Characterization of a carbon-carbon hydrolase from Mycobacterium tuberculosis involved in cholesterol metabolism

Article abstract of DOI:10.1074/jbc.M109.058081

In the recently identified cholesterol catabolic pathway of Mycobacterium tuberculosis, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (HsaD) is proposed to catalyze the hydrolysis of a carbon-carbon bond in 4,5-9,10-diseco-3-hydroxy-5,9,17-tri-oxoandrosta-1 (10),2-diene-4-oic acid (DSHA), the cholesterol meta-cleavage product (MCP) and has been implicated in the intracellular survival of the pathogen. Herein, purified HsaD demonstrated 4-33 times higher specificity for DSHA (k_cat/K_m = 3.3 ± 0.3 × 10⁴ M^-1 s^-1) than for the biphenyl MCP 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) and the synthetic analogue 8-(2-chlorophenyl)-2-hydroxy-5-methyl-6-oxoocta-2, 4-dienoic acid (HOPODA), respectively. The S114A variant of HsaD, in which the active site serine was substituted with alanine, was catalytically impaired and bound DSHA with a K_d of 51 ± 2 μM. The S114A·DSHA species absorbed maximally at 456 nm, 60 nm red-shifted versus the DSHA enolate. Crystal structures of the variant in complex with HOPDA, HOPODA, or DSHA to 1.8 -1.9 A indicate that this shift is due to the enzyme-induced strain of the enolate. These data indicate that the catalytic serine catalyzes tautomerization. A second role for this residue is suggested by a solvent molecule whose position in all structures is consistent with its activation by the serine for the nucleophilic attack of the substrate. Finally, the α-helical lid covering the active site displayed a ligand-dependent conformational change involving differences in side chain carbon positions of up to 6.7 A, supporting a two-conformation enzymatic mechanism. Overall, these results provide novel insights into the determinants of specificity in a mycobacterial cholesterol-degrading enzyme as well as into the mechanism of MCP hydrolases.

Full text of DOI:10.1074/jbc.M109.058081

Enzyme Catalysis and Regulation:
Characterization of a Carbon-Carbon
Hydrolase from Mycobacterium
tuberculosis Involved in Cholesterol
Metabolism
Nathan A. Lack, Katherine C. Yam, Edward
D. Lowe, Geoff P. Horsman, Robin L. Owen,
Edith Sim and Lindsay D. Eltis
J. Biol. Chem. 2010, 285:434-443.
doi: 10.1074/jbc.M109.058081 originally published online October 29, 2009
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 1, pp. 434–443, January 1, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Characterization of a Carbon-Carbon Hydrolase
from Mycobacterium tuberculosis Involved in
□
S
Cholesterol Metabolism*
Received for publication, August 28, 2009, and in revised form, September 25, 2009 Published, JBC Papers in Press, October 29, 2009, DOI 10.1074/jbc.M109.058081
Nathan A. Lack‡1, Katherine C. Yam§2, Edward D. Lowe¶, Geoff P. Horsman§, Robin L. Owenʈ, Edith Sim‡,
and Lindsay D. Eltis§3
From the Departments of ‡Pharmacology and ¶Molecular Biophysics, University of Oxford, Oxford OX1 3QT, United Kingdom, the
§Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada,
and the ʈSwiss Light Source, Paul Scherrer Institut, 5232 Villigen, Switzerland
In the recently identified cholesterol catabolic pathway of rial cholesterol-degrading enzyme as well as into the mecha-
Mycobacterium tuberculosis, 2-hydroxy-6-oxo-6-phenylhexa- nism of MCP hydrolases.
2,4-dienoate hydrolase (HsaD) is proposed to catalyze the hy-
drolysis of a carbon-carbon bond in 4,5–9,10-diseco-3-hydroxy-
5,9,17-tri-oxoandrosta-1(10),2-diene-4-oic acid (DSHA), the
cholesterol meta-cleavage product (MCP) and has been impli-
cated in the intracellular survival of the pathogen. Herein, puri-
Mycobacterium tuberculosis is the leading cause of bacterial
mortality, causing an estimated 2 million deaths/year (1). The
mechanisms underlying the remarkable ability of this pathogen
to survive for long periods of time within the host are poorly
understood (2). Although it was well known that saprophytic
mycobacteria could metabolize cholesterol (3), it was only
recently demonstrated that pathogenic strains can also utilize
this nutrient as a growth substrate (4, 5). Interestingly, choles-
terol has been found in high concentrations within caseating
granulomas in both humans and mice (6, 7), and bacteria have
been observed congregating around cholesterol foci (7). High-
lighting the importance of cholesterol in bacterial pathogene-
sis, the deletion of genes involved in cholesterol metabolism
reduces the virulence of M. tuberculosis (5, 8). Therefore, fur-
ther knowledge of cholesterol metabolism in M. tuberculosis is
crucial to our understanding of bacterial virulence.
fied HsaD demonstrated 4–33 times higher specificity for
؊1
DSHA (kcat/Km ؍ 3.3 ؎ 0.3 ؋ 104
M
s
؊1) than for the
biphenyl MCP 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic
acid (HOPDA) and the synthetic analogue 8-(2-chlorophenyl)-
2-hydroxy-5-methyl-6-oxoocta-2,4-dienoic acid (HOPODA),
respectively. The S114A variant of HsaD, in which the active site
serine was substituted with alanine, was catalytically
impaired and bound DSHA with a Kd of 51 ؎ 2 ␮M. The
S114AꢀDSHA species absorbed maximally at 456 nm, 60 nm
red-shifted versus the DSHA enolate. Crystal structures of the
variant in complex with HOPDA, HOPODA, or DSHA to
˚
1.8–1.9 A indicate that this shift is due to the enzyme-in-
duced strain of the enolate. These data indicate that the cat-
alytic serine catalyzes tautomerization. A second role for this
residue is suggested by a solvent molecule whose position in
all structures is consistent with its activation by the serine for
the nucleophilic attack of the substrate. Finally, the ␣-helical
lid covering the active site displayed a ligand-dependent con-
formational change involving differences in side chain car-
M. tuberculosis catabolizes cholesterol using a metabolic
pathway similar to that identified in Rhodococcus jostii RHA1
(4, 9). In this pathway, the aerobic degradation of the four-
ringed steroid nucleus occurs through the opening of ring B,
aromatization of ring A, and hydroxylation of the phenolic
metabolite. The resulting catechol undergoes oxygenolytic
cleavage, yielding 4,5–9,10-diseco-3-hydroxy-5,9,17-trioxoan-
drosta-1(10),2-diene-4-oic acid (DSHA),4 (see Fig. 1 for IUPAC
numbering) a meta-cleavage product (MCP; Fig. 1). The latter
is then hydrolyzed by the MCP hydrolase, HsaD, to yield 9,17-
dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid (DOHNAA)
˚
bon positions of up to 6.7 A, supporting a two-conformation
enzymatic mechanism. Overall, these results provide novel
insights into the determinants of specificity in a mycobacte-
* This work was supported by grants from the Wellcome Trust (to E. S.) and and 2-hydroxy-hexa-2,4-dienoic acid (HHD). HHD is then
the Canadian Institutes of Health Research (to L. D. E.).
