The catalytic serine of meta-cleavage product hydrolases is activated differently for C-O bond cleavage than for C-C bond cleavage

Article abstract of DOI:10.1021/bi300663r

meta-Cleavage product (MCP) hydrolases catalyze C-C bond fission in the aerobic catabolism of aromatic compounds by bacteria. These enzymes utilize a Ser-His-Asp triad to catalyze hydrolysis via an acyl-enzyme intermediate. BphD, which catalyzes the hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) in biphenyl degradation, catalyzed the hydrolysis of an ester analogue, p-nitrophenyl benzoate (pNPB), with a k_cat value (6.3 ± 0.5 s^-1) similar to that of HOPDA (6.5 ± 0.5 s^-1). Consistent with the breakdown of a shared intermediate, product analyses revealed that BphD catalyzed the methanolysis of both HOPDA and pNPB, partitioning the products to benzoic acid and methyl benzoate in similar ratios. Turnover of HOPDA was accelerated up to 4-fold in the presence of short, primary alcohols (methanol > ethanol > n-propanol), suggesting that deacylation is rate-limiting during catalysis. In the steady-state hydrolysis of HOPDA, k_cat/K_m values were independent of methanol concentration, while both k_cat and K_m values increased with methanol concentration. This result was consistent with a simple model of nucleophilic catalysis. Although the enzyme could not be saturated with pNPB at methanol concentrations of >250 mM, k_obs values from the steady-state turnover of pNPB at low methanol concentrations were also consistent with a nucleophilic mechanism of catalysis. Finally, transient-state kinetic analysis of pNPB hydrolysis by BphD variants established that substitution of the catalytic His reduced the rate of acylation by more than 3 orders of magnitude. This suggests that for pNPB hydrolysis, the serine nucleophile is activated by the His-Asp dyad. In contrast, rapid acylation of the H265Q variant during C-C bond cleavage suggests that the serinate forms via a substrate-assisted mechanism. Overall, the data indicate that ester hydrolysis proceeds via the same acyl-enzyme intermediate as that of the physiological substrate but that the serine nucleophile is activated via a different mechanism.

Full text of DOI:10.1021/bi300663r

Article
pubs.acs.org/biochemistry
The Catalytic Serine of meta-Cleavage Product Hydrolases Is
Activated Differently for C−O Bond Cleavage Than for C−C Bond
Cleavage
Antonio C. Ruzzini, Geoff P. Horsman,† and Lindsay D. Eltis*
Department of Biochemistry and Molecular Biology and Department of Microbiology and Immunology, University of British
Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada
S
* Supporting Information
ABSTRACT: meta-Cleavage product (MCP) hydrolases catalyze C−C bond fission in the aerobic catabolism of aromatic
compounds by bacteria. These enzymes utilize a Ser-His-Asp triad to catalyze hydrolysis via an acyl−enzyme intermediate. BphD,
which catalyzes the hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) in biphenyl degradation, catalyzed
the hydrolysis of an ester analogue, p-nitrophenyl benzoate (pNPB), with a kcat value (6.3 0.5 s−1) similar to that of HOPDA
(6.5 0.5 s−1). Consistent with the breakdown of a shared intermediate, product analyses revealed that BphD catalyzed the
methanolysis of both HOPDA and pNPB, partitioning the products to benzoic acid and methyl benzoate in similar ratios.
Turnover of HOPDA was accelerated up to 4-fold in the presence of short, primary alcohols (methanol > ethanol > n-propanol),
suggesting that deacylation is rate-limiting during catalysis. In the steady-state hydrolysis of HOPDA, kcat/Km values were
independent of methanol concentration, while both kcat and Km values increased with methanol concentration. This result was
consistent with a simple model of nucleophilic catalysis. Although the enzyme could not be saturated with pNPB at methanol
concentrations of >250 mM, kobs values from the steady-state turnover of pNPB at low methanol concentrations were also
consistent with a nucleophilic mechanism of catalysis. Finally, transient-state kinetic analysis of pNPB hydrolysis by BphD
variants established that substitution of the catalytic His reduced the rate of acylation by more than 3 orders of magnitude. This
suggests that for pNPB hydrolysis, the serine nucleophile is activated by the His-Asp dyad. In contrast, rapid acylation of the
H265Q variant during C−C bond cleavage suggests that the serinate forms via a substrate-assisted mechanism. Overall, the data
indicate that ester hydrolysis proceeds via the same acyl−enzyme intermediate as that of the physiological substrate but that the
serine nucleophile is activated via a different mechanism.
novel insights into the versatility of the Ser-His-Asp catalytic
triad.3
he meta-cleavage product (MCP) hydrolases are involved
Tin the aerobic degradation of aromatic compounds by
bacteria, cleaving a C−C bond of metabolites resulting from the
dioxygenase-mediated meta-ring cleavage of catechols. MCP
hydrolases are implicated in processes as divergent as the global
carbon cycle and human health. For example, BphD is a
determinant in the mineralization of polychlorinated biphenyls
(PCBs),1 while HsaD is a determinant in the catabolism of
cholesterol by Mycobacterium tuberculosis and related patho-
gens.2 In addition to their practical significance, MCP
hydrolases have proven to be surprisingly interesting with
respect to their catalytic mechanism and have recently provided
The MCP hydrolases belong to the α/β-hydrolase super-
family, which is defined by one of nature’s most widespread
protein folds. The α/β-hydrolase core brings a nucleophile,
histidine, and an acid group together, forming a catalytic triad.
Despite the members of the family sharing a common core,
family specific insertions and active site loop orientation
contribute greatly to the chemical diversity of this superfamily,
Received: May 21, 2012
Revised: June 27, 2012
Published: June 29, 2012
© 2012 American Chemical Society
5831
dx.doi.org/10.1021/bi300663r | Biochemistry 2012, 51, 5831−5840

Biochemistry
Article
Scheme 1. Proposed Mechanisms of MCP Hydrolase-Mediated C−C Bond Cleavage
which includes haloperoxidases, dehalogenases, cofactor-less
dioxygenases, and hydrolases of C−N, C−O, and C−C
bonds.4−7 Likewise, the respective catalytic mechanisms of
the various families show surprising diversity despite the
conserved hydrolytic machinery. Thus, while a nucleophilic
mechanism has been experimentally supported for the majority
of the α/β-hydrolases, at least three families utilize a general
base mechanism of catalysis: hydroxynitrile lyases,8 cofactor-less
dioxygenases,9 and enzymes such as enterobactin hydrolase that
contain a His-Asp dyad but no nucleophilic residue.10
include esterases. Nevertheless, differences in the chemical
nature of MCPs and esters ought to be reflected in the
mechanism of hydrolysis. For example, the proposed substrate-
assisted mechanism of serinate formation is dependent on the
ability of these enzymes to localize charge to C5 of the MCP’s
dienoate core. The corresponding position in the ester
substrates is an oxygen atom. More interestingly, not one of
the mechanisms proposed for the MCP hydrolases invokes
activation of the nucleophilic serine by the His-Asp dyad.
