Substrate specificity, substrate channeling, and allostery in BphJ: An acylating aldehyde dehydrogenase associated with the pyruvate aldolase BphI

Article abstract of DOI:10.1021/bi300407y

BphJ, a nonphosphorylating acylating aldehyde dehydrogenase, catalyzes the conversion of aldehydes to form acyl-coenzyme A in the presence of NAD ⁺ and coenzyme A (CoA). The enzyme is structurally related to the nonacylating aldehyde dehydrogenases, aspartate-β-semialdehyde dehydrogenase and phosphorylating glyceraldehyde-3-phosphate dehydrogenase. Cys-131 was identified as the catalytic thiol in BphJ, and pH profiles together with site-specific mutagenesis data demonstrated that the catalytic thiol is not activated by an aspartate residue, as previously proposed. In contrast to the wild-type enzyme that had similar specificities for two- or three-carbon aldehydes, an I195A variant was observed to have a 20-fold higher catalytic efficiency for butyraldehyde and pentaldehyde compared to the catalytic efficiency of the wild type toward its natural substrate, acetaldehyde. BphJ forms a heterotetrameric complex with the class II aldolase BphI that channels aldehydes produced in the aldol cleavage reaction to the dehydrogenase via a molecular tunnel. Replacement of Ile-171 and Ile-195 with bulkier amino acid residues resulted in no more than a 35% reduction in acetaldehyde channeling efficiency, showing that these residues are not critical in gating the exit of the channel. Likewise, the replacement of Asn-170 in BphJ with alanine and aspartate did not substantially alter aldehyde channeling efficiencies. Levels of activation of BphI by BphJ N170A, N170D, and I171A were reduced by ≥3-fold in the presence of NADH and ≥4.5-fold when BphJ was undergoing turnover, indicating that allosteric activation of the aldolase has been compromised in these variants. The results demonstrate that the dehydrogenase coordinates the catalytic activity of BphI through allostery rather than through aldehyde channeling. (Figure Presented).

Full text of DOI:10.1021/bi300407y

Article
pubs.acs.org/biochemistry
Substrate Specificity, Substrate Channeling, and Allostery in BphJ:
An Acylating Aldehyde Dehydrogenase Associated with the Pyruvate
Aldolase BphI
Perrin Baker, Jason Carere, and Stephen Y. K. Seah*
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
S
* Supporting Information
ABSTRACT: BphJ, a nonphosphorylating acylating aldehyde
dehydrogenase, catalyzes the conversion of aldehydes to form
acyl-coenzyme A in the presence of NAD+ and coenzyme A
(CoA). The enzyme is structurally related to the nonacylating
aldehyde dehydrogenases, aspartate-β-semialdehyde dehydro-
genase and phosphorylating glyceraldehyde-3-phosphate de-
hydrogenase. Cys-131 was identified as the catalytic thiol in
BphJ, and pH profiles together with site-specific mutagenesis
data demonstrated that the catalytic thiol is not activated by an
aspartate residue, as previously proposed. In contrast to the
wild-type enzyme that had similar specificities for two- or
three-carbon aldehydes, an I195A variant was observed to have
a 20-fold higher catalytic efficiency for butyraldehyde and
pentaldehyde compared to the catalytic efficiency of the wild type toward its natural substrate, acetaldehyde. BphJ forms a
heterotetrameric complex with the class II aldolase BphI that channels aldehydes produced in the aldol cleavage reaction to the
dehydrogenase via a molecular tunnel. Replacement of Ile-171 and Ile-195 with bulkier amino acid residues resulted in no more
than a 35% reduction in acetaldehyde channeling efficiency, showing that these residues are not critical in gating the exit of the
channel. Likewise, the replacement of Asn-170 in BphJ with alanine and aspartate did not substantially alter aldehyde channeling
efficiencies. Levels of activation of BphI by BphJ N170A, N170D, and I171A were reduced by ≥3-fold in the presence of NADH
and ≥4.5-fold when BphJ was undergoing turnover, indicating that allosteric activation of the aldolase has been compromised in
these variants. The results demonstrate that the dehydrogenase coordinates the catalytic activity of BphI through allostery rather
than through aldehyde channeling.
ldehyde dehydrogenases (ALDHs) are evolutionarily
mechanism whereby the release of the reduced nicotinamide
Adiverse enzymes present in both prokaryotic and cofactor occurs before the binding of coenzyme A and the
transthioesterification step.8−10 While mechanistic and struc-
eukaryotic organisms that are involved in numerous biological
tural aspects of hydrolytic ALDHs have been extensively
studied,11−17 considerably less is known about CoA-dependent
ALDHs.18
functions, including central metabolism, osmotic protection,
cellular differentiation, and detoxification pathways. They can
be classified as phosphorylating or nonphosphorylating
ALDHs, producing activated or nonactivated acid products.
Both groups of ALDHs share a common acylation step,
whereby the thiolate of cysteine acts as a nucleophile to attack
the carbonyl carbon of the aldehyde forming a thiohemiacetal
intermediate. Following the transfer of a hydride to NAD+, a
thioacylenzyme intermediate is produced. The deacylation step,
however, differs depending on the nature of the acyl
acceptor.1−3 In phosphorylating dehydrogenases, inorganic
phosphate acts as the acyl acceptor, while in nonphosphorylat-
ing dehydrogenases, the thioacylenzyme intermediate under-
goes nucleophilic attack by either water (CoA-independent/
hydrolytic) or coenzyme A (CoA-dependent/acylating),
leading to the formation of either nonactivated or CoA-
activated acids, respectively. Hydrolytic ALDHs undergo a
sequential kinetic mechanism with NAD(P)H dissociating
last,4−7 whereas CoA-dependent ALDHs exhibit a ping-pong
BphJ is a nonphosphorylating CoA-dependent ALDH from
the polychlorinated biphenyl (PCB) pollutant-degrading
bacterium Burkholderia xenovorans LB400 (Scheme 1).19,20
BphJ forms a stable complex with the aldolase, BphI, which
catalyzes the aldol cleavage of 4-hydroxy-2-oxoacids to pyruvate
and an aldehyde.21,22 Aldehyde intermediates, up to six carbons
in length, can be channeled directly from the active site of BphI
to BphJ, via a molecular tunnel, with an efficiency of >80%.19
Because aldehydes are reactive, substrate channeling sequesters
the aldehydes from cellular components and overcomes the
inefficiency of the dehydrogenase, BphJ, for processing
exogenous aldehydes because of high Km values.20 When
Received: March 30, 2012
Revised: May 8, 2012
Published: May 10, 2012
© 2012 American Chemical Society
4558
dx.doi.org/10.1021/bi300407y | Biochemistry 2012, 51, 4558−4567