S
metabolized to tricarboxylic acid cycle intermediates, whereas
□
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1 and S2.
the metabolic fate of DOHNAA is unknown. In this pathway,
The atomic coordinates and structure factors (codes 2wud, 2wue, 2wuf, and
2wug) have been deposited in the Protein Data Bank, Research Collaboratory
for Structural Bioinformatics, Rutgers University, New Brunswick, NJ 4 The abbreviations used are: DSHA, 4,5–9,10-diseco-3-hydroxy-5,9,17-tri-
(http://www.rcsb.org/).
oxoandrosta-1(10),2-diene-4-oic acid; HOPDA, 2-hydroxy-6-oxo-6-phe-
nylhexa-2,4-dienoic acid; HOPODA, 8-(2-chlorophenyl)-2-hydroxy-5-
methyl-6-oxoocta-2,4-dienoic acid; DHSA, 3,4-dihydroxy-9,10-seco-
nandrost-1,3,5(10)-triene-9,17-dione; DHDS, 2,3-dihydroxy-6-methyl-
7,8-dihydrostilbene; MCP, meta-cleavage product; HsaD, 2-hydroxy-6-
oxo-6-phenylhexa-2,4-dienoate hydrolase; DOHNAA, 9,17-dioxo-
1,2,3,4,10,19-hexanorandro-stan-5-oic acid; HHD, 2-hydroxy-hexa-2,4-
dienoic acid; ht, histidine-tagged; bis-tris, 1,3-bis(tris(hydroxylmethyl)-
methylamino) propane.
1 Recipient of a studentship from the Natural Sciences and Engineering
Research Council of Canada.
2 Recipient of studentships from the Natural Sciences and Engineering
Research Council and the Michael Smith Foundation for Health
Research.
3 To whom correspondence should be addressed: 2350 Health Sciences Mall,
Vancouver, BC V6T 1Z3, Canada. Tel.: 604-822-0042; Fax: 604-822-6041;
E-mail: leltis@interchange.ubc.ca.
434 JOURNAL OF BIOLOGICAL CHEMISTRY
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Crystal Structure and Kinetics of HsaD
FIGURE 1. The successive reactions catalyzed by HsaC and HsaD in the catabolism of cholesterol by M. tuberculosis and alternate substrates of HsaD.
DSHA is referred to as 4,5–9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-diene-4-oic acid in the steroid literature. However, the carbon atoms are
numbered here according to its IUPAC name, 2-hydroxy-5-methyl-8-(7a-methyl-1,5-dioxooctahydro-1H-inden-4-yl)-6-oxoocta-2,4-dienoic acid, to be consis-
tentwiththenumberinginHOPDAandHOPODA. Inthisstudy, DSHA, HOPDA, andHOPODAweregeneratedbyringcleavageofDHSA, 2,3-dihydroxybiphenyl,
and DHDS, respectively.
HsaD is of particular interest, because transposon mutagenesis 18O into a nonhydrolyzable substrate analogue (19). Subse-
has indicated that it is critical for the intracellular survival of M. quently, low amounts of this gem-diol intermediate were
tuberculosis (10).
detected using 13C NMR spectroscopy (20) in reactions cat-
MCP hydrolases are involved in the aerobic degradation of alyzed by MhpC and BphD. A general base mechanism is
aromatic compounds, hydrolyzing vinylogous 1,5-diketones more consistent with that of other ␣/␤-hydrolases that acti-
arising from the extradiol ring cleavage of catechols (11). Crys- vate small molecules (21).
tal structures of MCP hydrolases, including those of BphD from
Nevertheless, features of the MCP hydrolase mechanism
Burkholderia xenovorans LB400, MhpC from Escherichia coli, remain to be resolved. First, the nature of a transient interme-
and a ht-HsaD (12–14), reveal that these enzymes belong to the diate observed in the transformation of HOPDA by BphD,
␣/␤-hydrolase superfamily and contain a conserved Ser-His- E:Sred, is unclear (13). E:Sred possesses a red-shifted absorption
Asp catalytic triad as well as an “oxyanion” hole, similar to a spectrum (␭
ϭ 492 nm) with respect to that of the free
number of other hydrolytic enzymes (15). The substrate-bind- HOPDA enomlaatxe (␭max ϭ 434 nm). X-ray diffraction studies (13)
ing pocket comprises two subsites: a hydrophilic polar subsite of a similar intermediate (␭
ϭ 506 nm) trapped using the
max
that binds the dienoate moiety of the MCP and a hydrophobic S112A variant of BphD revealed torsion angles that were most
nonpolar subsite that accommodates the remainder of the consistent with a ketonized tautomer of HOPDA (E:Sk). Never-
MCP. In HsaD, the nonpolar subsite is at least twice as a large as theless, the refinements did not exclude the 2-enol tautomer,
that of other MCP hydrolases, possibly to fit the larger choles- and a strained enolate (E:Sse) more satisfactorily accounts for
terol metabolite DSHA (14).
the red-shifted absorption spectrum because steric hindrance
Despite the similar catalytic machinery of MCP hydrolases to planarity about a double bond raises the ground state energy
and serine proteinases, the mechanism of the former appears level but not the excited state (22). Second, the roles of catalytic
to differ from the nucleophilic mechanism of the latter in residues remain unclear. The active site serine has been vari-
two respects. First, the enolate substrate of MCP hydrolases ously proposed to deprotonate the nucleophilic water (19) and
undergoes tautomerization to a keto intermediate prior to to stabilize the oxyanion (23). Similarly, the active site histidine
hydrolysis (16). Second, the ketonized substrate (17) is has been proposed to catalyze tautomerization (23) and to dep-
believed to be hydrolyzed through a general base mechanism rotonate the catalytic water (23). Finally, despite evidence sup-
via a gem-diol intermediate (18, 19). Initial evidence for this porting a general base mechanism, in the structures of BphD
intermediate was obtained from the incorporation of 18O from B. xenovorans LB400 and MhpC from E. coli, no solvent
from H218O into the MCP of MhpC as well as the exchange of species have been detected near the active site of the enzyme.
