The lid domain of the MCP hydrolase DxnB2 contributes to the reactivity toward recalcitrant PCB metabolites

Article abstract of DOI:10.1021/bi400774m

DxnB2 and BphD are meta-cleavage product (MCP) hydrolases that catalyze C-C bond hydrolysis of the biphenyl metabolite 2-hydroxy-6-oxo-6-phenylhexa-2,4- dienoic acid (HOPDA). BphD is a bottleneck in the bacterial degradation of polychlorinated biphenyls (PCBs) by the Bph catabolic pathway due in part to inhibition by 3-Cl HOPDAs. By contrast, DxnB2 from Sphingomonas wittichii RW1 catalyzes the hydrolysis of 3-Cl HOPDAs more efficiently. X-ray crystallographic studies of the catalytically inactive S105A variant of DxnB2 complexed with 3-Cl HOPDA revealed a binding mode in which C1 through C6 of the dienoate are coplanar. The chlorine substituent is accommodated by a hydrophobic pocket that is larger than the homologous site in BphD_LB400 from Burkholderia xenovorans LB400. The planar binding mode observed in the crystalline complex was consistent with the hyper- and hypsochromically shifted absorption spectra of 3-Cl and 3,9,11-triCl HOPDA, respectively, bound to S105A in solution. Moreover, ES^red, an intermediate possessing a bathochromically shifted spectrum observed in the turnover of HOPDA, was not detected, suggesting that substrate destabilization was rate-limiting in the turnover of these PCB metabolites. Interestingly, electron density for the first α-helix of the lid domain was poorly defined in the dimeric DxnB2 structures, unlike in the tetrameric BphD_LB400. Structural comparison of MCP hydrolases identified the NC-loop, connecting the lid to the α/β-hydrolase core domain, as a determinant in the oligomeric state and suggests its involvement in catalysis. Finally, an increased mobility of the DxnB2 lid may contribute to the enzyme's ability to hydrolyze PCB metabolites, highlighting how lid architecture contributes to substrate specificity in α/β-hydrolases.

Full text of DOI:10.1021/bi400774m

Article
pubs.acs.org/biochemistry
The Lid Domain of the MCP Hydrolase DxnB2 Contributes to the
Reactivity toward Recalcitrant PCB Metabolites
Antonio C. Ruzzini,‡,∥ Shiva Bhowmik,†,∥ Katherine C. Yam,‡ Subhangi Ghosh,† Jeffrey T. Bolin,*,†
and Lindsay D. Eltis*,‡,§
†Purdue Cancer Research Center and Markey Center for Structural Biology, Department of Biological Sciences, Purdue University,
West Lafayette, Indiana, United States
§
‡Department of Biochemistry and Molecular Biology and Department of Microbiology and Immunology, University of British
Columbia, BC, Canada
S
* Supporting Information
ABSTRACT: DxnB2 and BphD are meta-cleavage product
(MCP) hydrolases that catalyze C−C bond hydrolysis of the
biphenyl metabolite 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic
acid (HOPDA). BphD is a bottleneck in the bacterial
degradation of polychlorinated biphenyls (PCBs) by the Bph
catabolic pathway due in part to inhibition by 3-Cl HOPDAs.
By contrast, DxnB2 from Sphingomonas wittichii RW1 catalyzes
the hydrolysis of 3-Cl HOPDAs more efficiently. X-ray
crystallographic studies of the catalytically inactive S105A
variant of DxnB2 complexed with 3-Cl HOPDA revealed a
binding mode in which C1 through C6 of the dienoate are
coplanar. The chlorine substituent is accommodated by a hydrophobic pocket that is larger than the homologous site in
BphDLB400 from Burkholderia xenovorans LB400. The planar binding mode observed in the crystalline complex was consistent
with the hyper- and hypsochromically shifted absorption spectra of 3-Cl and 3,9,11-triCl HOPDA, respectively, bound to S105A
in solution. Moreover, ESred, an intermediate possessing a bathochromically shifted spectrum observed in the turnover of
HOPDA, was not detected, suggesting that substrate destabilization was rate-limiting in the turnover of these PCB metabolites.
Interestingly, electron density for the first α-helix of the lid domain was poorly defined in the dimeric DxnB2 structures, unlike in
the tetrameric BphDLB400. Structural comparison of MCP hydrolases identified the NC-loop, connecting the lid to the α/β-
hydrolase core domain, as a determinant in the oligomeric state and suggests its involvement in catalysis. Finally, an increased
mobility of the DxnB2 lid may contribute to the enzyme’s ability to hydrolyze PCB metabolites, highlighting how lid architecture
contributes to substrate specificity in α/β-hydrolases.
In the Bph pathway, HOPDA is hydrolyzed by BphD to yield
benzoic acid and 2-hydroxy-2,4-pentadienoic acid (HPD).
HOPDA and related compounds result from the dioxygenase-
mediated cleavage of catechols and are known as meta-cleavage
products (MCPs); thus BphD is an MCP hydrolase. BphD was
identified as a key determinant in the transformation of PCBs
based on at least three observations: (i) yellow-colored
metabolites, 3-Cl HOPDAs, accumulated during bacterial
growth in the presence of 2,4′-chlorobiphenyls;7 (ii) accumu-
lation of 3-Cl HOPDAs was strain-independent;6 and (iii) 3-Cl
HOPDAs inhibit BphD.8,9 Accordingly, the identification of
DxnB2, an MCP hydrolase from Sphingomonas wittichii RW1
that possesses 8- to 80-fold greater specificity for 3-Cl HOPDA
relative to bona fide BphD orthologs, represents a promising
lead in the expansion of the cometabolic potential of the Bph
pathway.10
olychlorinated biphenyls (PCBs) are aromatic xenobiotic
Pcompounds that were widely used in the twentieth century
in a variety of industrial applications. While bans on the
production and use of PCBs began during the 1970s,
approximately 10% of all PCBs (∼2.8 × 108 kg) remain in
mobile environmental reservoirs.1 PCB exposure has been
linked to a range of human diseases including cancers and
developmental and neurological disorders.2 Improved environ-
mental bioremediation strategies would minimize continued
exposure to these toxic bioaccumulative compounds. To this
end, aerobic, biphenyl-utilizing bacteria, first reported in 1970,3
have been studied for their ability to cometabolize PCBs.4
Although the ability of bacteria to transform different PCB
congeners is largely strain-dependent, reflecting the substrate
specificities of the first four enzymes of the biphenyl (Bph)
catabolic pathway,5 the strain-independent accumulation of
particularly recalcitrant metabolites has been observed.6 The
latter include 3-chloro-2-hydroxy-6-oxo-6-phenylhexa-2,4-di-
enoic acids (3-Cl HOPDAs).