Moreover, it has been proposed that C−O bond hydrolysis by
the MCP hydrolases proceeds via a general base mechanism in
which the active site serine acts as a hydrogen bond donor to a
gem-diolate intermediate during bond cleavage.19,21 This
proposal was largely based on a Hammett analysis of the
BphD-mediated hydrolysis of para-substituted p-nitrophenyl
benzoates (pNPBs).
Herein, we used BphD to investigate the catalytic mechanism
of C−C and C−O bond cleavage in MCP hydrolases. Steady-
state kinetics and high-performance liquid chromatography
(HPLC) analysis of reaction products were performed using
the enzyme’s physiological substrate, HOPDA, and an ester,
pNPB, in the presence of small primary alcohols. The observed
methanol-dependent kinetic behavior and product ratios were
linked to simple kinetic models. Finally, rapid-scanning, visible
absorbance spectrophotometry monitoring the pre-steady-state
turnover of pNPB by the wild-type and variant enzymes was
used to better define the mechanism of C−O bond hydrolysis.
Together, the experiments provide new insight into the MCP
hydrolase esterase activity. The mechanisms of C−C and C−O
bond hydrolysis are discussed within the context of this
remarkable family of enzymes.
For more than a decade, it had been argued that MCP
hydrolases utilize a general base mechanism, with hydrolysis
proceeding via a gem-diolate intermediate.11,12 However,
chemical quench and mass spectrometry recently provided
direct evidence of an acyl−enzyme intermediate in a wild-type
enzyme, indicating that MCP hydrolases utilize a nucleophilic
mechanism of catalysis (Scheme 1).3 These data were obtained
using BphD of Burkholderia xenovorans LB400, which hydro-
lyzes the 5,6-bond of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic
acid (HOPDA) in biphenyl degradation with a Ser112-His265-
Asp237 catalytic triad. The Ser112-benzoyl species was also
trapped in crystals of a BphD H265Q variant incubated with
HOPDA. Accordingly, a substrate-assisted mechanism of
nucleophilic catalysis has been proposed in which Ser112 is
acylated during turnover. More generally, binding of the MCP
to the enzyme is thought to perturb the substrate’s delocalized
system of π-electrons, in part through bond strain, to catalyze
the enol(ate)-to-keto tautomerization of the MCP’s dienoate
core (Scheme 1).13,14 This process, which may also activate the
serine for nucleophilic attack, generates a 2,6-dioxo isomer,
providing an electron sink that can support carbanion
formation during bond cleavage.15,16 The mechanism of
tautomerization is poorly understood, despite the latter’s
importance to the reaction. Remarkably, the active site of the
MCP hydrolases contains five conserved residues (Asn111,
Phe175, Arg190, Cys263, and Trp266) in addition to the triad,
all of which directly contact the bound substrate, with the
exception of Cys263.14 Mutational analysis of these residues in
either BphD or MhpC, a homologue from Escherichia coli, has
shown that the high degree of conservation is important for C−
C bond hydrolysis.14,17 Indeed, evolutionary maintenance of
the active site may reflect the difficulty associated with
hydrolyzing a C−C bond, and the need for substrate
tautomerization.
MATERIALS AND METHODS
■
Chemicals and Enzyme Preparation. HOPDA was
enzymatically generated from 2,3-dihydroxybiphenyl using
2,3-dihydroxybiphenyl dioxygenase as previously described.1
All other chemicals were of analytical grade. All enzymes were
purified as previously described22 except that BphD was
overproduced in Rosetta(DE3)pLysS cells (EMD4Biosciences)
at 30 °C.
Enzyme Activity Measurements. Steady-state kinetic
assays were performed in 1 mL of potassium phosphate (I =
0.1 M, pH 7.5) at 25 °C, unless otherwise stated. Initial reaction
velocities were measured using a Cary 5000 spectrophotometer
equipped with a thermostated cuvette holder. HOPDA
hydrolysis was monitored at 434 nm (ε434 nm = 25.7 mM−1
cm−1), and p-nitrophenol production was monitored at 400 nm
MCP hydrolases also mediate C−O bond hydrolysis in
vitro.18−21 This esterase activity is not surprising considering
the conserved catalytic machinery of the α/β-hydrolases, which
5832
dx.doi.org/10.1021/bi300663r | Biochemistry 2012, 51, 5831−5840

Biochemistry
Article
(ε400 nm = 12.8 mM−1 cm−1). Steady-state rate equations were
fit to individual data sets using the least-squares and dynamic
weighting options of LEONORA.23 Rate equations for specific
kinetic models (vide infra) were fit using SCIENTIST
(Micromath, St. Louis, MO).
Alcoholysis was measured at 25 and 100 mM methanol,
ethanol, n-propanol, and 2-propanol using 4 nM BphD and 10
μM HOPDA. The methanol dependence of the steady-state
kinetic turnover of HOPDA by BphD was investigated by
varying the alcohol concentration between 0 and 5% (v/v; or
from 0 to 1240 mM). Steady-state kinetic parameters for the
BphD-mediated hydrolysis of pNPB were determined using up
to 8 μM pNPB in the buffer described above supplemented
with 0.2% acetone to solubilize the ester. Similarly, the
methanol dependence on v0/[E]T (kobs) for pNPB turnover
was measured at 2 and 4 μM ester, concentrations that were
saturating in the absence of methanol.
HPLC Analysis of BphD Cleavage Products. Reactions
were analyzed using a Waters 2695 HPLC system (Waters
Corp., Milford, MA) equipped with a Hewlett-Packard ODS
Hypersil C18 column (5 μM, 125 mm × 4 mm) operating at a
flow rate of 1 mL/min and equilibrated at 10% solvent B
(methanol) in solvent A (0.5% H3PO4 in H2O). Reactions were
quenched with H3PO4 (0.5%, v/v) and mixtures passed
through a 0.45 μm filter before injection. Product ratios or
percentages of acid product were calculated by measuring the
amount of benzoate produced in each reaction relative to
controls performed without alcohol. Control reactions in
potassium phosphate (I = 0.1 M, pH 7.5) with or without
0.2% acetone were considered to be 100% hydrolytic, and the
amount of benzoate was quantified by integrating the eluted
peak volume using Empower 3 (Waters Corp.). All reactions
analyzed using HPLC were performed in triplicate: the mean
and standard deviation are reported.
shots and was analyzed using single- and double-exponential
equations using Pro-Data Software (Applied Photophysics
Ltd.). The fitted parameters from corresponding replicates were
averaged, and the errors are reported as standard deviations.
Similar reactions performed using the S112A variant (8 μM
BphD and 2 μM pNPB) were monitored using a Cary 5000
spectrophotometer. Finally, reactions performed using excess
ester (0.1, 0.25, or 0.5 μM BphD and 2 μM pNPB) were also
analyzed at 400 nm using the stopped-flow instrument. Burst
and double-exponential equations were fit to replicate data sets.