JANUARY 1, 2010•VOLUME 285•NUMBER 1
JOURNAL OF BIOLOGICAL CHEMISTRY 435

Crystal Structure and Kinetics of HsaD
In this study, we determined the specificity of HsaD for var- mutated hsaD was subcloned into pEMBL18 downstream of
ious MCPs including its proposed physiological substrate, the Plac promoter as an XbaI fragment, yielding pEMHSA.
HsaD was produced and purified from E. coli strain GJ1158
containing pT7HD1. The cells were grown at 30 °C in salt-de-
pleted Luria-Bertani broth (10 g of tryptone extract, 5 g of yeast
extract/liter) supplemented with 100 ␮g/ml ampicillin. The
culture was grown to an optical density at 600 nm of ϳ0.5, at
which point NaCl was added to a final concentration of 300 mM.
The cultures were incubated for an additional 20 h, and then the
cells were harvested by centrifugation. The S114A variant was
produced using E. coli strain DH5␣ containing pEMHSA. The
cells were grown at 30 °C in LB broth supplemented with 100
␮g/ml ampicillin. The culture was grown to an optical density
at 600 nm of ϳ0.5, and isopropyl-1-thio-␤-D-galactopyranoside
was added to a final concentration of 0.5 mM. The culture was
incubated for an additional 20 h before the cells were harvested.
The cell pellet obtained from 4 liters of culture was resus-
pended in 40 ml of 20 mM HEPES, pH 7.5, and disrupted using
three passages through a Emulsiflex C-5 cell disrupter (ATA
Scientific, Sutherland, Australia) operating at a pressure of
15,000 p.s.i. The cell debris was removed by ultracentrifugation
at 120,000 ϫ g for 60 min. The supernatant was removed and
filtered through a 0.45-␮m cellulose filter to yield ϳ45 ml of
raw extract. The raw extract was loaded onto 28 ml of Source
15Q anion exchange resin (GE Healthcare) packed in an AP-2
column (Waters Corp., Milford, MA) equilibrated with 20 mM
HEPES, pH 7.5. The proteins were eluted using a linear gradient
of 70–220 mM NaCl in 280 ml. HsaD eluted at ϳ140 mM NaCl.
Fractions of 10 ml containing activity (in the case of the wild-
type enzyme) or the protein of the expected size (in the case of
the variant) were pooled and concentrated to 10 ml using an
Amicon stirred cell concentrator equipped with an YM10 ultra-
filtration membrane (Millipore, Billerica, MA). The HsaD-con-
taining solution was brought to 1 M (NH4)2SO4, and the sample
was briefly centrifuged to harvest the pellet. The cloudy white
precipitate was washed twice with 1 M (NH4)2SO4 solution;
exchanged into 20 mM HEPES, pH 8.5, to resolubilize the
enzyme; concentrated to Ͼ20 mg/ml; flash frozen as beads in
liquid nitrogen; and stored at Ϫ80 °C. Coomassie Blue-stained
denaturing gels revealed that preparations of the wild-type and
variant proteins were Ͼ97% HsaD.
DSHA. To elucidate the interactions between enzyme and
substrates, a S114A variant of HsaD was generated to slow
substrate turnover, and the crystal structures of the
variantꢀsubstrate complexes were analyzed. These results pro-
vide insights into the catalytic mechanism of MCP hydrolases.
EXPERIMENTAL PROCEDURES
Chemicals—HOPDA used for crystallization was synthe-
sized as previously described (24). 2,3-Dihydroxybiphenyl and
2,3-dihydroxy-6-methyl-7,8-dihydrostilbene (DHDS) were
synthesized by a combined directed ortho metalation cross-cou-
pling strategy as described elsewhere (8, 25). 3,4-Dihydroxy-9,10-
seco-nandrost-1,3,5(10)-triene-9,17-dione (DHSA) was obtained
by incubating a culture of the ⌬hsaC mutant of R. jostii RHA1
with cholesterol as described previously (8). All other chemicals
were of analytical grade.
Preparation and Characterization of MCPs—MCPs were
produced by dissolving the corresponding catechol in a small
volume of ethanol and diluting to the desired volume with 100
mM potassium phosphate, pH 7.5 (ethanol constituted less than
0.1% of the solution). To this solution a sufficient amount of
HsaC was added to completely transform DHDS and DHSA to
HOPODA and DSHA, respectively. Upon the addition of HsaC
to a diluted sample, complete cleavage of DHDS and DHSA was
verified by ensuring that there was no further increase in
absorbance of the ring-cleaved products at 396 nm (⑀ ϭ 6.8 and
3.8 mMϪ1 cmϪ1 for HOPODA and DSHA, respectively). MCPs
used for steady-state kinetic studies were prepared enzymati-
cally using 100 mM potassium phosphate, pH 7.5, and used
within 2 h.
To determine pKa values, HOPODA and DSHA were pre-
pared as described above in unbuffered water. HsaC was
removed by ultrafiltration using a stirred cell equipped with an
YM10 membrane (Amicon). Solutions of ϳ0.5 mM MCP were
acidified to pH 3 using 12 N HCl. This solution was titrated with
aliquots of 50 mM NaOH, and the pH was determined after the
addition of each aliquot. The pKa values were determined from
plots of pH versus the amount of base added.
The nonenzymatic transformation of MCPs in solution (100
mM potassium phosphate, pH 7.5, at 25 °C) was determined by
monitoring the absorption spectra. The experiments were per-
formed using two concentrations of each MCP (0.03, 0.3 mM).