Biochemistry
Article
a
Scheme 1. Reaction Catalyzed by BphJ
a
Nucleophilic attack by cysteine on the aldehyde results in the formation of a thiohemiacetal intermediate. Hydride transfer reduces the NAD+
cofactor to NADH and leads to the formation of a thioacylenzyme intermediate. NADH leaves the active site, and the deacylation step occurs by the
nucleophilic attack of coenzyme A on the thioacylenzyme intermediate, leading to the formation and release of acyl-CoA product.
bound to BphJ, the nicotinamide cofactor increases the aldol
cleavage activity of BphI by ∼5-fold.19,20
into plasmid pET28a, and bphI was cloned into pBTL4-T7.20
Site-directed mutagenesis was completed according to a
modified Quikchange (Stratagene) method that uses non-
overlapping primers (Table S1 of the Supporting Informa-
tion).29 DNA of putative mutants was sequenced at the Guelph
Molecular Supercenter to confirm the presence of desired
mutations and the absence of secondary mutations.
Protein Expression and Purification. Expression and
purification of BphI-BphJ was completed as previously
described.20 Sodium dodecyl sulfate−polyacrylamide gel
electrophoresis was performed, and gels were stained with
Coomassie Blue to assess purity. The purified enzyme was
aliquoted and stored in 20 mM HEPES buffer (pH 8.5) with 10
mM DTT at −80 °C. The concentration of enzyme was
determined using the Bradford method.30
Dehydrogenase Activity Assays. The substrate specificity
of the dehydrogenase was determined using 0.4 mM NAD+ and
0.1 mM coenzyme A unless stated otherwise, and aldehyde
concentrations were varied from at least 0.1Km to 5Km.
Concentrations of aldehyde stocks were determined by
measuring the stoichiometric oxidation of NADH using alcohol
dehydrogenase. Cofactor specificities were determined under
similar conditions in which the acetaldehyde concentrations
were held at 100 mM and concentrations of NAD+ and CoA
were varied between 0.1Km and 5Km.
The effects of pH on kcat and kcat/Km were studied in the pH
range of 6.0−9.0 using a constant-ionic strength buffer
containing 0.1 M Tris, 0.05 M acetic acid, and 0.05 M MES.
All other conditions were the same as those of standard enzyme
assays with the substrate acetaldehyde concentration varied
from at least 0.1Km to 5Km. Data were plotted using eq 1 by
nonlinear regression in Leonora31
The crystal structure of DmpG-DmpF [Protein Data Bank
(PDB) entry 1NVM], an ortholog of BphI-BphJ from the
phenol degradation pathway of Pseudomonas putida CF600, is
available.23 Interestingly, DmpF is not structurally related to
methylmalonate-semialdehyde dehydrogenase (PDB entry
1T90) from Bacillus subtilis, which is the only other acylating
aldehyde dehydrogenase with a known structure.24 Instead, it
was reported to structurally resemble glyceraldehyde-3-
phosphate dehydrogenase, a phosphorylating hydrolytic de-
hydrogenase.23 It was further demonstrated through hydro-
gen−deuterium exchange mass spectrometry experiments that
NAD+ and coenzyme A share the same binding site.25 While
other enzymes, such as succinic synthetases, utilize the
Rossmann folds to bind coenzyme A,26−28 DmpF and related
enzymes appear to be unique in that they utilize this domain to
bind both the nicotinamide cofactor and coenzyme A.
Conversely, it has been suggested that NAD+ and CoA in the
unrelated acylating ALDH, methylmalonate semialdehyde
dehydrogenase, bind to separate domains of the enzyme.18
Here active site residues in BphJ were replaced by site-
specific mutagenesis. The catalytic thiol and residues important
for substrate specificity, coenzyme binding, and allosteric
regulation were identified. Our results shed light on the
molecular basis for the divergence of function in related
ALDHs.
EXPERIMENTAL PROCEDURES
■
Chemicals. All aldehydes, NAD+, NADH, L-lactate
dehydrogenase (LDH, rabbit muscle), all aldehydes, and
alcohol dehydrogenase (Saccharomyces cerevisiae) were from
Sigma-Aldrich (Oakville, ON). Ni-NTA Superflow resin was
obtained from Qiagen (Mississauga, ON). 4(S)-Hydroxy-2-
oxopentanoate was synthesized using BphI according to
previously described methods.21 Aldehyde dehydrogenase
from S. cerevisiae was obtained from Calzyme (San Luis
Obispo, CA). All other chemicals were analytical grade and
were obtained from Sigma-Aldrich or Fisher Scientific (Nepean,
ON).
C
v =
1 + H/Ka1
(1)
where C is the pH-independent value of kcat or kcat/Km, H is the
proton concentration, and Ka1 is the ionization constant of
groups involved in the reaction.
Aldolase Activity Assays. The aldolase cleavage activity
was determined continuously or discontinuously by coupling
the production of pyruvate to LDH and monitoring the
oxidation of NADH.20 All assays were performed in at least
DNA Manipulation. DNA was purified, digested, and
ligated using standard protocols. bphJ was previously cloned
4559
dx.doi.org/10.1021/bi300407y | Biochemistry 2012, 51, 4558−4567