RESULTS
■
BphD Cleaved HOPDA and pNPB with the Same kcat.
To investigate C−O bond hydrolysis by the MCP hydrolases,
we measured the ability of BphD to hydrolyze pNPB, an ester
that shares a chemical substructure with HOPDA. The BphD-
mediated hydrolysis of pNPB obeyed Michaelis−Menten
kinetics, and the resulting fitted parameters were as follows:
k
cat = 6.3 0.5 s−1, kcat/Km = 19 3 μM−1 s−1, and Km = 0.34
0.02 μM. Under nearly identical conditions (with or without
0.2% acetone), BphD hydrolyzed HOPDA at essentially the
same rate (kcat = 6.5 0.5 s−1).
While the essentially identical kcat values for the hydrolysis of
HOPDA and pNPB could have a variety of origins, we
hypothesized that it was due to the rate-limiting breakdown of a
shared acyl−enzyme intermediate. To test this hypothesis, we
tested the ability of BphD to utilize methanol as an alternative
acceptor. Products resulting from the turnover of 40 μM
HOPDA or 4 μM pNPB in the presence of 25 mM methanol
were analyzed by HPLC. In both reactions, equal amounts of
benzoate and methyl benzoate were produced: benzoic acid
accounted for 48 2 and 49 1% of the benzoyl-containing
product from the cleavage of HOPDA and pNPB, respectively
(Figure S1 of the Supporting Information).
Alcoholysis reactions were performed in 400 μL of potassium
phosphate (I = 0.1 M, pH 7.5) at 25 °C containing 40 μM
HOPDA and were initiated by adding BphD to a final
concentration of 4 nM. The completeness of the reactions was
judged spectrophotometrically before quenching. Samples were
eluted using the following solvent gradients: (i) 10 to 30% B
from 0 to 20 min, (ii) 30 to 60% B from 20 to 30 min, and (iii)
60 to 100% B from 30 to 40 min. Product elution times were as
follows: 5.5 min for HPD, 13.7 for benzoate, 25.5 for methyl
benzoate, 29.9 for ethyl benzoate, and 32.4 for propyl benzoate.
The retention times of the alcoholytic products matched those
of purchased standards.
Experiments that aimed to assess the methanol dependence
of BphD-mediated hydrolysis of HOPDA and pNPB were
conducted via elution using similar gradients: (i) 10 to 30% B
from 0 to 20 min, (ii) 30 to 60% B from 20 to 30 min, and (iii)
60 to 100% B from 30 to 32 min. Because of the relatively low
solubility of the ester, reaction volumes were increased to 1 mL
and mixtures contained 4 μM pNPB. Moreover, 500 μL was
injected to compensate for the reduced substrate and/or
product concentration.
Short-Chain, Primary Alcohols Accelerated the Turn-
over of HOPDA. To further characterize the observed
mechanism of alcoholysis and the event associated with kcat,
we tested the ability of BphD to utilize other alcohols during
the steady-state turnover of HOPDA. The effects of several
alcohols on the initial velocity (v0) of BphD-catalyzed turnover
of 10 μM HOPDA were measured. In the absence of alcohol, v0
was 25 1 μM s−1 [kobs ∼ 6 s−1 (Table 1)]. Concentration-
dependent increases in v0 were apparent upon the addition of
either methanol, ethanol, or n-propanol. The observed effects
ranged from an approximate 3.2-fold increase in the presence of
100 mM methanol to a more modest 1.2-fold increase at the
Table 1. Effects of Alcohol on BphD-Mediated C−C Bond
a
Hydrolysis
[alcohol] (mM)
v0 (μM s−1
25
45.8 0.7
76
)
acid:ester product ratio
methanol
0
25
1
1.06 0.01
0.24 0.01
3.78 0.05
0.95 0.02
42.8 0.8
3.57 0.07
100
25
1
ethanol
29.4 0.9
45.9 0.7
26.0 0.6
Stopped-Flow Spectrophotometry. Experiments were
conducted using an SX.18MV stopped-flow reaction analyzer
(Applied Photophysics Ltd., Leatherhead, U.K.) maintained at
25 °C by circulating water. Single turnovers of 2 μM pNPB
were monitored at 400 nm using either 2, 4, or 8 μM wild-type
BphD or the H265Q or H265A variant at 8 μM. Reactions
were performed in triplicate using freshly prepared reagents for
each replicate. Each replicate averaged the results of at least five
100
25
n-propanol
2-propanol
100
25
29
1
b
25.8 0.6
25.5 0.3
ND
b
100
ND
a
Distinct experiments were used to measure v0 (10 μM HOPDA) and
b
product ratios (40 μM HOPDA). Not detected.
5833
dx.doi.org/10.1021/bi300663r | Biochemistry 2012, 51, 5831−5840

Biochemistry
Article
same concentration of n-propanol. Inspection of the series
indicates that the values of v0 reflect the pKa of the substituting
nucleophile. Importantly, v0 was not significantly affected by the
addition of 25 or 100 mM 2-propanol, suggesting that the
tested alcohols compete to replace the nucleophilic water and
do not contribute any allosteric effects that accelerate the
reaction.
In addition to the observed kinetic phenomena, HPLC
analysis of the reaction products was used to correlate the
observed accelerations in v0 directly to alcohol utilization by
BphD (Table 1). Specifically, we observed that partitioning
between the hydrolytic and alcoholytic reaction pathways was
linked to common v0 measurements. For instance, using either
25 mM methanol or 100 mM ethanol, the acid:ester ratio was
∼1 and v0 ∼ 46 μM s−1. Similarly, the same v0 was observed
using 25 mM ethanol and 100 mM n-propanol (∼29 μM s−1),
and the acid:ester product ratio was ∼3.6.
Methanolysis Provided Indirect Evidence of a Shared
Intermediate. The remarkably similar kcat values for the
hydrolysis of HOPDA and pNPB and the results of the
alcoholysis experiments suggested that the BphD-catalyzed
reactions involve the rate-determining breakdown of a shared
catalytic intermediate, such as a benzoyl−enzyme intermediate.
To investigate this possibility, we studied the effect of variable
methanol concentrations on the steady-state kinetic parameters
and product ratios for the BphD-catalyzed transformation of
HOPDA (Figure 1). Significantly, kcat and Km increased with
nucleophilic mechanism correctly predicts the methanol
independence of the kcat/Km values, as kcat/Km = k2/KS. In
contrast, the general base mechanism predicts that this
parameter should increase with methanol concentration.