The half-lives were determined by fitting equations to the decay
at 396 nm using Excel.
Kinetic Measurements—The enzyme-catalyzed hydrolysis of
the MCP was monitored using a Varian Cary 5000 spectropho-
tometer equipped with a thermostatted cuvette holder (Varian
Canada, Mississauga, Canada) maintained at 25.0 Ϯ 0.5 °C. The
amount of enzyme used in each assay was adjusted so that the
progress curve was linear for at least 2 min. Initial velocities
were determined from a least squares analysis of the progress
curves using the kinetics module of the Cary WinUV software.
Specificity experiments were performed in a total volume of
1.0 ml of 100 mM ionic strength potassium phosphate, pH 7.5, at
25.0 Ϯ 0.1 °C. The reaction was initiated by adding 5 ␮l of an
appropriately diluted enzyme preparation to the reaction
cuvette. The reactions were monitored at the wavelengths cor-
responding to the enolate. Initial velocities were determined
over the following ranges of substrate concentration: 0.5–30
Protein Production and Purification—Ser114 of HsaD was
substituted with alanine to generate the S114A variant using
the QuikChange II-E site-directed mutagenesis kit (Stratagene)
as per the manufacturer’s instructions. In this reaction, the
template was pT7HD1, a previously constructed pT7Ϫ7 deriv-
ative carrying hsaD under control of the T7 promoter (4). The
primers were 5Ј-CTGGTGGGCAACGCGTTGGGCG-
GGG-3Ј and 5Ј-CCCCGCCCAACGCGTTGCCCACCAG-3Ј.
The nucleotide sequence of the mutated gene was confirmed
using an ABI 373 Stretch DNA sequencer (Applied Biosystems, ␮M HOPDA, 10–300 ␮M HOPODA, and 2–60 ␮M DSHA.
Foster City, CA) and a Big-Dye Terminator v3.1 kit. The Steady-state kinetic parameters were evaluated by fitting the
436 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 1•JANUARY 1, 2010

Crystal Structure and Kinetics of HsaD
TABLE 1
Properties of MCPs
appropriate equations to the data using the least squares and
dynamic weighting options of LEONORA (26).
Half-lifea
pKa
b
Substrate
␭
⑀
Kd Determination—The dissociation constant of the
S114AꢀDSHA complex was determined by titrating a 200-␮l
solution of 100 mM ionic strength potassium phosphate, pH 7.5,
at 25.0 Ϯ 0.1 °C containing 5 ␮M DSHA with S114A and mon-
itoring the change in absorbance at 456 nm. The spectra were
recorded using a Cary 5000 spectrophotometer. The dissocia-
tion constant was evaluated by fitting the binding equation (27)
to the data using the curve fitting program and the nonlinear
statistical analyses of R.
max
Ϫ1
nm
mMϪ1 cm
h
HOPDA
HOPODA
DSHA
434
396
396
25.7
58c
25
7.3c
7.5
6.8
6.8
3.8
15.4
a Half-lives were determined in potassium phosphate buffer (I ϭ 0.1 M), pH 7.5, at
25 °C.
b The pKa values of the respective enolates were determined in unbuffered water.
c Data shown are from Ref. 17.
TABLE 2
Steady-state kinetic parameters of HsaD with HOPDA, HOPODA, and
DSHA
The half-life of S114AꢀDSHA was determined by monitoring
the decay of the complex at 456 nm. The experiment was car-
ried out in a total volume of 150 ␮l of 100 mM ionic strength
potassium phosphate, pH 7.5, at 25.0 Ϯ 0.1 °C containing 14 ␮M
S114A and 6 ␮M DSHA. A single-exponential equation was fit
to the data.
The experiments were performed using potassium phosphate buffer (I ϭ 0.1 M), pH
7.5, at 25 °C. The values in parentheses indicate standard deviations.
Substrate
Km
kcat
s
k
cat/Km
Ϫ1
Ϫ1
␮M
mMϪ1 s
HOPDA
HOPODA
DSHA
8 (1)
310 (70)
17 (2)
0.066 (0.005)
0.33 (0.06)
0.55 (0.02)
9 (1)
1.06 (0.06)
33 (3)
Crystallization of S114A and Preparation of Substrate
Complex—Protein crystals were grown by sitting drop vapor
diffusion with equal volumes (1 ␮l) of S114A (8 mg/ml) and
precipitant (200 mM KSCN, 24% polyethylene glycol 3.35 K, and C-2-OH group. Moreover, these compounds undergo a nonen-
100 mM bis-tris propane, pH 7.0). Disphenoid S114A crystals zymatic transformation in aqueous buffer (20). The half-lives of
typically grew in 3–5 days at 19 °C. Complexes between S114A HOPODA and DSHA were less than 30 h, significantly less than
and HOPDA, HOPODA, and DSHA were obtained by soaking that of HOPDA. These data are summarized in Table 1 together
S114A crystals in 10 ␮l of precipitant supplemented with 15 mM with the pKa values of the enolic hydroxyl groups as determined
of each ligand for 30–60 min.
by titration and the extinction coefficients of the respective
Diffraction Data Measurement and Processing—The crystals enolate anions.
were briefly transferred to a cryoprotectant solution (1:4 (v/v)
Substrate Specificity of HsaD—Steady-state kinetic studies
mixture of glycerol and precipitant) and then flash-frozen in were conducted to assess the substrate specificity of HsaD.
liquid nitrogen. For enzyme-ligand complexes, the cryopro- Over the substrate concentration ranges studied, HsaD dis-
tectant solution was supplemented with the ligand (15 mM).
played Michaelis-Menten behavior with all three MCPs. Con-
S114A crystal x-ray data were collected in-house using a sistent with the role of the enzyme in cholesterol degradation,
Bruker MicroStar x-ray generator with a Smart6000 CCD HsaD had the highest specificity (kcat/Km) for DSHA, which was
detector and integrated with the commercial software SAINT ϳ33 times higher than for HOPODA but only ϳ4 times higher
(Bruker-AXS). X-ray data from S114AꢀHOPDA crystals were than for HOPDA (Table 2). Although HsaD had a higher spec-
collected at the Swiss Light Source on Beamline X06SA with a ificity for DSHA over HOPODA, the enzyme turned over these
Pilatus 6M detector and integrated with MOSFLM (28). X-ray substrates with similar kcat values.