Biochemistry
Article
Figure 1. Superimposition of the nonphosphorylating acylating aldehyde dehydrogenase DmpF with structurally similar aldehyde dehydrogenases
glyceraldehyde-3-phosphate dehydrogenase and aspartate-semialdehyde dehydrogenase. (A) Superimposition of the structure of DmpF with
ASADHs (rmsd of 2.5−2.7 Å). (B) Superimposition of the structure of DmpF with GAPDHs (rmsd of 2.4−2.7 Å). DmpF from P. putida CF600
(PDB entry 1NVM) is colored pink. ASADH from Vibrio colerae (PDB entry 2R00) is colored green. ASADH from Streptococcus pneumoniae (PDB
entry 2GZ3) is colored teal. Phosphorylating GAPDH from Bacillus stearothermophilius (PDB entry 3CMC) is colored yellow. GAPDH from
Arabidopsis thaliana (PDB entry 3K2B) is colored tan. (C) Active site residues are not conserved with the exception of the putative catalytic
cysteines. All residues in the active site of DmpF are conserved in BphJ with the exception of Met-198 that is replaced with Leu-197. DmpF from P.
putida CF600 has carbon atoms colored magenta, GAPDH from B. stearothermophilus carbon atoms colored yellow, and ASADH from S. pneumoniae
carbon atoms colored cyan. Oxygen and nitrogen atoms are colored red and blue, respectively.
duplicate, at 25 °C in a total volume of 1 mL using a Varian
Cary 3 spectrophotometer with a thermojacketed cuvette
holder. Standard continuous assays contained 0.4 mM NADH,
1 mM MnCl2, and 19.2 units of LDH in 100 mM HEPES buffer
(pH 8.0). The concentration of 4(S)-hydroxy-2-oxopentanoate
(HOPA) was varied from 0.1Km to 10Km. Oxidation of NADH
was monitored continuously at 340 nm with an extinction
coefficient of 6200 M−1 cm−1. To assess the activity of BphI in
the absence of NADH, a discontinuous assay was utilized. Assay
mixtures contain 1 mM MnCl2, HOPA concentrations that
varied from 0.1Km to 10Km, and 20 μg (80 μg for N170A and
N170D) of BphI-BphJ in 100 mM HEPES buffer (pH 8.0).
After 10 min, the reaction was quenched with 20 mM EDTA
(pH 8.5) and 0.4 mM NADH. The amount of pyruvate
produced was measured by an end point assay using LDH.
Test of Substrate Channeling. The amount of aldehyde
channeled directly from the aldolase to the dehydrogenase
without export to the bulk solvent was assessed using an
enzyme competition assay previously described.19 Unless
otherwise stated, assays contained NAD+ at a concentration
of at least 5Km, coenzyme A at a concentration of least 3Km, 1
mM MnCl2, and 5 μg of BphI-BphJ with 20 units of aldehyde
dehydrogenase (ALDH), which converts aldehydes to acids and
reduces NAD+ to NADH. Reactions were quenched after 5 min
with 24 μL of 3 N HCl and mixtures centrifuged for 3 min at
21000g to pellet the denatured enzyme. A 500 μL aliquot of the
reaction mixture was subjected to high-performance liquid
̈
chromatography (HPLC) using an AKTA Explorer 100
(Amersham Pharmacia Biotech, Baie d’Urfe, QC) equipped
with a HyPURITY C18 column (Thermo Scientific). The
4560
dx.doi.org/10.1021/bi300407y | Biochemistry 2012, 51, 4558−4567