Received: June 17, 2013
Revised: July 20, 2013
Published: July 23, 2013
© 2013 American Chemical Society
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The MCP hydrolases catalyze C−C bond cleavage through
an unusual nucleophilic mechanism that is dependent on a
substrate ketonization (Figure 1).11,12 As members of the α/β-
ESred, an intermediate that is susceptible to protonation and,
subsequently, bond cleavage.
The ability of DxnB2 to overcome the apparent inhibition of
3-Cl HOPDAs is poorly understood. In this work, we
investigated the ability of DxnB2 to hydrolyze 3-Cl and
3,9,11-triCl HOPDA, the latter of which is a biologically
relevant PCB metabolite. Crystal structures of DxnB2, DxnB2
S105A, and a DxnB2 S105A:3-Cl HOPDA binary complex
were utilized for a comparative study of structural determinants
for variations in catalytic efficiency. Transient-state kinetic
analyses of the DxnB2-mediated turnover of 3-Cl HOPDAs
were performed under single turnover conditions to identify
reaction intermediates and the rate-determining step in
catalysis. A structural basis for the PCB-metabolite specificity
of DxnB2 is forwarded.
MATERIALS AND METHODS
■
Chemicals. 3-Chloro (3-Cl) and 3,9,11-trichloro (3,9,11-
triCl) HOPDAs were generated enzymatically from 4-chloro-
2,3-dihydroxybiphenyl and 4,3′,5′-trichloro-2,3-dihydroxybi-
phenyl, respectively, using 2,3-dihydroxybiphenyl dioxygenase,
as previously described.8 All other chemicals were of analytical
grade.
Figure 1. Ketonization and hydrolysis of 3,9,11-triCl HOPDA
catalyzed by the MCP hydrolases.
Mutagenesis, Protein Production, and Purification.
Site-directed mutagenesis was performed using a 5′-phosphory-
lated primer, 5′-PO4-CCCTGATCGGCAACGCCATGGG-3′
to amplify a single-stranded plasmid carrying the desired codon
substitution followed by the use of a QuikChange Multi Site-
Directed Mutatgenesis Kit (Stratagene, La Jolla, CA). DxnB2
was produced in Escherichia coli as previously described.13
Harvested cells overproducing the recombinant enzymes were
resuspended in 20 mM HEPES, pH 7.5 (Buffer A) and lysed at
4 °C using an Emulsi Flex-C5 homogenizer (Avestin Inc.,
Ottawa, ON). Debris was removed by ultracentrifugation at
50000 g for 45 min at 4 °C. The supernatant was passed
through a 0.45 μm filter before being loaded onto a 28 mL
Source 15Q column, which was equilibrated in Buffer A
containing 35 mM NaCl and operated at 15 mL/min. Proteins
were eluted using a linear gradient from 35 to 200 mM NaCl in
15 column volumes. Fractions containing DxnB2, which
centered near 130 mM NaCl, were pooled and concentrated
using an Amicon stirred cell concentrator fitted with an YM30
membrane (Millipore, Billerica, MA). The concentrated
fractions were then brought to 1 M (NH4)2SO4 at pH 7.5
and stirred for 12 h at 4 °C. The resulting precipitate was
washed with three 25 mL volumes of Buffer A containing 1 M
(NH4)2SO4 before dissolution in 20 mM TAPS, pH 8.5. The
purified DxnB2 preparations (>99% homogeneity based on
SDS-PAGE) were then exchanged into potassium phosphate
buffer (I = 0.1 M), pH 7.5, concentrated to >20 mg/mL, and
flash frozen in liquid nitrogen. Approximately 80 mg of purified
DxnB2 was obtained per liter of cell culture, and all enzyme
preparations were stored at −80 °C. The enzyme concentration
used in each assay was determined using the molar absorptivity
of DxnB2 (ε280 nm = 36.2 mM−1 cm−1), calculated from the A280
nm of a sample whose concentration was determined by amino
acid analysis at the Advanced Protein Technology Centre
(Hospital for Sick Children, Toronto, ON). BphD17 and
HsaD18 were produced and purified as previously described.
Enzyme−Substrate Complex Formation. ES complexes
were formed by mixing 40 μM DxnB2 S105A with 10 μM 3-Cl
or 3,9,11-triCl HOPDA in potassium phosphate buffer (I = 0.1
M), pH 7.5. Electronic absorption spectra of the substrates
hydrolase superfamily, the MCP hydrolases possess a Ser-His-
Asp triad and an ‘oxyanion hole’ that catalyze the hydrolytic
reaction. This catalytic machinery also spatially divides the
active site into two subsites. The non-polar (NP) subsite
accommodates the variable MCP moiety: a phenyl ring in the
case of HOPDAs. The polar (P) subsite includes five conserved
residues: Gly35, Asn43, Asn104, Arg180, and Trp256 (DxnB2
numbering). These interact with the substrate’s dienoate
moiety and have been implicated in the ketonization reaction.13
The MCP hydrolases possess a lid domain consisting of 4 α-
helices (α5−α8) inserted between β6 and α9 of the α/β-
hydrolase core. Both the α/β-hydrolase core and the MCP
hydrolase lid domains are required to furnish the complete suite
of catalytic residues.
The mechanism of inhibition by 3-Cl HOPDA has been
studied extensively for BphD from Burkholderia xenovorans
LB400 (BphDLB400). Steady-state kinetic studies revealed that
3-Cl HOPDA competitively inhibits BphDLB400 (Kic = 0.57
μM) and is ultimately hydrolyzed 470-times slower than
HOPDA.8 A crystal structure of the BphDLB400 S112A:3-Cl
HOPDA binary complex revealed a bound conformation of the
HOPDA in which C1 through C6 of the dienoate were
coplanar.14 This non-productive binding mode is apparently
stabilized by an interaction between a hydrophobic pocket and
the chlorine substituent. This contrasts to the productive, non-
planar conformation observed in the BphDLB400 S112A:HOP-
DA binary complex,15 which has been correlated to an enzyme-
destabilized species that is characterized by a bathochromically
shifted absorption spectrum observed both in solution and in
the crystalline state.15 This orange-colored species (λmax
≈
470−500 nm) resembles a catalytically relevant intermediate
named ESred that precedes substrate ketonization.11,15,16 Under
no tested conditions has a similarly red-shifted species been
observed for the 3-Cl HOPDAs. However, kinetic studies on a
3-F HOPDA revealed a small population of ESred during
turnover.14 Taken together, the studies on BphDLB400 indicate
that the 3-Cl substituent inhibits access to the destabilized
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alone and in complexes were recorded using a Cary5000
spectrophotometer equipped with a thermostatted cuvette
holder maintained at 25 °C.