Further inspection reveals that the model based on a
nucleophilic mechanism is better able to account for the
experimental data in several other respects. In this model, kcat
for HOPDA depletion is described by eq 4, with the following
fitted values: k2 = 30 3 s−1, k3[H2O] = 8 3 s−1, and k4 = 230
80 M−1 s−1 (Scheme 2). These rate constants allow the
calculation of kcat values specific for acid and ester formation,
which were used to predict product ratios at each methanol
concentration (Table 2). Indeed, the product ratios predicted
by the model are in good agreement with the experimentally
measured amounts of benzoate produced during steady-state
turnover of HOPDA (Table 2 and Figure S1 of the Supporting
Information). The kcat/Km value, ∼25 μM−1 s−1, also provides
an estimate of KS for the nucleophilic mechanism, allowing a
comparison of predicted and experimental Km values.
Simplifying kcat/Km for HOPDA to k2/KS and substituting a
k2 of 30 s−1 yields a KS of 1.2 μM. Subsequent predictions of
the methanol-dependent Km values from KS are consistent with
the experimental values (Table 2).
To further investigate the mechanism of C−O bond
cleavage, we also measured the methanol dependence of
pNPB turnover by BphD (Table 3 and Figure 2).
Unfortunately, the relatively low solubility of pNPB precluded
reliable determination of the steady-state kinetic parameters in
the presence of methanol. Despite this limitation, the rate of
BphD-catalyzed C−O bond cleavage at 2 and 4 μM pNPB
provided some mechanistic insight. As we observed for the
transformation of HOPDA, the rate of pNPB turnover
increased with methanol concentration. However, the kobs
value also decreased at methanol concentrations >250 mM,
reflecting an increase in the Km pNPB as observed for HOPDA
turnover. At lower methanol concentrations, the kobs values for
pNPB turnover were within error of the kcat values determined
for HOPDA. Furthermore, the methanol-dependent product
formation, as measured by HPLC, indicated that the
partitioning ratio was equal to that observed for HOPDA
(Table 3).
Activation of the Serine Nucleophile for C−O Bond
Hydrolysis. While the alcohol-dependent steady-state kinetic
data suggest that both the C−O and C−C hydrolytic reactions
proceed through an acyl−enzyme intermediate, they do not
establish the mechanism of nucleophile activation. Accordingly,
we further probed the mechanism of ester bond hydrolysis
using stopped-flow, visible spectrophotometry to monitor the
hydrolysis of pNPB by BphD and three catalytic triad variants:
H265Q, H265A, and S112A. The turnover of 2 μM pNPB by 1
or 2 equivalents of WT BphD resulted in the biphasic
production of pNP (Table 4 and Figure 3) reminiscent of
HPD formation during HOPDA turnover.13 Interestingly,
turnover of pNPB by a 4-fold excess of enzyme resulted in
monophasic formation of pNP at a rate approximately 5-fold
faster than the second phase of turnover observed at equimolar
concentrations of enzyme and pNPB. Even at equal reactant
concentrations, k2 was approximately 7-fold faster than kcat,
suggesting that an additional process, downstream of pNP
release, is rate-determining but cannot be observed at 400 nm
during a single turnover. Again, these results support the rate-
limiting breakdown of an acyl−enzyme intermediate during
BphD-catalyzed C−O bond cleavage.
Figure 1. Dependence of kcat on methanol concentration for the
BphD-catalyzed transformation of HOPDA. The solid line represents
the fit of the steady-state rate equation for the nucleophilic mechanism
(Scheme 3, eq 4) to the experimentally determined kcat values for
■
HOPDA depletion ( ). The fitted parameters were as follows: k2 = 30
3 s−1, k3[H2O] = 8 3 s−1, and k4 = 230 80 M−1 s−1. Values from
the quantification of benzoate at the varied methanol concentrations
□
are shown in white ( ). The area of each peak was calculated as a
function of the voltage per second and then converted to milli-
absorbance units per second.
methanol concentration while kcat/Km remained unchanged
(Table 2). The experimentally determined values were
compared to those predicted from kinetic models based on
each of the general base and nucleophilic mechanisms. Steady-
state rate expressions for partitioning in each of the two
mechanisms are shown in Schemes 224 and 3,25 respectively. A
cursory inspection of the rate equations reveals that only the
5834
dx.doi.org/10.1021/bi300663r | Biochemistry 2012, 51, 5831−5840

Biochemistry
Article
Table 2. Experimentally Observed and Calculated Steady-State Kinetic Parameters for the Methanol-Dependent BphD-
a
Catalyzed Transformation of HOPDA
b
experimental
kcat/Km (μM−1 s−1
calculated
[MeOH] (mM)
kcat (s−1
)
Km (μM)
)
% acid
kcat (s−1
)
kcat acid (s−1
)
kcat ester (s−1
)
Km (μM)
% acid
0
25
6.5 0.5
9.4 0.5
13
0.20 0.05
0.4 0.1
0.60
32
27
22
19
26
31
21
14
0
48
27
17
10
2
2
2
1
1
6.5
9.6
13
6.5
5.7
4.7
3.7
2.6
1.6
1.4
0.8
0
3.9
8.2
13
18
22
24
26
0.26
0.38
0.52
0.66
0.82
0.96
1.0
0
59
37
23
13
7
62
124
247
494
618
1240
16
0.83
17
21
26
21
28
4
0.8 0.2
0.85
21
5.8 0.3
4.9 0.3
3.0 0.2
24
5
4
0.99 0.07
2.0 0.4
25
5
27
1.1
3
a
Standard errors are reported for kinetic parameters; values without an error represent a single measurement. Errors for product analysis are reported
b
as standard deviations calculated from three independent samples. Calculated using the equations for the nucleophilic mechanism in Scheme 2: k2 =
30 s−1, k3[H2O] = 8.3 s−1, k4 = 230 M−1 s−1, and KS = 1.2 μM.
Scheme 2. Steady-State Rate Expressions for a Simple Model of Nucleophilic Catalysis
Scheme 3. Steady-State Rate Expression for a Simple Model of General Base Catalysis
Table 3. Experimentally Observed Methanol-Dependent
Rates (kobs = vi/[E]T) of pNPB Transformation by BphD
kobs (s−1
)
[MeOH] (mM)
2 μM pNPB
4 μM pNPB
% acid at 4 μM pNPB
0
25
6.4 0.3
9.9 0.5
12.8 0.2
6.5 0.1
10.7 0.5
14.6 0.3
18.4 0.1
0
49
29
20
1
1
1
1
62
124
247
618
1240
16
17
14
11
1
2
1
1
21
1
14.4 0.7
5.7 0.3
3.7 0.2
20.1 0.8
17.0 0.4
For the sake of simplicity, subsequent experiments were
performed using a 4-fold excess of enzyme to substrate.
Substitution of Ser112 with alanine resulted in the near
abrogation of C−O bond cleavage (k ∼ 10−4 s−1). Furthermore,
the rates of ester bond hydrolysis were reduced by at least 3
orders of magnitude for the His265 variants. Specifically, the
H265A and H265Q variants cleaved pNPB at rates of 0.06 and
0.014 s−1, respectively (Table 4).