data from HOPODA and S114AꢀDSHA crystals were collected
Characteristics of S114A in Solution—The catalytic serine of
at the Diamond Light Source on Beamline I03 with a Quantum HsaD was substituted with alanine to generate a catalytically
ADSC CCD detector and integrated with MOSFLM (28).
impaired variant. The addition of a 60-fold excess of S114A to a
Structure Determination and Refinement—The initial phases 5 ␮M solution of DSHA produced a species with an absorbance
of S114A were determined by molecular replacement using ht- maximum of 456 nm (Fig. 2), 60 nm red-shifted with respect to
HsaD (Protein Data Bank code 2vf2), refined to 2.3 Å, as a that of the DSHA enolate tautomer. This species had a half-life
search template (14) with the program MOLREP (29). For the of 3 h (supplemental Fig. S1), similar to that of the BphD
enzyme-ligand complexes the phases were determined as S112AꢀHOPDA complex (34). Based on this, the upper limit of
described above, using the refined S114A structure as a search the kcat value of S114A for DSHA was 8.5 (Ϯ 0.9) ϫ 10Ϫ5 s
,
Ϫ1
template. Simulated annealing refinement was followed with 6500-fold lower than that of the wild-type enzyme. This slow
iterative cycles of manual density fitting with COOT (30) and turnover facilitated further study of the S114AꢀDSHA
refinement with PHENIX (31). PHENIX eLBOW (31) was used complex.
to calculate the geometric restraints for HOPDA, HOPODA,
Titration of the DSHA with S114A yielded a dissociation
and DSHA. The stereochemical properties and quality of the constant (Kd) of 51 Ϯ 2 ␮M (Fig. 2, inset). In comparison, titra-
final model were assessed with the program MOLPROBITY tion of a 2 ␮M solution of HOPDA with the variant enzyme
(32). All of the structural figures, alignments, and graphical ren- resulted in modest spectral shifts; in the presence of a 10-fold
derings were done using PYMOL (33).
excess of S114A, most of the HOPDA (ϳ90%) was present as
the free enolate.
RESULTS
Crystallography—To characterize the interactions of HsaD
Physical Properties of HOPODA and DSHA—The absorption with its substrate, crystallization studies were undertaken using
spectrum of MCPs are strongly pH-dependent because of the the S114A variant. The variant failed to crystallize using condi-
JANUARY 1, 2010•VOLUME 285•NUMBER 1
JOURNAL OF BIOLOGICAL CHEMISTRY 437

Crystal Structure and Kinetics of HsaD
FIGURE 2. Characterization of the S114AꢀDSHA complex by UV-visible absorption spectroscopy. A, spectra of S114AꢀDSHA and DSHA in solution are
shown by dotted and solid lines, respectively. The complex absorbs maximally at 456 nm. B, the curve represents the fit of the binding equation to ⌬A456
observed upon titrating DSHA with S114A (Kd ϭ 51 Ϯ 2 ␮M). The experiments were performed using potassium phosphate (I ϭ 0.1 M, pH 7.5) at 25 °C.
TABLE 3
Summary of data collection and refinement statistics
The values in parentheses are for the highest resolution shell.
S114
S114AꢀHOPDA
S114AꢀHOPODA
S114AꢀDSHA
X-ray source
Cu-K␣
Swiss light source (X06SA)
Diamond (I03)
Diamond (I03)
Space group
F 222
F 222
F 222
F 222
Unit cell parameters
a ϭ 112.3, b ϭ 118.8,
c ϭ 182.6, ␣ ϭ ␤ ϭ ␥ ϭ 90
a ϭ 111.9, b ϭ 118.0,
c ϭ 180.9, ␣ ϭ ␤ ϭ ␥ ϭ 90
a ϭ 111.9, b ϭ 118.0,
c ϭ 180.9, ␣ ϭ ␤ ϭ ␥ ϭ 90
a ϭ 111.9, b ϭ 118.0,
c ϭ 180.9, ␣ ϭ ␤ ϭ ␥ ϭ 90
Data collection statistics
Resolution (Å)
No. of unique reflections
R␴
59.8-2.1 (2.2-2.1)
35,484 (4560)
0.0753 (0.2759)
11.7 (3.8)
29.49-1.80 (1.80-1.90)
55,165 (7928)
0.080 (0.390)
16.8 (3.3)
40.66-1.80 (1.80-1.90)
55,043 (8086)
0.069 (0.413)
16.0 (3.1)
40.59-1.90 (2.00-1.90)
46,107 (6736)
0.094 (0.419)
9.9 (2.0)
I/␴ (I)
Completeness (%)
99.5 (99.2)
98.6 (97.8)
99.1 (100)
98.4 (99.3)
Multiplicity
4.2 (3.8)
4.2 (4.2)
5.0 (5.0)
3.5 (3.5)
Refinement and model statistics
Resolution (Å)
49.7789-2.1 (2.11-2.10)
29.49-1.80 (1.90-1.80)
36.34-1.80 (1.90-1.80)
40.59-1.90 (2.00-1.90)
No. reflections used (work ϩ test)
35,439
0.192
54,360
53,203
0.194
42,140
a
a
Rwork
0.167
0.209
Rfree
0.239
0.192
0.210
0.231
No. of residues (chain A/chain B)
No. of water molecules
281/281
275
282/282
281/282
334
281/281
280
183
Additional molecules
2 thiocynate
4990
1 glycerol, 1 thiocynate
2 thiocynate
4775
2 glycerol, 1thiocynate
Total No. of atoms
4871
0.008
0.783
16.5
4725
0.013
0.933
28.5
Root mean square deviation bond lengths (Å)
Root mean square deviation bond angles (°)
Wilson B-factor (Å2)
0.009
0.008
0.746
0.716
16.6
21.1
Mean B-factor (Å2)
21.0
21.6
27.3
34.4
Ramachandran statistics (%)
Core region
98.04
1.96
0.00
98.40
1.60
0.00
98.06
1.94
0.00
97.84
2.16
0.00
Additional allowed region
Disallowed
a Rwork and Rfree ϭ ⌺h͉͉Fo(h)͉ Ϫ ͉Fc(h)͉͉/⌺h͉͉ for the working set and test set (5%) of reflections, where the Fo(h) and Fc(h) are the observed and calculated structure factor
amplitudes for reflection h.