Stopped-Flow Spectrophotometry. Experiments were
conducted using an SX.18MV stopped-flow reaction analyzer
(Applied Photophysics Ltd., Leatherhead, U.K.) outfitted with a
The structure of WT DxnB2 was determined using programs
from the CCP4 program package.20 Initial phases were
obtained by the molecular replacement method. An initial
search model was derived from the structure of a homologue,
CarC (PDB ID: 1J1I).21 The sequence of DxnB2 was
incorporated using the CCP4 program CHAINSAW, which
pruned nonconserved residues to the last common atom.
Molecular replacement calculations performed with the
program MOLREP22 using reflections to 3.5 Å resolution
placed one monomer in the asymmetric unit (initial R =
50.9%). After preliminary rigid body and restrained atomic
parameter refinement R and Rfree were 36% and 42%,
respectively. The electron density was discontinuous for
residues 140−158 of the lid domain. A revised search model
was prepared for a second round of molecular replacement by
deleting the entire lid domain, incorporating distal atoms from
nonconserved residues, and rebuilding other residues. The
structure of WT served as the search model for phasing of the
S105A variant by molecular replacement.
Initial solutions obtained from molecular replacement
underwent rigid body refinement and multiple cycles of
restrained atomic parameter refinement using REFMAC.23
Residues and substrates were manually built into the electron
density utilizing molecular graphics programs O24 and
COOT.25 Coordinates for 3-Cl HOPDA and sulfate were
generated using PRODRG.26 Refinement restraint libraries
were generated using LIBCHECK from the CCP4 package.
Comparison of MCP Hydrolases. Superposition of MCP
hydrolase structures was performed using the STAMP
algorithm implemented in Multiseq, a plugin available in
VMD27−29 or tools available in COOT. The common names,
biological sources and PDB codes of the aligned enzymes are:
BphDLB400 from B. xenovorans LB400 (2OG1, chain A),16
BphDRHA1 from Rhodococcus jostii RHA1 (1C4X),30 CarC from
Janthinobacterium sp. strain J3 (1J1I),21 CumD from
Pseudomonas fluorescens IP01 (1IUP),31 DxnB2 from Sphingo-
monas witichii RW1 (this study), HsaD from Mycobacterium
tuberculosis H37Rv (2VF2, chain A),32 and MhpC from E. coli
W3110 (1U2E, chain A).33
circulating water bath maintained at 25
0.1 °C. Kinetic
measurements were performed using potassium phosphate
buffer (I = 0.1 M) 30% sucrose (w/v), pH 7.5. A photodiode
array (PDA) detector was initially used to determine the λmax of
detectable intermediates formed during each reaction. The
reported rate data are derived from single wavelength
measurements using a 4.96 nm/mm bandpass monochromator
with the slit width set at 0.5 mm. Single turnover experiments
were performed using a 4:1 ratio of DxnB2 to substrate: 16 μM
DxnB2 was reacted with 4 μM 3-Cl HOPDA or 3,9,11-triCl
HOPDA. All reactions were performed using freshly prepared
reagents for each replicate. Each replicate was an average of at
least five shots from the stopped-flow syringes. The Applied
Photophysics Pro-Data Software was employed for nonlinear
curve fitting and the kinetic parameters are reported as the
average of and standard deviation between three replicates.
Size Exclusion Chromatography Multiangle Light
Scattering (SEC-MALS). SEC-MALS experiments were
̈
performed using an AKTA Purifier (GE Healthcare) fitted
with Wyatt Technology Corporation SEC column and guard
column (WTC-030S5 and WTC-030S5G). The protein
concentrations used were between 1.5 and 2.5 mg/mL,
prepared in 20 mM HEPES, pH 7.5. Multiangle light scattering
and refractive index were measured using the miniDAWN
TREOS and Optilab T-rEX detectors (Wyatt Technology
Corporation, Santa Barbara, CA). Calibration was performed
using bovine serum albumin.
Crystallization and Manipulation of Crystals. Crystals
were grown using the sitting drop vapor diffusion method at 20
°C. Protein samples, 8 to 9 mg/mL in 20 mM HEPES, pH 7.5,
were mixed 1:1 with the reservoir and equilibrated against 500
μL of the reservoir solution. Wild-type (WT) DxnB2 crystals
were obtained from a grid of ammonium sulfate (1.0−2.5 M)
containing 0.1 M Tris, pH 8.2 and 6−10% ethanol. Crystals of
the S105A variant were obtained from a grid of sodium
malonate (1.5−2.4 M), pH 6.5/7.0. Crystalline ES complexes
were prepared by incubating crystals of S105A in a 30 μL
droplet of reservoir solution containing 3-Cl HOPDA at a
concentration of ∼15 mM for 15 to 20 min at 20 °C. Prior to
flash freezing, crystals were transferred into 30 μL droplets of
reservoir solution augmented with a suitable cryoprotectant.
The cryoprotectants employed for WT and S105A were 20%
glycerol and 2.1 M sodium malonate, respectively. Cryopro-
tectant solutions for ES complexes contained ∼5 mM 3-Cl
HOPDA.
Structure Determination and Refinement. Diffraction
experiments were performed under cryogenic conditions (100
K) at the Advanced Photon Source, Argonne National
Laboratory, Argonne, IL, USA: beamlines 14-BM-C and 23-
ID-D were used for the WT and S105A crystals, respectively.