Biphasic Pre-Steady-State Formation of pNP. To
investigate the biphasic product release observed during
single-turnover experiments, we monitored the turnover of 4-,
8-, and 20-fold excesses of pNPB by BphD. In all cases, the
kinetic traces monitoring multiple pNPB turnovers were
Figure 2. Methanol-dependent BphD-mediated cleavage of pNPB.
□
The experimentally observed rates (kobs) represent v0/[E]T at 2 ( )
■
and 4 μM pNPB ( ).
biphasic (Figure 4) and could be modeled by either burst or
double-exponential equations (Table 5 and Figure S2 of the
Supporting Information). Fitting a burst equation to the data
5835
dx.doi.org/10.1021/bi300663r | Biochemistry 2012, 51, 5831−5840

Biochemistry
Article
Table 4. Kinetic Data for pNPB Turnover by Wild-Type
BphD and Its Variants
[E]:[pNPB]
k1 (s−1) (% Amp)
k2 (s−1) (% Amp)
1 WT
210 40 (18)
170 10 (38)
206 5...
41 5 (82)
74 2 (62)
2 WT
4 WT
4 H265Q
0.014 0.002
0.06
a
4 H265A
b
4 S112A
0.00016 0.00002
a
b
Data reported for a single replicate. Value measured using a Cary
5000 spectrophotometer.
Figure 4. Representative stopped-flow experiments demonstrating the
pre-steady-state formation of pNP by BphD in potassium phosphate
buffer (I = 0.1 M, pH 7.5) and 0.2% (v/v) acetone at 25 °C. The
hydrolysis of 2 μM pNPB was monitored using 0.1 (light gray), 0.25
(dark gray), and 0.5 μM BphD (black).
of k2 from the fit of a double-exponential equation to the
multiple-turnover data, from 0.8 to 2.8 s−1, demonstrated a
dependence on enzyme concentration and were significantly
lower than the experimentally determined kcat value of 6.5 s−1.
The “burst-like” kinetic behavior observed for pNPB turnover is
consistent with rate-limiting deacylation. Nevertheless, the
magnitude of the pre-steady-state burst of pNP formation
appears to be substoichiometric, but significantly higher than
the value of 50% predicted from the two-conformation model
proposed for C−C bond hydrolysis.
DISCUSSION
■
The alcoholysis experiments presented herein provide addi-
tional evidence of covalent catalysis in the MCP hydrolases,
supporting the idea that the hydrolysis of esters by these
enzymes also involves an acyl−enzyme intermediate. Thus, the
similar kcat values for the hydrolysis of HOPDA and pNPB and
the similar partitioning of their products to benzoate and
methyl benzoate indicate that both C−C and C−O bond
cleavage proceed through a shared intermediate. Moreover, the
steady-state kinetic analysis of methanolysis and propanolysis
provides indirect evidence that the shared intermediate is an
acyl−enzyme intermediate. This builds on the direct observa-
tion of a Ser112−benzoyl species by chemical quench MS in
WT BphD, its observation in the H265Q variant using
crystallography,3 and indirect evidence of its occurrence,
including the stoichiometric incorporation of 18O from H218O
during turnover and the appearance of a pre-steady-state kinetic
burst. Finally, the methanolysis and propanolysis data further
indicate that kcat represents the rate-limiting breakdown of the
acyl−enzyme intermediate.
The observed accelerations in the initial velocities and the
ability of BphD to utilize longer alcohols, such as n-propanol,
are consistent with covalent catalysis. The crystal structure of
BphD S112A in complex with HOPDA revealed a complex
suite of interactions,17,26 reflecting the high degree of active site
residue conservation. Little residual volume is present within
the occupied active site, suggesting that acylation and release of
HPD precede the binding of additional nucleophiles such as
Figure 3. Representative single turnover of pNPB by a 4-fold excess of
BphD. (A) For WT, k ∼ 200 s−1. (B) For H265A, k ∼ 0.06 s−1 (light
gray). For H265Q, k ∼ 0.014 s−1 (dark gray). For S112A, k ∼ 0.0002
s−1 (black).
revealed that the rates of pNP formation during the burst phase
(kburst ∼ 42 s−1) and the steady-state phase (kss ∼ 8 s−1) were
independent of enzyme concentration. This analysis also
indicated that the amount of pNP formed during the burst
phase corresponded to ∼70% of the enzyme present in the
assay. Similarly, analysis using a double-exponential equation
implied that ∼60% of the enzyme was active during the pre-
steady-state phase (k1) which proceeded at a rate of ∼50 s−1, in
relatively good agreement with kburst and k2 during a single
turnover of pNPB at a 1:1 ratio with BphD. In contrast, values
5836
dx.doi.org/10.1021/bi300663r | Biochemistry 2012, 51, 5831−5840

Biochemistry
Article
a
Table 5. Pre-Steady-State Kinetic Parameters Derived from Monitoring pNB Formation at 400 nm
Fits to a Burst Equation
[E] (μM)
kburst (s−1
)
Ampburst (ΔA400
)
[pNP]burst (μM)
kss (ΔA400 s−1
)
kss (s−1
)
0.10
0.25
0.50
42
41
44
3
4
5
0.00077 0.00004
0.0022 0.0001
0.0044 0.0003
0.060 0.003
0.17 0.01
0.35 0.02
0.0101 0.0005
0.024 0.001
0.046 0.001
7.9 0.4
7.4 0.4
7.1 0.2
Fits to a Double-Exponential Equation
b
[E] (μM)
k1 (s−1) (% Amp)
[pNP]k (μM)
k2 (s−1) (% Amp)
[pNP]total (μM)
1
0.10
0.25
0.50
44 2 (5)
47 6 (8)
60 10 (12)
0.059 0.004
0.152 0.007
0.27 0.02
0.8 0.1 (90)
1.4 0.1 (92)
2.8 0.4 (88)
1.2 0.2
1.8 0.1
2.2 0.3
a
b
Errors associated with amplitudes were <12%. [pNP]total was calculated on the basis of the fitted amplitudes.
Scheme 4. Nucleophilic Mechanism for C−C and C−O Bond Cleavage
water or alcohols. Indeed, product inhibition studies support a
mechanism in which HPD is released before benzoate during
turnover of HOPDA.13 In contrast, significant movement of the
MCP hydrolase lid domain (residues 146−211) would be
required to bind additional molecules to a HOPDA-occupied
active site. Considering the full suite of interactions within the
enzyme−substrate complex, it is difficult to conceive how such
lid movement would not disrupt productive substrate binding.