␣
tions previously developed for ht-HsaD (14). After screening 0.26 Å, C ) except for the substituted active site residue. Briefly,
and optimization, diffracting disphenoid crystals of S114A this structure comprises a core ␣/␤ hydrolase domain with a
could be reproducibly grown. Using these conditions, the struc- four-helix lid domain, inserted between residues 137 and 233 in
tures of S114A were determined alone or in complex with HsaD, which “caps” the active site (supplemental Fig. S2). As
HOPDA, HOPODA, or DSHA at resolutions between 1.8 and with other MCP hydrolases (12, 13), the residues of the lid
2.1 Å. The crystals have space group F222 (a ϭ 112Å, b ϭ 118Å, domain in substrate-free S114A had an elevated temperature
c ϭ 181Å) with two protomers/asymmetric unit. There was factor (26.2 Ϯ 0.5 Å2 S.E.) compared with the rest of the struc-
clear electron density in all structures for at least residues ture (19.7 Ϯ 0.2 Å2 S.E.), with the highest B-factor found in helix
7–288. For all of the models, Ͼ98% of the residues were in the ␣L4 (residues 199–212; 36.2 Ϯ 1.2 Å2 S.E.) (Fig. 3). The substi-
favored region, and none were in the disallowed region of Ram- tuted active site residue is found within a tightly turning
achandran plots as defined by MolProbity (32). The statistics of “nucleophilic elbow” (Gly112, Asp113, Ala114, Leu115, and
the data collection and refinement for these structures are sum- Gly116) similar to other ␣/␤ hydrolases. This strand-turn-helix
marized in Table 3.
motif introduces unfavorable main chain torsional angles but
Structure of S114A—The structure of S114A is essentially contributes to the “oxyanion hole” formed by the backbone
identical to that of ht-HsaD (14) (root mean square deviation, nitrogen atoms from Leu115 and Gly45. His269 of the catalytic
438 JOURNAL OF BIOLOGICAL CHEMISTRY
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Crystal Structure and Kinetics of HsaD
triad forms a hydrogen bond via N␦1 with O␦2 of Asp241 (2.7 Å)
and likely also interacts with O␦1 of this residue (3.2 Å).
Structures of S114AꢀMCP Complexes—In the structure of
S114A crystals soaked with HOPDA, there was clear electron
density in the active site, supporting the presence of HOPDA in
both protomers A and B (Fig. 4A). However, in crystals soaked
with either DSHA or HOPODA, there was well defined electron
density only in the active site of protomer B (Fig. 4, B and C). In
protomer A of DSHA-soaked crystals, there was density sup-
porting the presence of a ligand, but it was not sufficiently well
resolved for accurate placement. Similar to previously pub-
lished work, attempts to fit the 3E,5Z,2E,4E and 2Z,4Z 2-enol or
the monoanionic (Z) 2-keto tautomers of the ligand were
unsuccessful (34). The ligands were therefore initially refined as
both the 2Z,4E enol and monoanionic (E) 2-keto tautomers.
Protomer B of S114AꢀHOPDA was refined first, because this
structure had the clearest ligand electron density. Although
both tautomers fit the electron density of HOPDA equally well,
the refinement of the ligand introduced considerable deviations
of the torsion angles for the enol tautomer. Specifically, the
refinement of the enol tautomer introduced a 67° deviation
from planarity of the torsion angle about the C-4–C-5 bond
(Table 4). By contrast, the keto tau-
FIGURE3. SecondarystructureandB-factorofS114A. Thesecondarystruc-
ture of S114A was color-coded according to C␣ B-factor from blue (lowest
B-factor: 6.0 Å2) to red (highest B-factor: 67.4 Å2).
tomer refined without significant
deviations in the expected torsional
angle of the C-3ϭC-4 double bond.
A similar trend was seen in pro-
tomer A of the S114AꢀHOPDA, pro-
tomer B of S114AꢀHOPODA, and
S114AꢀDSHA structures, in which
the C-4–C-5 bond refined with 51,
77, and 67° deviations from planar-
ity, respectively (Table 4). There-
fore, all of the ligands were refined
as the monoanionic (E) 2-keto
tautomers.
In each of the three S114AꢀMCP
structures, the ligands orientated
similarly in the active sites; the
dienoate was bound to the polar
subsite, and the rings (bicycloal-
kanone in the case of DSHA and
phenyl in the case of HOPODA and
HOPDA) were found in the nonpo-
lar subsite (Fig. 5). More specifically,
the C-6-oxo group of each ligand
forms hydrogen bonds with the
backbone amides of Gly45 (2.7–2.9
Å) and Leu115 (2.8–3.1 Å) (Fig. 6A).
This interaction keeps the ligands in
close proximity with active site res-
idue 114. The C-5-methyl group of
both DSHA and HOPODA was
located within
a
hydrophobic
pocket lined by the side chains of
Phe173, Met177, and Leu158. When in
the presence of a ligand, Arg192 was
found to adopt two conformations:
FIGURE 4. Section of electron density map of S114A in complex with MCP. Stereo view showing 2Fo Ϫ Fc
(blue, contour level ϭ 1 ␴) and Fo Ϫ Fc (green, contour level ϭ 2.5␴). The electron density maps are shown for
the ligand HOPDA (A), DSHA (B), and HOPODA (C). The protein structures are shown as white, whereas the
ligandsareshownaspink(HOPDA), purple(DSHA), orcyan(HOPODA). Allofthestructuresareshowninballand
stick representation, with the nitrogen, oxygen, and sulfur atoms colored blue, red, and yellow, respectively.
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Crystal Structure and Kinetics of HsaD
one similar to that observed in the absence of ligand and a with Asn54 (3.1–3.4 Å). Although likely too distant to form
3
second conformation involving a rotation of ϳ90° about ␹
such that the guanidium group is in closer contact with ligand.