Typical data sets included 300 frames, collected with a 0.5°
rotation per frame at an X-ray wavelength of 1.0 Å. Exposure
times were 6 s per degree. Diffraction images were recorded
using MarMosaic 300 CCD detectors (Mar USA, Inc.,
Evanston, IL). Programs from the HKL2000 suite were utilized
to process the images and scale and merge intensities.19
RESULTS
■
Stable Enzyme−Substrate Complexes. Substitution of
the catalytic serine to alanine in the MCP hydrolases results in a
catalytically impaired enzyme. However, these variants bind
substrates in a manner that closely reproduces spectroscopic
signatures of a reaction intermediate that precedes chemistry in
the wild type enzyme. For example, the binding of HOPDA to
catalytic serine-to-alanine variants of BphDLB400 and DxnB2
resulted in bathochromic shifts of the enolate spectra by 72 and
66 nm, respectively, which mirrored the 58 and 53 nm
perturbations that characterized ESred under single turnover
conditions.11,15,16 Accordingly, DxnB2 S105A was used to
investigate the ability of this enzyme to destabilize 3-Cl
HOPDAs. The electronic absorption spectra of 10 μM 3-Cl or
3,9,11-triCl HOPDA was measured in the presence or absence
of 40 μM DxnB2 S105A in potassium phosphate buffer (I = 0.1
M), pH 7.5 at 25 °C. Binding of 3-Cl HOPDA to DxnB2
S105A resulted in a hyperchromically shifted spectrum (Figure
2A), whereas the DxnB2 S105A:3,9,11-triCl HOPDA complex
resulted in a 21 nm hypsochromic shift from 438 to 417 nm
(Figure 2B). Neither complex revealed a detectable population
of a red-shifted species associated with substrate destabiliza-
tion.16,18
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Figure 2. Electronic absorption spectra of 3-Cl HOPDAs in solution
or in complex with 40 μM DxnB2 S105A in potassium phosphate
buffer (I = 0.1 M), pH 7.5 at 25 °C. (A) 10 μM 3-Cl HOPDA and (B)
10 μM 3,9,11-triCl HOPDA.
Figure 3. Results from representative stopped-flow experiments
demonstrating the turnover of 4 μM 3-Cl HOPDAs by 16 μM
DxnB2 in potassium phosphate buffer (I = 0.1 M), pH 7.5 at 25 °C.
(A) Hydrolysis of 3-Cl HOPDA monitored at 432 nm. (B) Hydrolysis
of 3,9,11-triCl HOPDA monitored at 417 and 438 nm. For each
substrate, the accumulation of intermediates as revealed by monitoring
time-dependent changes to the visible spectrum from 300 to 600 nm is
inset.
Single Turnover of 3-Cl HOPDAs by DxnB2. In order to
gain kinetic insight into the ability of DxnB2 to overcome the
apparent inhibitory or stabilizing effect attributed to the 3-
chloro substituent, hydrolysis of 3-Cl and 3,9,11-triCl HOPDA
was monitored under single turnover conditions. Specifically,
16 μM DxnB2 was reacted with 4 μM substrate in potassium
phosphate buffer (I = 0.1 M), pH 7.5 at 25 °C. A PDA detector
was utilized to identify intermediates (Figure 3, inset). For
more accurate rate determination, reactions were monitored at
single wavelengths. While decay of the HOPDA enolates (λmax
432 or 438 nm) was readily monitored, the formation of 3-Cl-
HPD (λmax ≈ 244 nm) could not be distinguished from other
underlying processes, and the resulting multiphasic kinetic
traces could not be reliably interpreted (Supplementary Figure
S1). To distinguish intra- and intermolecular events, additional
reactions were performed in the presence of a viscogen, 30%
sucrose (w/v).
The hydrolysis of both substrates by DxnB2 was biphasic
(Table 1; Figure 3). The first observable kinetic phase, 1/τ1,
was attributed to ES complex formation, which occurred at
rates of 220 and 26 s−1 for the mono- and trichlorinated
substrate, respectively. In both cases, the presence of sucrose
slowed this intermolecular event by 1.3- to 2-fold. For 3-Cl
HOPDA, ES complex formation was accompanied by a small
hypochromic perturbation, whereas the spectrum of 3,9,11-
triCl HOPDA was hypsochromically shifted by 21 nm as in the
stable S105A complex. Following accumulation of ES
complexes, the respective signals decayed at rates of 0.152
and 0.056 s−1. The observed insensitivity of 1/τ2 to sucrose and
the relatively large loss in amplitude (∼90%), corresponding to
the dissipation of the HOPDA enolate chromophore, support
the assignment of this kinetic step to on-enzyme chemistry. A
more precise assignment of 1/τ2 to acylation limited by the rate
of substrate ketonization was recently described for DxnB2.11
However, the absence of ESred from the kinetic traces suggests
that formation of this intermediate is rate-limiting to the
hydrolysis of 3-Cl HOPDAs.
Overall Structure and Oligomeric State of DxnB2. The
stable DxnB2 S105A ES complexes and kinetic behavior of the
wild type enzyme paralleled studies on BphDLB400 and therefore
provided limited insight into the apparent increased specificity
for 3-Cl HOPDAs. These results, together with the
classification of DxnB2 and BphDLB400 into phylogenetically
distinct MCP hydrolase subfamilies or classes,10 inspired the
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a
Table 1. Kinetic Values for the DxnB2-Mediated Hydrolysis of 3-Cl and 3,9,11-triCl HOPDA
b
substrate
λ (nm)
k1 (s−1) [Amp (%)]
k2 (s−1) [Amp (%)]
kcat
3-Cl
432
432
417
438
417
438
434
220 20 [11.5 0.3]
100 10 [7.4 0.3]
26 1 [12.2 0.6]
22 2 [32 1]
0.152 0.001 [88 2]
0.152 0.001 [92.6 0.7]
0.056 0.001 [88 2]
0.056 0.001 [68 1]
0.054 0.001 [88 2]
0.054 0.001 [67 2]
0.25 0.1
+ 30% sucrose
3,9,11-triCl
0.13 0.1
+ 30% sucrose
18 2 [12 1]
16.9 0.2 [32.9 0.8]
c
HOPDA
>500
69.4 0.1
a
b
c
Values are reported as an average of 3 replicates standard deviation. Data from ref 10. Data from ref 11.