The alcohol-dependent acceleration of v0 provides novel
insight into the MCP hydrolase catalytic cycle inasmuch as it
suggests that the breakdown of the acyl−enzyme intermediate
is rate-limiting. To date, experiments in MhpC and BphD have
assigned kcat to either release of the noncovalently bound
product16,19 or a conformational change associated with
chemistry.13 The observed increase in the rate of turnover in
the presence of alternate acceptors suggests breakdown of the
acyl−enzyme intermediate rather than product release limits
catalysis. Indeed, the methanol-induced rate acceleration (28
s−1 vs 6.5 s−1) is similar in magnitude to that reported for the
turnover of an ester substrate by α-chymotrypsin (482 s−1 vs
144 s−1), a reaction that is also thought to proceed via rate-
limiting hydrolysis of an acyl−enzyme intermediate.27 The
alcoholytic reactions also highlight the ability of the His-Asp
dyad to activate water or an alcohol. While the data do not
unambiguously refute the possibility that product release is
rate-determining, the observation of similar accelerations of v0
by different alcohols suggests that this scenario is unlikely.
Specifically, the limiting of turnover by product release would
imply that methyl, ethyl, and propyl benzoates are released
from the active site at an equal rate that is faster than that of
benzoic acid. By contrast, assigning a chemical dependence to
The similar kcat values for HOPDA and pNPB hydrolysis
together with the similar partitioning of these reactions
between the hydro- and alcoholytic pathways (Scheme 4)
mirror what has been reported for other hydrolases that utilize
a covalent mechanism. Thus, constant partition ratios have
been reported in serine proteases, such as chymotrypsin,27 and
non-serine hydrolases, such as prostatic acid phosphatase,28 all
of which are proposed to proceed via acyl−enzyme
intermediates. For the BphD-mediated methanol-dependent
turnover of HOPDA, a simple kinetic model for a nucleophilic
mechanism correctly predicted the observed increase in kcat and
Km as well as the independence of kcat/Km. Furthermore, the
experimentally determined product ratios from the turnover of
both HOPDA and pNPB also matched the values predicted by
the nucleophilic model. Collectively, the data suggest that the
distinct substrates are cleaved through a shared, Ser112−
benzoyl intermediate. This conclusion contradicts an earlier
proposal that Ser112 acts as a hydrogen bond donor during C−
O bond hydrolysis. This previously assigned role for Ser112
was deduced from a Hammet analysis using para-substituted
pNPBs19 and further supported using substrate docking
experiments.21 In particular, the Hammet plot was nonlinear,
a feature that was attributed to a change in the rate-limiting step
from ester bond cleavage to product release for substrates for
which σ ≥ 0. An alternative explanation for the nonlinearity is
that the activation of water for nucleophilic attack on the
Ser112−benzoyl ester may become rate-limiting for substrates
for which σ ≥ 0. This alternate explanation is consistent not
only with the dependence of kcat on alcohol but also with the
aforementioned lack of viscosity effect and the presence of a
sKIE during turnover of esters.
k
cat is consistent with the reported absence of a solvent viscosity
Transient-state kinetic analyses of catalytic triad variants of
BphD indicate that the mechanism of nucleophile activation
differs in pNPB and HOPDA hydrolysis, consistent with the
different leaving groups of the two substrates. Thus, serinate
effect during HOPDA turnover,13 and the presence of a solvent
kinetic isotope effect (sKIE) during BphD-mediated ester bond
hydrolysis.19
5837
dx.doi.org/10.1021/bi300663r | Biochemistry 2012, 51, 5831−5840

Biochemistry
Article
formation during C−C bond cleavage was recently proposed to
occur through a substrate-assisted mechanism, facilitated by an
enzymatic perturbation of the electron-rich MCP dienoate.3
Consistent with this proposal, substitution of His265 with Ala
or Gln reduced the rate of acylation by only 5−40-fold during
HOPDA hydrolysis, while breakdown of the acyl−enzyme
intermediate remained rate-limiting.3 In contrast, these same
substitutions reduced the rate of acylation by more than 3
orders of magnitude for pNPB hydrolysis such that acylation
becomes rate-limiting. This significant reduction in the rate of
catalysis is similar to those observed in Ser-His-Asp triad
variants of other α/β-hydrolases such as esterases29,30 and
serine proteases.31 Both of the latter classes of hydrolases utilize
covalent mechanisms of catalysis and use the His-Asp dyad for
serinate formation. Our results, together with the apparent
abrogation of chemistry upon substitution of Ser112 in BphD,
are also consistent with a previous steady-state study of MhpC
in which the S110A and H263A variants did not possess
detectable esterase activity against monoethyl adipate.18
Overall, the reduction in the rate of pNP production in the
His265 variants implicates the His-Asp dyad in the activation of
the serine for C−O bond hydrolysis in MCP hydrolases.
The steady-state kinetic studies of the methanol dependence
of C−C bond cleavage provide additional evidence of a two-
conformation model of catalysis. This model was previously
proposed on the basis of biphasic product formation in BphD
observed in stopped-flow analyses13 and half-site reactivity in
the wild-type enzyme.3 According to the proposal, one form of
the enzyme catalyzes C−C bond cleavage and HPD release
(E), and a second, inactive form (E′ in Scheme 5) requires a
high methanol concentrations, the sum of the transit times
(reciprocal rates) for two acylation steps yields a kcat of ∼25 s−1
{i.e., 1/[1/(50 s−1) + 1/(50 s−1)]}, in reasonable agreement
with the fitted value of 30 s−1.
The transient-state kinetic studies of pNPB hydrolysis further
support the occurrence of active sites with different reactivities
in BphD. Specifically, the BphD-mediated production of pNP
was biphasic at 2:1 and 1:1 enzyme:substrate ratios. Amplitude
analyses indicate that the relative proportion of “fast-acting”
enzyme, characterized by acylation rates of 200 s−1, diminished
with an increasing active site occupancy and suggested that the
occupancy of more than a single active site within the tetramer
leads to a 5-fold reduction in the rate of pNP production from
individual catalytic sites (k2 ∼ 40 s−1). Nevertheless, qualitative
inspection of the kinetic traces did not reveal a textbook pre-
steady-state burst phase even at the highest concentration of
BphD tested. The poorly defined burst phase may be due, in
part, to the relatively small difference in observed rates: kburst
(=k2) ∼ 40 s−1, and kss ∼ 8 s−1. However, it may also reflect the
different manner in which pNPB interacts with the active site of
BphD as compared to HOPDA, which must be tautomerized
prior to hydrolysis. Indeed, turnover of pNPB did not strictly
conform to the two-conformation model of catalysis: the
turnover was substoichiometric, and pNP production was
asynchronous. Thus, during the first turnover, 60−70% of the
protomers were fast-acting and the visible signal originating
from the remaining 30−40% of the active sites likely
contributed to a poorly defined pre-steady-state phase. Overall,
these results, together with the single-turnover experiments at
varying enzyme:substrate ratios, highlight a catalytic outcome of
disrupting the well-conserved MCP hydrolase−substrate
interactions. In similar studies monitoring the BphD-catalyzed
hydrolysis of p-nitrophenyl acetate, no pre-steady-state kinetic
burst was reported.19 However, acetylation of Ser112 may be
rate-limiting for this substrate as it shares no chemical
substructure with HOPDA.