This movement allowed the formation of a salt bridge (2.7–3.4
Å) between N␩1 of Arg192 and the carboxylic acid group of the
substrate. In addition, the substrate carboxylic acid interacts
hydrogen bonds with the C-2-oxo group of HOPDA,
HOPODA, and DSHA, the NH2 of Trp270 (3.6–4.1 Å), N␦2 of
Asn113 (3.7–4.0 Å), and N⑀2 of His269 (3.5–4.0 Å) may contrib-
ute to a polar environment important for substrate binding and
positioning (Fig. 6A). In all structures, a solvent species was
present 3.2–3.4 Å from the C-6-carbonyl (Fig. 6B) and in two of
them, S114AꢀDSHA and S114AꢀHOPODA, formed an angle
(water O–C-6–O-6) that was within 10–20° of the Bu¨rgi-Dun-
itz angle of 107°. Stabilized by hydrogen bonds with N␦2 of
Asn244 (3.0–3.1 Å), this water molecule has a relatively low
B-factor (30.8 Ϯ 12.3 Å2), suggesting essentially 100% occu-
pancy. A similarly positioned solvent species was also present in
the ligand-free S114A structure. The bicycloalkanone and phe-
nylchloro moieties of DSHA and HOPODA, respectively, were
located within the nonpolar subsite in a hydrophobic region
formed by Phe212, Met208, Leu115, and Val155. However, the ring
moieties of all MCPs were found to have elevated B-factors
compared with the remainder of the ligand, suggesting that
these groups are mobile. As such, there is weak electron density
for these groups (Fig. 4, B and C).
TABLE 4
Torsional angles of the refined 2-enol and 2-keto tautomers of
HOPDA, HOPODA, and DSHA in S114A
2-Enol
2-Keto
C2-C3 C3-C4 C4-C5 C2-C3 C3-C4 C4-C5
Bonding
Double Single Double Single Double Single
HOPDAchainB
HOPDAchainA
HOPODA
DSHA
174
164
173
159
175
168
165
167
113
129
103
113
177
149
176
158
177
173
165
175
116
139
98
97
Comparison of the five ligand-containing protomers with the
three ligand-free protomers of S114A revealed two conforma-
tions of the ␣L4 helix within the lid domain. In the presence of
the ligand, the lid domain is observed in a “closed” orientation,
whereas the lid domain of the ligand-free protomers is in an
“open” orientation. As displayed in Fig. 7, for the DSHA-con-
taining protomer B, these two conformations differ in the posi-
␣
tion of C (Gly209) by up to 2.8–3.3 Å, and the position of
individual side chain carbons (Phe212) differs by up to 4.2–6.7 Å
(Fig. 7). The net effect of the implied movement places Met208
and Phe212 closer in the nonpolar subsite of the binding pocket.
Although neither residue is likely to directly influence the bind-
ing of HOPDA, both could form hydrophobic interactions with
the ring moieties of DSHA and HOPODA, respectively. The
conformation of the ␣-helix in the ligand-free protomers is
FIGURE 5. Overlay of the MCP substrates HOPDA, HOPODA, and DSHA in
complex with S114A. The protein (white) is shown in with the side chains
involved in ligand binding shown as sticks. The ligands are represented as
sticks and are colored pink (HOPDA), cyan (HOPODA), and purple (DSHA). In all
structures the nitrogen, oxygen, and sulfur atoms colored blue, red, and yel-
low, respectively.
FIGURE 6. Interaction of DSHA with S114A. A, the structure of S114A (white) in complex with DSHA (purple) is shown in cartoon representations with the
aminoacidsidechainsinvolvedinbindingandDSHAdisplayedinstickrepresentation. TheinteractionsbetweenS114AandtheC-2oxoandC-1carboxylicacid
are shown by yellow dashes, whereas the interactions between C-6 oxo and S114A are shown by red dashes. B, the structure of S114A (white) in complex with
DSHA (purple). The water molecule (HOH89) is shown by a red star. All of the distances are in angstroms.
440 JOURNAL OF BIOLOGICAL CHEMISTRY
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Crystal Structure and Kinetics of HsaD
tors than the remainder of the protein (12, 37) as observed here
for HsaD. Second, structures of BphD obtained in the different
states revealed similar but smaller differences in lid positions
(34, 38). The presence of malonate in the active site cleft of
substrate-free BphD may have reduced the observed differ-
ences in this enzyme. The conformational differences seen in
the lid domain of HsaD are lesser in magnitude than those
observed in another ␣/␤ hydrolase, the lipase from Rhizomucor
miehei (39, 40), however, they are remarkably similar in loca-
tion and in net effect.
The observation of two active site conformations in HsaD is
consistent with the half-site mechanism proposed on the basis
of the MphC structure (37) as well as from kinetic studies of
BphD and the ordered release of the two reaction products (13).
According to the half-site mechanism, the hydrolase cycles
between two interconverting conformations: one that is enzy-
matically inactive and one that is enzymatically active. More
particularly, benzoate dissociation followed that of the dieno-
ate, was nondiffusive, and occurred at a rate that was roughly
equivalent to the kcat (13). Consistent with this model, the elec-
tron density of the bound substrate in the S114AꢀMCP com-
plexes was always better defined in one protomer (B) than the
other. Moreover, in the structure with the weakest ligand elec-
tron density, S114AꢀHOPODA, the ␣L4 helix was in a mixed
conformation, with one protomer in a open conformation and
FIGURE 7. Conformation of the ␣L4 helix in the presence of the ligand
DSHA. The backbone of ligand-free S114A (blue) overlaid with that of the
S114AꢀDSHA complex (gray). The side chains Phe212, Met208, and also DSHA
(purple) are shown in stick representation.
essentially identical to that observed in protomers A and B of
ht-HsaD (14).
DISCUSSION
The relatively high specificity of HsaD from M. tuberculosis another in a closed conformation. Although speculative, the
for DSHA (kcat/Km ϭ 33,000 MϪ1 sϪ1) indicates that the enzyme movement of the ␣L4 helix could assist in the nondiffusive
is involved in cholesterol catabolism. Similarly, the structural release of the second product: benzoate and DOHNAA in
data indicate that the substrate-binding pocket is best adapted the cases of HOPDA and DSHA, respectively. Because the
to the steroid substrate. Although the specificity of HsaD is release of the second product is rate-limiting, the increased
ϳ2–3 orders of magnitude lower than other MCP hydrolases distance between the ␣L4 helix and the non-dienoate portion of
for their physiological substrates (13, 23, 35), this is consistent HOPDA compared with that of DSHA and HOPODA (Fig. 5)
with what has been reported for other M. tuberculosis enzymes could explain the 6-fold reduction of the kcat of HOPDA. How-
involved in cholesterol metabolism, including KshAB (36) and ever, further data are required to determine the effect of the C-5
HsaC (8).