Table 2. Properties of the Crystals, Diffraction Data, and Refinement Statistics
Crystal Properties and Diffraction Data
structure
PDB ID
DxnB2
DxnB2 S105A
4LYD
S105A:3 Cl-HOPDA
4LXG
4LXH
a
resolution range (Å)
space Group
36.5−2.2
P6522
57.4−2.2
P6522
57.6−2.0
P6522
cell dimensions (Å)
unique reflections
a = b = 67.0, c = 327.7
21,400
a = b = 66.3, c = 331.6
21,860
a = b = 66.6, c = 338.2
28,184
a
multiplicity
9.9 (9.6)
11.5 (6.9)
93.5 (54.3)
7.8 (52.7)
29.1 (2.0)
7.2 (6.5)
a
completeness (%)
99.5 (98.3)
9.8 (32.2)
21.0 (6.6)
98.6 (90.8)
9.0 (50.0)
10.8 (1.9)
a
Rsymm (%)
a
mean I/σ2
Refinement
R
factor/Rfree
0.20/0.23
0.21/0.26
0.21/0.26
model content (atoms)
non-hydrogen atoms
protein
2185
2095
10/0
80
2115
2053
0/0
62
2220
2114
1/17
88
b
precipitant/HOPDA
water oxygens
average B factors (Å2)
all atoms
protein
52.9
48.0
48
49.5
52.9
49.1
b
ligands/HOPDA
waters
59.5/NA
50.9
NA/NA
46.5
0.010
1.5
57.9/81.5 (0.8)
52.9
0.023
1.8
c
rmsd bond lengths (Å)
0.006
c
rmsd bond angles (deg)
1.0
a
b
Values for highest resolution bin in parentheses. Each ligand was modeled at full occupancy unless otherwise stated in parentheses; one molecule
c
of sulfate = 5 atoms, HOPDA = 16 atoms + substituent atoms. rmsd is the root-mean-square deviation from restraint targets.
Figure 4. (A) Ribbon drawings of the BphDLB400 protomer (PDB ID: 2OG1, chain A). The regions that are involved in either the A-B′ and/or A-A′
tetrameric interfaces, which include the NC-loop and lid domain helices, are colored green. (B) Superposition of the DxnB2 S105A:3-Cl HOPDA
binary complex and BphDLB400 demonstrating differences in the NC-loop. (C) The apolar phenylalanine cluster is conserved among MCP hydrolase
tetramers. The cluster comprises the F145 residue from each protomer within the tetrameric assembly. The dimer pairs (A-B pairs) are color coded
blue and yellow.
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Figure 5. (A) Ball-and-stick representation of the polar interactions between the side chain and backbone atoms of the poorly conserved loop
(white) extending from β6 and the α/β-hydrolase core (gray) of DxnB2. (B) Combined ribbon drawings and ball-and-stick representation of the
structural superposition of representatives from each MCP hydrolase class: CumD (blue), DxnB2 (white), and BphDLB400 (green). The lid domains
and relevant residues contributing to the structural divergence are shown and numbered. Possible polar interactions are drawn as dashed lines.
investigation of the tertiary and quaternary structure of DxnB2
using X-ray crystallography (Table 2) and SEC-MALS analysis.
SEC-MALS analysis indicated that DxnB2 exists as a dimer
structurally characterized MCP hydrolases were superposed,
and a structure-based sequence alignment was established
(Supplementary Figure S3). As noted previously,16 all of the
crystal structures present a 2-fold symmetric dimer mediated in
part by antiparallel β-interactions between edge strands (β8) of
the monomer’s core domains; we define the associated protein
with a molecular weight of 58
2 kDa in solution
(Supplementary Figure S2). The DxnB2 structures reported
herein contain one monomer per asymmetric unit in space
group P6522, but the crystal structure includes a 2-fold
symmetric dimer stabilized by an extensive interface centered
on an antiparallel β interaction between the monomers’ β8
strands, as observed in other MCP hydrolases.16
surfaces as elements of the dimeric interface, the A-B interface
16
identified for BphDLB400
.
Of interest here are the distinctions
in sequence and structure that mediate assembly of the
BphDLB400, HsaD, and MphC tetramers through additional
interfaces we label as tetrameric interfaces; in this context,
tetrameric refers to the A-B′ and A-A′ interfaces16 that build the
tetramer and does not necessarily imply interactions between
four monomers at the same interface.
Prior to the NC-loop, a conserved LMG sequence (DxnB2
127−9) completes the β6 strand in all known structures. The
glycine residue, at position i, has a left-handed α conformation,
which creates a sharp turn as the backbone departs from the
core. The next highly conserved sequence positions are a pair of
branched non-polar residues at positions j and j+3 (DxnB2 146
and 149); the segment between i and j encompasses 12−17
residues in the known structures. In all structures except that of
CarC (wherein these residues are disordered), the side chains
of residues j and j+3 project from a helical segment of the lid
domain into the NP-substrate binding subsite. Residues
between positions i and j are prominent at the tetrameric
The DxnB2 monomer, as other MCP hydrolases, is
composed of the α/β-hydrolase core (residues 1−129 and
204−277) and an α-helical lid domain (residues 138−203)
inserted between β6 and α9 of the core. For some α/β
hydrolase families,34,35 the term NC-loop has been used to refer
to a protein segment of variable length and structure
connecting β6 to the first structurally conserved helix in the
lid domain. Herein NC-loop refers to residues 130−145 of
DxnB2 and residues 137−152 of BphDLB400. For the three
reported structures, electron density maps clearly defined the
backbone for all residues of the core domain except the C-
terminal residue (Asp277). With the exception of a few solvent-
exposed residues, the electron density for core domain side
chains was also clear in all structures. In contrast, the electron
density was much less distinctive for several residues in the NC-
loop and the beginning of the lid domain. Thus the maps for all
structures clearly place residues 1−139 and residues 155−276,
but the quality of the density and the content of the model
varied among the crystal structures for residues 140−154. It is
unclear whether this disorder reflects a functionally significant
aspect of the α-helical lid in DxnB2.
interfaces, and the sequences and structures of BphDLB400
,
HsaD, and MhpC present features distinguishing them from
the dimeric MCP hydrolases.