Scheme 5. Two-Conformation Model of Catalysis by the
MCP Hydrolases
The structural basis of the half-site reactivity of MCP
hydrolases remains unexplained. Although the orientation of
active site residues such as Phe175, Phe239, and Trp266 can be
correlated with active site occupancy,13,32,33 it is unclear
whether these different orientations alter the reactivity of the
active site. Nevertheless, the crystal structures of MhpC and
HsaD both revealed half-of-sites occupancy in the presence of
model substrates or inhibitors,32,33 consistent with subpopula-
tions of active sites possessing different substrate affinities.
Despite the structural variation, determination of a dissociation
constant for the S114A variant of HsaD with a steroid-derived
substrate suggested that the binding was stoichiometric.33
Interestingly, the spectrum of a red-shifted catalytic inter-
mediate, named ESred, whose lifetime is extended in serine
variants,13,17,33 provides some evidence of the occurrence of
two distinct active site environments in MCP hydrolases
despite full occupancy. Thus, the spectrum of ESred in BphD
has λmax values at 473 and 492 nm, suggesting contributions
from two enzyme-bound species.13 Ongoing efforts are aimed
at elucidating the structural basis of half-site reactivity as well as
the nature of ESred.
conformational change associated with a catalytic event in a
linked, active subunit. Thus, the two-conformation model
requires C−C bond cleavage (k2) to occur in E (half the
enzyme sites) before a conformational change converts E′ (the
other half of the active sites) to E, thereby allowing catalysis to
proceed in the remaining “stalled” active sites. The steady-state
kinetic analysis reported herein is consistent with this two-
conformation model inasmuch as the apparent rate of acylation
observed at high methanol concentrations (k2 ∼ 30 s−1) is
significantly lower than that observed via single-turnover
stopped-flow experiments (k2 ∼ 50 s−1).13 Whereas acylation
was directly observed in the latter experiment, it is inferred
from a methanol-induced change in the rate-determining step
observed in the former experiments. At low methanol
concentrations, kcat primarily reflects rate-determining deacyla-
tion (∼8 s−1), the extent of which increases with methanol
concentration until acylation becomes rate-determining. There-
fore, in the simplified kinetic Scheme 2, when kdeacylation
>
ASSOCIATED CONTENT
* Supporting Information
Representative HPLC chromatograms and fitting of pre-steady-
state kinetic data. This material is available free of charge via the
Internet at http://pubs.acs.org.
kacylation (high MeOH concentration), kcat should be similar to
the rate of acylation. However, in the two-conformation model
depicted in Scheme 5, each substrate molecule (after the first
turnover) will remain enzyme-bound through two acylation−
deacylation cycles. Thus, if we assume kdeacylation > kacylation at
■
S
5838
dx.doi.org/10.1021/bi300663r | Biochemistry 2012, 51, 5831−5840

Biochemistry
Article
(11) Fleming, S. M., Robertson, T. A., Langley, G. J., and Bugg, T. D.
(2000) Catalytic mechanism of a C-C hydrolase enzyme: Evidence for
a gem-diol intermediate, not an acyl enzyme. Biochemistry 39, 1522−
1531.
(12) Li, J. J., Li, C., Blindauer, C. A., and Bugg, T. D. (2006)
Evidence for a gem-diol reaction intermediate in bacterial C-C
hydrolase enzymes BphD and MhpC from 13C NMR spectroscopy.
Biochemistry 45, 12461−12469.
(13) Horsman, G. P., Ke, J., Dai, S., Seah, S. Y., Bolin, J. T., and Eltis,
L. D. (2006) Kinetic and structural insight into the mechanism of
BphD, a C-C bond hydrolase from the biphenyl degradation pathway.
Biochemistry 45, 11071−11086.
(14) Li, C., Li, J. J., Montgomery, M. G., Wood, S. P., and Bugg, T. D.
(2006) Catalytic role for arginine 188 in the C-C hydrolase catalytic
mechanism for Escherichia coli MhpC and Burkholderia xenovorans
LB400 BphD. Biochemistry 45, 12470−12479.
(15) Lam, W. W., and Bugg, T. D. (1997) Purification, character-
ization, and stereochemical analysis of a C-C hydrolase: 2-Hydroxy-6-
keto-nona-2,4-diene-1,9-dioic acid 5,6-hydrolase. Biochemistry 36,
12242−12251.
(16) Henderson, I. M., and Bugg, T. D. (1997) Pre-steady-state
kinetic analysis of 2-hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid 5,6-
hydrolase: Kinetic evidence for enol/keto tautomerization. Biochem-
istry 36, 12252−12258.
(17) Horsman, G. P., Bhowmik, S., Seah, S. Y., Kumar, P., Bolin, J. T.,
and Eltis, L. D. (2007) The tautomeric half-reaction of BphD, a C-C
bond hydrolase. Kinetic and structural evidence supporting a key role
for histidine 265 of the catalytic triad. J. Biol. Chem. 282, 19894−
19904.
(18) Li, C., Montgomery, M. G., Mohammed, F., Li, J. J., Wood, S. P.,
and Bugg, T. D. (2005) Catalytic mechanism of C-C hydrolase MhpC
from Escherichia coli: Kinetic analysis of His263 and Ser110 site-
directed mutants. J. Mol. Biol. 346, 241−251.
(19) Li, J. J., and Bugg, T. D. (2007) Investigation of a general base
mechanism for ester hydrolysis in C-C hydrolase enzymes of the α/β-
hydrolase superfamily: A novel mechanism for the serine catalytic
triad. Org. Biomol. Chem. 5, 507−513.
(20) Li, C., Hassler, M., and Bugg, T. D. (2008) Catalytic
promiscuity in the α/β-hydrolase superfamily: Hydroxamic acid
formation, C−C bond formation, ester and thioester hydrolysis in
the C−C hydrolase family. ChemBioChem 9, 71−76.
(21) Zhou, H., Qu, Y., Kong, C., Wu, Y., Zhu, K., Yang, J., and Zhou,
J. (2012) Promiscuous esterase activities of the C-C hydrolases from
Dyella ginsengisoli. Biotechnol. Lett. 34, 1107−1113.
(22) Seah, S. Y., Terracina, G., Bolin, J. T., Riebel, P., Snieckus, V.,
and Eltis, L. D. (1998) Purification and preliminary characterization of
a serine hydrolase involved in the microbial degradation of
polychlorinated biphenyls. J. Biol. Chem. 273, 22943−22949.
(23) Cornish-Bowden, A. (1995) Analysis of enzyme kinetic data,
Oxford University Press, New York.
(24) Li, J., Szittner, R., Derewenda, Z. S., and Meighen, E. A. (1996)
Conversion of serine-114 to cysteine-114 and the role of the active site
nucleophile in acyl transfer by myristoyl-ACP thioesterase from Vibrio
harveyi. Biochemistry 35, 9967−9973.
