peak wavelength. The successful structure determination was due, in part, to the high redundancy and quality of the data (Table I) and to the anomalous contributions from both arsenic and selenium. The derivative with arsenic could be applicable to phasing of other esterase/protease structures, since arsenic is a broad scope inhibitor of serine hydrolases (3, 4). Moreover, deposited crystal structures in the Protein Data Bank also demonstrate specific reaction of arsenic with cysteine (6). Hence, arsenic may be generally exploited as a useful heavy metal for either multiple isomorphous replacement (MIR)/single isomorphous replacement (SIR) or multiple wavelength anomalous diffraction (MAD)/single wavelength anomalous diffraction (SAD) phasing.
The acetyl esterase structure is the first to demonstrate reactivity of an As(V)-containing compound in a serine esterase active site and the first example in the Protein Data Bank of covalent modification of serine by arsenic. The apparent dehydration that occurs in the active site is most likely a ligand substitution reaction, where arsenic remains in the As(V) oxidation state throughout. The pKa of dimethylarsinic acid is ~6.4, so inhibition likely occurs optimally at acidic pH in the protonated form, as an oxyanion is a poor leaving group. Although it is impossible to distinguish between an associative (SN2-like) or disassociative (SN1-like) mechanism from the crystal structure alone, we can predict a likely reaction pathway given the geometrical restraints of the active site.
Because hydroxide is also a poor leaving group, the cacodylate
hydroxyl must be protonated during the reaction (Fig.
6). Hence, when the Ser-160 nucleophile
attacks, the hydroxyl leaving group is likely positioned closest to
His-290 for optimal proton transfer in a transient, trigonal
bipyramidal-like transition state with Ser-160 O and one of the
methyl substituents at the apical positions (Fig. 6). In this
transition state, the formerly divalent oxygen becomes monovalent,
forming three stabilizing hydrogen bonds in the oxyanion hole, whereas
the other methyl substituent points into the acyl pocket (Fig. 6).
Importantly, the trigonal bipyramidal geometry of this proposed
transition state is consistent with the apparently strained tetrahedral
geometry observed in the adduct structure. In the last step, the
monovalent oxyanion collapses to resume divalency, the water molecule
is expelled, and the arsenic adduct adopts the strained tetrahedral
geometry with the apical methyl group of the transition state, shifting
to its pro-R position in close contact with His-290.
Finally, the proposed mechanism would be fully reversible, which is
also consistent with the refined occupancy of 0.61 for the adduct and
the relatively high Ki of ~12 mM.
Hence, this crystal structure fills a gap in the classic body of
literature on catalytic triads (31) and on inhibition of serine
hydrolases by arsenic (3, 4).
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CONCLUSION |
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A detailed understanding of this acetyl esterase now gives a foundation for the structure-based iterative design of second generation heroin biosensors with improved sensitivity. Moreover, at least two human carboxylesterases (hCE-1,2) (32) mediate the breakdown of 6-acetylmorphine to morphine, but no structures of these human enzymes have been reported. Hence, HerE provides a glimpse of an active site known to accept heroin as a substrate. The acyl binding pocket is quite small, thus optimally accommodating the acetyl substituent of heroin. It will be interesting to see if this small acyl binding pocket will be a conserved feature of the corresponding human carboxylesterases and whether this pocket could be expanded by site-directed mutagensis to accept larger substrates. Additionally, the active site complex identifies three hydrogen bond interactions in the oxyanion hole, similar to those seen in acetylcholinesterase and several other lipases. Although only a handful of serine hydrolases are known to have three rather than the more common two hydrogen bonds, as more hydrolase structures become available this tripartite oxyanion hole is likely to emerge as a common theme and perhaps provide an alternative rational for categorizing serine hydrolase enzymes.
Our crystal structure also demonstrates, for the first time, covalent
modification of the catalytic triad serine by an arsenic compound,
dimethylarsinic acid. Arsenic-containing compounds comprise a known
class of serine hydrolase inhibitors. Dimethylarsinic acid is of
particular interest, because it is a major metabolite formed upon
chronic exposure to inorganic arsenic in drinking water. Based on our
structural data, it is tempting to speculate that this compound may,
therefore, interact with serine hydrolases in vivo. Whether
or not this interaction accounts mechanistically for any of the toxic
effects of arsenic requires further study. However, the relatively high
Ki for dimethylarsinic acid would suggest that the
compound would only interfere with normal serine hydrolase function at
sufficiently high concentrations of arsenic.
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ACKNOWLEDGEMENTS |
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We thank the Stanford Synchrotron Radiation Laboratory. We are also grateful to Xiaoping Dai for assistance on synchrotron trips and Andreas Heine, Donmienne Leung, Benjamin F. Cravatt, Chi-Huey Wong, and Richard A. Lerner for helpful discussions. We also thank Ben List for generous use of his UV plate reader.
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FOOTNOTES |
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* The work was supported by National Institutes of Health Grant GM38273 (to I. A. W.) and the Biotechnology and Biological Sciences Research Council (to N. C. 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 1LZL and 1LZK) 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 Molecular
Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd. BCC206, La Jolla, CA, 92037. Tel.: 858-784-9706; Fax: 858-784-2980; E-mail: wilson@scripps.edu.
Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M210103200
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ABBREVIATIONS |
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The abbreviations used are: 2- BES, [bis(2-hydroxyethyl)amino]ethanesulfonic acid; SAD, single wavelength anomalous diffraction.
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