In particular, the tetrameric MCP hydrolases of known
crystal structure all share an F-X-P-X-P motif that begins at
position i+9 and contributes to the tetrameric interface that
associates a pair of lid domains, the A-B′ interface. In fact, Fi+9
makes a visually striking contribution to both tetrameric
interfaces as illustrated in Figure 4A and C. Thus Fi+9 lies at the
apex of a loop that protrudes away from the hydrolase and lid
domains, and Fi+9 residues from all four monomers cluster to
form a buried apolar core at the junction of the A-B′ and A-A′
interfaces (Figure 4C). Residues immediately preceding Fi+9 as
Comparison of the Tertiary and Quaternary Struc-
tures of MCP hydrolases. SEC-MALS analysis indicates
DxnB2 is a dimer, whereas BphDLB400 and HsaD are tetramers
(Figure 4A). CumD, CarC, and MhpC have also been
described as dimers. However, the crystal structure of MhpC
displays a tetrameric assembly of the same form as BphDLB400
and HsaD. BphDRHA1 purified as an octamer. To investigate the
basis of the differences in oligomeric state, the seven
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Biochemistry
Article
well as residues in the linker and the helix (α10) that follows
the triad Asp also contribute to tetramer formation but do not
belong to sequence motifs that distinguish dimers and
tetramers. BphDLB400, HsaD, and MhpC belong to the same
class, class I, on the basis of amino acid sequences.10 The
sequences of other homologues in this class (such as PcbD
from Pseudomonas stain sp. DJ12 and Ro09014 from
Rhodococcus jostii RHA1) show variations at the first position
of the F-X-P-X-P motif. However, the oligomeric states are not
known to us.
In contrast to the protruding structure observed for the
tetramers, the first few residues of the NC-loop in DxnB2
associate with helix α10 of the hydrolase core domain as well as
residues just prior to helix α9 via several hydrogen bonds
(Figure 5A). The NC-loop of CumD is five residues shorter
than that of DxnB2 and, while it follows a similar path, is not
involved in the same suite of H-bonding interactions. However,
CumD and several other class II enzymes share an F-P-X-P-Q-
X4-D motif that disrupts helix α8 in CumD and places the Gln
side chain from this motif in position to hydrogen bond with
the backbone carbonyl and amide of NC-loop i+6 and i+8
residues, respectively.
In general, the beginning and end of the lid show greatest
variability among the known MCP hydrolase crystal structures
(Figure 5B). Thus in many pairwise or multiprotein
comparisons it can be difficult to structurally align residues
from the NC-loop and α5 and/or residues within α8 and the
segment that links α8 to α9. In addition, the NC-loop and α5
are prone to disorder and are sensitive to the occupancy of
ligands in the active site. These uncertainties should be
considered in evaluating sequence alignments, including those
based on structures (Supplementary Figure S3). By contrast,
the segment of the lid spanning helices α6 and α7, residues
155−186 of DxnB2 and residues 164−196 of BphDLB400, is
more readily superposed and aligned.
Figure 6. (A) Ball-and-stick representation of the DxnB2 (white) and
BphDLB400 (PDB ID: 2OG1 chain A, green) active site (DxnB2
numbering). For simplicity, only the polar contacts among the catalytic
triad residues or involving Arg180 are drawn as dashed lines. The
sulfate and water molecules (salmon) that occupy the DxnB2 active
site are also shown. (B) Ball-and-stick representation of the stable 3-Cl
HOPDA binding mode observed in the crystalline DxnB2 S105A
complex. The 2Fo − Fc map (green) for the substrate is shown,
contoured at 1σ. Polar contacts ≤3.4 Å are drawn as dashed lines.
DxnB2 WT and S105A Active Sites. As described for
16
BphDLB400
,
the DxnB2 substrate binding site can be divided
into polar (P) and non-polar (NP) subsites that merge at the
locations of the catalytic serine, Ser105, and oxyanion hole
residues, Met106 and Gly34. The NP-subsite is largely formed
by the side chains of residues from both the core domain and
the lid. Because of disorder in the NC-loop and α5 (see above)
and perhaps the absence of substrate, the NP-subsite is not fully
defined by the electron density of the S105A structure. Thus
Leu146 and Val149 from α5, which correspond to residues in
contact with the substrates in structures of BphDLB400 and
HsaD,15,16,18 are not included in the model. These residues are
present in the model for the WT structure, and all others
predicted by sequence alignment to be involved in substrate
binding in the NP site are present in both structures. The P site
residues are well-defined in both structures.
The structure of the DxnB2 WT active site includes a sulfate
ion that binds at the heart of the active site within hydrogen
bonding distance of the catalytic serine, the backbone amides of
the oxyanion hole, the triad histidine (His255), Trp256, and
Asn104 (Figure 6A). The influence of the sulfate is extended by
bound water molecules. For example, two water molecules
form a bridge between the sulfate and conserved P-site Arg180,
and one of these also hydrogen bonds with Asn43. Similarly
two additional water molecules contribute to the interactions of
the sulfate with Asn104 and Ser105. Despite the presence of
the sulfate, the catalytic triad residues are within H-bonding
distances of each other: the Ser105 Oγ is 2.9 Å from the His255
Nε2, and the His255 Nε1 is 2.6 Å and 3.2 Å from the Asp227
carboxylate oxygens.
The ordered active site residues in the DxnB2 S105A model
generally superpose on their WT counterparts with high fidelity
and well within the expected positional error with the exception
of a slight difference in the side chain conformation of Met229
(see below). The final model for S105A does not include a
multiatom ligand or waters bound near the catalytic residues.
Nevertheless, it remains possible that the active site is organized
by a poorly ordered ligand inasmuch as the final difference map
includes a nondefinitive density feature above 0.15 e/Å3 and
large enough to accommodate an aspartate side chain. At one
end, the density predicts an atom within 3.0 Å of the oxyanion
hole amides. At the other end, the density extends to within
non-bonded contact distance of the side chain of Met229,
which is modeled into relatively poor electron density. Thus the
presence of some multiatom ligand of variable structure could
explain the slightly different conformation and nondefinitive
density of Met229; disorder in α5 could also contribute.
Malonate is the prime candidate for the poorly ordered ligand
because it is present at high concentration in the crystals.
The location and interactions of conserved P-subsite residue
Arg180 are unexpectedly and remarkably different in DxnB2
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compared to the architecture observed in BphDLB400 and HsaD.
In the latter, the guanidinium groups of the corresponding Arg
residues are positioned to interact with the substrate’s
carboxylate group and with residues at the apex of the
connector between β3 and α1 (DxnB2 residues 35 and 36). In
DxnB2, Arg180 interacts with residues from the α1 helix; the
shortest contact, 3.2 Å, is between Nη2 and the carbonyl O of
Ser42. Relative to BphDLB400, the guanidinium group of Arg180
is displaced by ∼3.5 Å. The orientation of Arg180 is shared
between the structures of the WT and S105A variant and
therefore cannot be attributed to the presence of sulfate in the
former.