(25) Nazi, I., and Wright, G. D. (2005) Catalytic mechanism of
fungal homoserine transacetylase. Biochemistry 44, 13560−13566.
(26) Bhowmik, S., Horsman, G. P., Bolin, J. T., and Eltis, L. D.
(2007) The molecular basis for inhibition of BphD, a C-C bond
hydrolase involved in polychlorinated biphenyls degradation: Large 3-
substituents prevent tautomerization. J. Biol. Chem. 282, 36377−
36385.
(27) Fastrez, J., and Fersht, A. R. (1973) Demonstration of the acyl-
enzyme mechanism for the hydrolysis of peptides and anilides by
chymotrypsin. Biochemistry 12, 2025−2034.
AUTHOR INFORMATION
■
Corresponding Author
*Address: 2350 Health Sciences Mall, Vancouver, British
Columbia V6T 1Z3, Canada. Telephone: (604) 822-0042.
Fax: (604) 822-6041. E-mail: leltis@mail.ubc.ca.
Present Address
†Department of Chemistry, Wilfrid Laurier University, Water-
loo, Ontario, Canada.
Funding
A.C.R. is a recipient of a studentship from the National Science
and Engineering Research Council (NSERC) of Canada. This
work was supported by an NSERC Discovery Grant to L.D.E.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Jeffrey T. Bolin for helpful discussion.
ABBREVIATIONS
■
DxnB2, 2,8-dihydroxy-6-oxo-6-phenylhexa-2,4-dienoate 5,6-hy-
drolase; BphD, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate 5,6-
hydrolase; MhpC, 2-hydroxy-6-oxonona-2,4-diene-1,9-dioic
acid 5,6-hydrolase; HOPDA, 2-hydroxy-6-oxo-6-phenylhexa-
2,4-dienoate; pNPB, p-nitrophenyl benzoate; pNP, p-nitro-
phenol; v0, initial velocity; sKIE, solvent kinetic isotope effect.
REFERENCES
■
(1) Seah, S. Y., Labbe, G., Nerdinger, S., Johnson, M. R., Snieckus, V.,
and Eltis, L. D. (2000) Identification of a serine hydrolase as a key
determinant in the microbial degradation of polychlorinated biphenyls.
J. Biol. Chem. 275, 15701−15708.
(2) Rengarajan, J., Bloom, B. R., and Rubin, E. J. (2005) Genome-
wide requirements for Mycobacterium tuberculosis adaptation and
survival in macrophages. Proc. Natl. Acad. Sci. U.S.A. 102, 8327−8332.
(3) Ruzzini, A. C., Ghosh, S., Horsman, G. P., Foster, L. J., Bolin, J.
T., and Eltis, L. D. (2012) Identification of an Acyl-Enzyme
Intermediate in a meta-Cleavage Product Hydrolase Reveals the
Versatility of the Catalytic Triad. J. Am. Chem. Soc. 134, 4615−4624.
(4) Ollis, D. L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F.,
Franken, S. M., Harel, M., Remington, S. J., Silman, I., Schrag, J., et al.
(1992) The α/β-hydrolase fold. Protein Eng. 5, 197−211.
(5) Nardini, M., and Dijkstra, B. W. (1999) α/β-Hydrolase fold
enzymes: The family keeps growing. Curr. Opin. Struct. Biol. 9, 732−
737.
(6) Jiang, Y., Morley, K. L., Schrag, J. D., and Kazlauskas, R. J. (2011)
Different active-site loop orientation in serine hydrolases versus
acyltransferases. ChemBioChem 12, 768−776.
(7) Holmquist, M. (2000) α/β-Hydrolase fold enzymes: Structures,
functions and mechanisms. Curr. Protein Pept. Sci. 1, 209−235.
(8) Gruber, K., Gartler, G., Krammer, B., Schwab, H., and Kratky, C.
(2004) Reaction mechanism of hydroxynitrile lyases of the α/β-
hydrolase superfamily: The three-dimensional structure of the
transient enzyme-substrate complex certifies the crucial role of
Lys236. J. Biol. Chem. 279, 20501−20510.
(9) Frerichs-Deeken, U., Ranguelova, K., Kappl, R., Huttermann, J.,
and Fetzner, S. (2004) Dioxygenases without requirement for
cofactors and their chemical model reaction: Compulsory order
ternary complex mechanism of 1H-3-hydroxy-4-oxoquinaldine 2,4-
dioxygenase involving general base catalysis by histidine 251 and
single-electron oxidation of the substrate dianion. Biochemistry 43,
14485−14499.
(10) Larsen, N. A., Lin, H., Wei, R., Fischbach, M. A., and Walsh, C.
T. (2006) Structural characterization of enterobactin hydrolase IroE.
Biochemistry 45, 10184−10190.
(28) Ostrowski, W., and Barnard, E. A. (1973) Evidence for a
phosphoryl-enzyme intermediate in the catalytic reaction of prostatic
acid phosphatase. Biochemistry 12, 3893−3898.
(29) Padhi, S. K., Fujii, R., Legatt, G. A., Fossum, S. L., Berchtold, R.,
and Kazlauskas, R. J. (2010) Switching from an esterase to a
5839
dx.doi.org/10.1021/bi300663r | Biochemistry 2012, 51, 5831−5840

Biochemistry
Article
hydroxynitrile lyase mechanism requires only two amino acid
substitutions. Chem. Biol. 17, 863−871.
(30) Lai, K. K., Stogios, P. J., Vu, C., Xu, X., Cui, H., Molloy, S.,
Savchenko, A., Yakunin, A., and Gonzalez, C. F. (2011) An inserted α/
β subdomain shapes the catalytic pocket of Lactobacillus johnsonii
cinnamoyl esterase. PLoS One 6, e23269.
(31) Corey, D. R., and Craik, C. S. (1992) An investigation into the
minimum requirements for peptide hydrolysis by mutation of the
catalytic triad of trypsin. J. Am. Chem. Soc. 114, 1784−1790.
(32) Dunn, G., Montgomery, M. G., Mohammed, F., Coker, A.,
Cooper, J. B., Robertson, T., Garcia, J. L., Bugg, T. D., and Wood, S. P.
(2005) The structure of the C-C bond hydrolase MhpC provides
insights into its catalytic mechanism. J. Mol. Biol. 346, 253−265.
(33) Lack, N. A., Yam, K. C., Lowe, E. D., Horsman, G. P., Owen, R.
L., Sim, E., and Eltis, L. D. (2010) Characterization of a carbon-carbon
hydrolase from Mycobacterium tuberculosis involved in cholesterol
metabolism. J. Biol. Chem. 285, 434−443.
5840
dx.doi.org/10.1021/bi300663r | Biochemistry 2012, 51, 5831−5840