It is unclear why HOPODA is a poorer substrate for HsaD
methyl substituent on the kcat of HsaD.
The spectroscopic (Fig. 2) and structural data (Fig. 4 and
than HOPDA; the chloro-phenyl moiety of HOPODA was Table 4) of the S114AꢀMCP complexes are very similar to those
designed to mimic the bulk of the bicycloalkanone portion of reported for the equivalent S112AꢀMCP complexes of BphD.
DSHA, and the structures of the enzymeꢀsubstrate complexes Specifically, the orientation and the torsional angles of the
indicate that the dienoate moiety of the three MCPs were iden- MCPs in the BphD structure suggest that the substrate had
tically positioned in the active site of S114A (Fig. 5). Neverthe- undergone ketonization, yielding an E:Sk species. However, the
less, the Km of HOPODA was 30-fold higher than that of spectroscopic data indicated that the bound MCPs were conju-
HOPDA or DSHA. The increased Km and kcat/Km is in contrast gated because the disrupted ␲-conjugation of E:Sk is predicted
to the high specificity of BphDs for HOPDA variants bearing to result in a blue shift. Interestingly, the 60-nm red shift in
chloro substituents on the phenyl ring, particularly in the meta- S114AꢀDSHA is similar in magnitude to those observed in
and para-positions (17). Although chlorophenyl HOPDAs are S112AꢀHOPDA (72 nm) (12, 13) and in the L photointermedi-
thought to stabilize the negatively charged tetrahedral interme- ate of the retinal chromophore of bacteriorhodopsin (60 nm)
diate at C-6, the chlorophenyl of HOPODA is unlikely to con- (41). The latter has been explained on the basis of twist-about
tribute to stabilization because of the saturated two carbon double bonds, a distortion of the chromophore that has
bridge between the chlorophenyl and the dienoate moieties. recently been observed crystallographically (42). The dihedral
Redesigning the chlorophenyl moiety of the substrate analogue angles of the twisted double bond in the L photointermediate of
may improve HsaD-analogue interactions and provide clues to bacteriorhodopsin were up to 40° from the resting state mole-
the binding mechanism.
cule, similar to the values observed with the 2-enol tau-
This study provides the clearest evidence to date that the tomers of the S114AꢀMCP structures (Table 4). Although it
active sites of MCP hydrolase exist in two conformations: open is possible that different species of S114AꢀDSHA occur in
in the absence of ligand and closed in its presence. Two obser- solution and in crystallo, a strained enolate, E:Sse, is most
vations in previous structures of MCP hydrolases had suggested consistent with the structure and red-shifted absorption
this possibility. First, the ␣-helix lid domains had higher B-fac- spectrum of S114AꢀDSHA. Moreover, the similar electronic
JANUARY 1, 2010•VOLUME 285•NUMBER 1
JOURNAL OF BIOLOGICAL CHEMISTRY 441

Crystal Structure and Kinetics of HsaD
FIGURE8. ProposedmechanismofHsaD. UponbindingoftheMCP, His269 deprotonatesthehydroxylatC-2, generatingastrainedenolateintermediate, E:Sse.
Protonation of C-5 by Ser114 drives tautomerization of the substrate to generate a keto intermediate, E:Sk. Ser114 is positioned to activate water for attack at the
C-6 carbonyl to form the gem-diolate. The collapse of the tetrahedral intermediate releases HHD, which triggers a conformational change in the lid domain and
allows subsequent release of DOHNAA.
absorption spectra of S114AꢀDSHA and E:Sred indicate the strate is initiated by deprotonation of the C-2 hydroxyl by
occurrence of a strained enolate species as a catalytic inter- His269. The bulk of the latter helps to generate a strained eno-
mediate and imply that the serine residue plays a significant late intermediate, E:Sse. C-5 is then protonated by Ser114, induc-
role in tautomerizing the bound MCP.
ing tautomerization of the substrate and further rotation
In MCP hydrolases, the enzymatic cleavage of the ligand has around the C-4–C-5 to yield a ketonized intermediate, E:Sk.
been proposed to occur through a general base mechanism Subsequently, Ser114 deprotonates a nearby water molecule,
(19). The structures of the S114AꢀMCP complexes are most which attacks the C-6 carbonyl to yield a gem-diol intermediate.
consistent with the catalytic serine acting as a general base to The tetrahedral intermediate collapses, releasing HHD and
activate a water molecule for nucleophilic attack of the C-6 triggering a conformational change of the lid domain, which
carbonyl of the substrate. Notably, a solvent species is located releases DOHNAA. This proposal provides a basis for further
3.2–3.6 Å from the carbonyl and 3.7–4.1 Å from the C␤ of study on the mechanism of MCP hydrolases.
Ala114 (Fig. 6B). Although the location of the water molecule
Acknowledgments—We thank the beamline scientists at the Diamond
Light Source (Oxfordshire, UK) and Swiss Light Source (Villigen, Swit-
zerland) for valuable technical support, Jie Liu for help constructing
the S114A variant, Jeffrey T. Bolin for critically reading the manu-
script, and Isaac Westwood for expert crystallography advice.
may differ in the native enzyme, the overlaid structure of ht-
HsaD with S114AꢀMCP places the O␥ from Ser114 within ϳ3 Å
from the water and 2.4 Å from the C-6 carbonyl in the overlaid
structures. However, the O␥–C-6–O-6 angle of 46° deviates
significantly from the Bu¨rgi-Dunitz angle. Finally, His269 is over
5 Å away from the closest water molecule (Fig. 6B).
In conclusion, the results presented herein suggest a dual role
for the catalytic serine of MCP hydrolases and enable further
refinement of the proposed catalytic mechanism for these
enzymes. As summarized in Fig. 8, hydrolysis of the bound sub-
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