DxnB2 S105A:3-Cl HOPDA Complex. To define DxnB2-
specific interactions with recalcitrant substrates, S105A crystals
were incubated with 3-Cl HOPDA, resulting in a structure of
the binary complex at a resolution of 2.0 Å. Similar to the
S105A structure in the absence of HOPDA, electron density
was poorly defined for residues 140−151. Although the model
includes these residues, only the course of the backbone is clear
from the density. The electron density for the 3-Cl HOPDA
ligand was also incomplete in early maps such that the distal
atoms of the phenyl moiety (CB3, CB4, and CB6) and the C1-
carboxylate could not be confidently positioned. Nevertheless,
the density clearly indicated that the C3-Cl and C2-O atoms
are in a cis-conformation around the C2-C3 bond and that the
carbon atoms of the dienoate moiety are coplanar (Figure 6B).
Ultimately, the 3-Cl HOPDA molecule was modeled as a
(2Z,4E)-2-oxido-6-oxo- isomer based on the available electron
density and the yellow color of the crystals, which is indicative
of a dianionic species (3-Cl HOPDA pKa2 = 6.18). Other
isomers of HOPDA may be consistent with the electron
density. In line with the spectrophotometric data, the binding
mode of 3-Cl HOPDA differed significantly from those
observed in BphDLB400 S112A:HOPDA and DxnB2
S105A:HOPDA or 5,8-diF HOPDA binary complexes, which
have been correlated to ESred.11,15
Figure 7. Protein surface representation of the hydrophobic pocket
that stabilizes the planar 3-Cl HOPDA binding mode in (A) DxnB2
S105A and (B) BphDLB400 S112A (PDB ID: 2RHT). Leu156 of the
BphDLB400 lid domain is the positional equivalent of Val149 in DxnB2,
which is further removed from the MCP bound in the dimeric
homologue. Other DxnB2 lid domain residues illustrated include
Ile162 (BphDLB400 Met171) and Leu166. The remaining residues that
help to form the hydrophobic pocket are part of the α/β-hydrolase
core domain.
Although 3-Cl HOPDA binds to DxnB2 S105A in a planar
conformation with the C6-O in the oxyanion hole, as previously
observed for BphDLB400, enzyme−substrate interactions in the
P-subsite differ markedly. In particular, the guanidinium group
of Arg180 does not interact with the substrate’s carboxylate
group and its C2-O atom as in the BphDLB400 complex.
Although it appears that the Arg180 side chain could be placed
in an appropriate location to bind the carboxylate, it maintains
the same slightly removed location and conformation as in the
unliganded structures. Interactions of the carboxylate with
Trp256 Nε1, Asn104 Nδ2, and a water molecule bridging to
Arg180 replace the direct HOPDA:Arg190 interactions
observed in the BphDLB400 complex.
The interactions of the 3-Cl substituent also vary between
the complexes. In BphDLB400, the Cl binding pocket includes
atoms within 4.4 Å from five residues, and it is capped by side
chains from a well-ordered and close-packed α5 helix, which
supplies a 3.7 Å contact with the side chain of Leu156 that is
backed by the side chain of a second-shell residue Ile153
(Figure 7A). By contrast, the DxnB2 pocket includes only three
side chain interactions within 4.4 Å (Figure 7B). In addition, α5
is poorly ordered, as noted above, and the Cα atoms of the
DISCUSSION
■
The single turnover data together with the proposed catalytic
mechanism of MCP hydrolases indicate that the higher
specificity of DxnB2 for 3-Cl HOPDAs (kcat/Km) versus
BphD is due to higher rate of ESred formation in the former.
More specifically, the hydrolysis of HOPDAs by DxnB211 and
14−16
BphDLB400
is defined by three observable steps for which
the rates have been determined: (i) ESred formation (>500 s−1);
(ii) ESred decay (50 to 70 s−1), which is coupled to acylation/
HPD formation and limited by the ketonization reaction; and
(iii) deacylation (kcat ≈ 0.4 to 6.5 s−1). In contrast, the
hydrolysis of 3-Cl HOPDAs by these enzymes is characterized
by just two observable processes (Table 1): (i) formation of an
alternative, presumably nonproductive, binding mode named
14
ESe in BphDLB400 and characterized by a hypo- and/or
hypsochromically shifted absorption spectrum in DxnB2; and
(ii) ESe decay, which is coupled to HPD formation and
corresponds to kcat (Table 1). The failure to detect ESred during
3-Cl HOPDA cleavage indicates that it is consumed at a rate
that precludes detection. Thus, the similarity between the
observed rates of HPD formation and kcat values indicate that
ESred formation is rate-limiting to catalysis.10 For 3-Cl HOPDA,
this rate (1/τ2) is at least 3000-fold slower than for HOPDA in
DxnB2.17 However, formation of ESred from 3-Cl HOPDA in
DxnB2 (0.152 s−1) is 20-fold faster than in BphDLB400 (0.0077
residues corresponding to Leu156 and Ile153 in BphDLB400
,
DxnB2 Val149 and Leu146, are 1.4 Å and 3.1 Å further
removed from the chlorine atom.
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s−1), accounting for the increased specificity of the former for
recalcitrant PCB metabolites. Interestingly, ESred formation
during turnover of 3,9,11-triCl HOPDA was only ∼3-fold
slower than 3-Cl HOPDA. This small reduction is in line with
the general observation that ring substituents are minor
contributors to the substrate specificity of BphDLB400 and
DxnB2.8,10
The structural analyses of the MCP hydrolases further
support a significant contribution of the NC-loop to catalysis.
This loop, which joins the core and lid domains, has been
proposed to contribute to catalysis in several other α/β-
hydrolase families including the lipases,35 haloalkane dehaloge-
nases,36 and epoxide hydrolases.37 Furthermore, substitution of
the NC-loop was required to convert an esterase into an
epoxide hydrolase.38 In the MCP hydrolases, domain-swapping
experiments between BphD (BphDLA4) and MfphA from Dyella
ginsengisoli LA-4 further established that the identity of the lid
domain is a strong determinant of substrate specificity,39 and a
single amino acid substitution located in the NC-loop of
BphDLA4 reduced kcat/Km for HOPDA by nearly 300-fold.40
Molecular dynamics simulations have also been used to argue
the relevance of NC-loop flexibility in the haloalkane
dehalogenases,36 and significant α5 helix motions were
observed in the Candida antarctica lipase B (CALB).41
Interestingly, while the CALB and MCP hydrolase lid domains
are distinct, the first helix of each, which follows the NC-loop,
occupies very similar three-dimensional space in relation to the
α/β-hydrolase core. Indeed, the sum of the available data for
the α/β-hydrolases sets a precedent for the involvement of the
NC-loop in substrate binding and catalysis by the MCP
hydrolases.
The identification of the NC-loop as a determinant of MCP
hydrolase oligomeric state provides a further example of the
ability of the α/β-hydrolase core to accommodate family
specific insertions that determine substrate and chemical
specificity.42,43 Thus, comparison of the seven structurally
characterized MCP hydrolases representing three different
oligomeric states revealed high structural variation for the NC-
loop. The NC-loop residues appear to be most highly
conserved among the class I enzymes and at least partially
mediate tetramer formation. In particular, the NC-loop
contributes to two distinct tetrameric interfaces, and the
phenylalanine residue of its F-X-P-X-P motif lies at the core of
the biological assembly. Moreover, differences in the NC-loop
are propagated to the lid domain architecture: the position of
and interactions involving the first helix of the MCP hydrolase
lid, α5, are not conserved between the phylogenetically distinct
classes. Each class possesses a distinguishing set of intralid
interactions, which are likely to mitigate substrate specificity.
The variable disorder in the α5 helix in crystal structures of
DxnB2 suggests that this region has increased mobility relative
to that of other MCP hydrolases with the exception of CarC.
Such movement is probably restricted in the tetrameric
enzymes based on the location of the tetrameric interfaces.
Interestingly, superposition of three DxnB2 S105A:HOPDA
binary complexes reveals large difference in the position of the
α5 helix, which varies up to ∼6 Å based on placement of the
Cα atoms (Figure 8). The position of α5 helix drastically affects
the form of the NP-subsite. Specifically, the α5 helix carries
Leu146 and Val149, which are further removed from the
Figure 8. Comparison of DxnB2 S105A binary complexes reveals
differences in the lid domains. The three structures include S105A in
complex with 3-Cl HOPDA (green), HOPDA (white), and 5,8-diF
HOPDA (gray).
the relative increase in hydrolytic activity toward recalcitrant
PCB metabolites. Thus, the possible mobility of this helix may
account for the increased accessibility to ESred in DxnB2 versus
BphDLB400
.
The 3-Cl HOPDA binding mode observed in DxnB2
suggests that significant conformational changes must occur
in order to reach a productive substrate-binding mode. Many of
14
these changes have been highlighted by studies in BphDLB400
.
The DxnB2 S105A:3-Cl HOPDA complex is particularly
intriguing since in addition to the distance observed between
the NP-subsite and the substrate, it is also missing a number of
polar contacts at the P-subsite. Thus, neither Asn43 nor Arg180
contact the dienoate moiety in the DxnB2 S105A:3-Cl HOPDA
binary complex. These contrast with the direct interactions
observed in other crystalline complexes that were correlated to
ESred,11,15 and those that potentially form in the BphDLB400
S112A:3-Cl HOPDA complex.14 These observed differences in
stable, planar ESe complexes may also contribute to the ability
of DxnB2 to form ESred faster than BphDLB400
.
The high structural variance of the NC-loop contributes to
the ability of DxnB2 to overcome the stabilization of 3-Cl
HOPDAs in nonproductive, planar dienoate binding modes.
Moreover, the NC-loop is prominent at MCP hydrolase
tetrameric interfaces, suggesting that it at least partially
stabilizes the oligomeric state of class I family members. The
attributes of this loop have implications for both environmental
bioremediation and the design of therapeutics. Replacement of
BphDLB400 (or other bona fide BphD enzymes) by DxnB2 in
the Bph pathway may alleviate the metabolic bottleneck caused
by 3-Cl HOPDAs. The dimeric MCP hydrolases could also
represent better starting points for future enzyme engineering
efforts. The plasticity of the NC-loop should also be considered
in designing inhibitors of HsaD, which appears to be required
for survival of Mycobacterium tuberculosis in macrophages.44
Finally, while the results further establish the catalytic
adaptability of the α/β-hydrolase superfamily and highlight
the influence of lid architecture within a specific enzyme family,
the correlation of loop and lid movements to catalysis is
relevant to a broader understanding of enzyme dynamics.45
substrate in crystal structures of DxnB2 than in BphDLB400
.
Accordingly, the comparatively limited restraint placed by the
DxnB2 NP-site on the substrate is also likely to contribute to
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ASSOCIATED CONTENT
* Supporting Information
HPLC analysis of reaction products, SEC-MALS, and sequence
alignment. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*E-mail: jtb@purdue.edu; leltis@mail.ubc.ca.
■
Present Address
⊥Department of Integrative Structural and Computational
Biology, The Scripps Research Institute, 10550 North Torrey
Pines Road, La Jolla, CA 92037.
Author Contributions
∥These authors contributed equally to this work.
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This research was supported by a Discovery grant from the
Natural Sciences and Engineering Research Council of Canada
to L.D.E., and A.C.R. is an NSERC PGS-D scholar. Use of the
Advanced Photon Source was supported by the U.S. Depart-
ment of Energy, Office of Science, Office of Basic Energy
Sciences, under Contract No. W-31-109-Eng-38. Some of the
diffraction data were obtained using GM/CA at the APS
facilities. GM/CA at the APS has been funded in whole or in
part with Federal funds from the National Cancer Institute (Y1-
CO-1020) and the National Institute of General Medical
Sciences (Y1-GM-1104). Use of BioCARS Sector 14 was
supported by grants from the National Center for Research
Resources (5P41RR007707) and the National Institute of
General Medical Sciences (8P41GM103543) from the National
Institutes of Health.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge Dr. Michael Murphy and Marek Kobylarz for
access to and technical assistance with the SEC-MALS
instrument, respectively.
ABBREVIATIONS
■
Bph, biphenyl; HOPDA, 2-hydroxy-6-oxo-6-phenylhexa-2,4-
dienoic acid; HPD, 2-hydroxy-2,4-pentadienoic acid; MCP,
meta-cleavage product; PCB, polychlorinated biphenyl; PDA,
photodiode array
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NOTE ADDED AFTER ASAP PUBLICATION
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This manuscript published ASAP on August 9, 2013. The
image for Figure 5 was replaced and the revised version was
reposted on August 20, 2013.
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