Biochemistry
Article
Figure 2. Active site of DmpF showing differences in the apo and NAD+-bound structures. (A) Apo form of DmpF with the proposed catalytic thiol
(Cys-132) oriented toward Asp-209 through hydrogen bonding with a bridging water molecule 4.0 Å from Cys-132 and 2.6 and 3.4 Å from Asp-209.
(B) Holo form of DmpF depicting Cys-132 in a second conformation oriented toward the nicotinamide moiety of NAD+, which is in turn hydrogen
bonding to Asn-171. In this conformation, both Ile-172 and Met-198 change their rotameric configuration in comparison to the apoenzyme
structure. Numbers in parentheses correspond to the residue numbers of BphJ. All residues are conserved with the exception of Met-198 that is
replaced with Leu-197 in BphJ.
sample was eluted with a 50 mM sodium phosphate (pH 5.3)/
acetonitrile mixture (47:3). The channeling efficiency was
calculated by comparing the concentration of acetyl-CoA
produced (determined using HPLC) to the concentration of
4-hydroxy-2-oxopentanoate utilized by BphI based on the
amount of NADH produced by BphJ and ALDH (eq 2).19
root-mean-square deviations (rmsds) of 2.4 and 2.5 Å,
respectively. Superimposition of these structures revealed that
catalytic cysteines of these enzymes are equivalent in position
to Cys-132 in DmpF. None of the other active site residues are
conserved among the three enzymes (Figure 1C).
Significant movement in the Rossmann fold occurs between
the apo and NAD+-bound forms of DmpF, with an average
rmsd of 1.8 Å. The active site, located between the NAD+-
binding domain and the dimzerization domain, contains the
putative catalytic thiol, Cys-132 (Cys-131 in BphJ), on the N-
terminal end of α6.25 This cysteine residue adopts two
rotameric configurations in the DmpG-DmpF crystal structure
(Figure 2). In the apo form, the thiol orients toward Asp-209
with a bridging water molecule 4.0 Å from the sulfur atom of
Cys-132 and 2.6 and 3.4 Å from the carboxylic oxygens of Asp-
209. In the NAD+-bound form, the thiol is perpendicular to the
nicotinamide moiety of NAD+. It was previously proposed that
changes in the conformation of Cys-132 and the bridging water
are required to activate the thiol for nucleophilic attack on the
carbonyl carbon of the aldehyde substrate.23,36
Three hydrophobic residues, Ile-172, Ile-196, and Met-198
(Ile-171, Ile-195, and Leu-197, respectively, in BphJ), form part
of the interface between DmpF and the aldolase DmpG and
also form the exit of the aldehyde channel connecting the two
enzymes.19,23 These residues have been proposed to act as a
gate to control the passage of aldehydes that are channeled
from the aldolase to the dehydrogenase.23,36 This is partly
supported by alteration of the rotameric configuration of Ile-
172 and Ile-196 between the NAD+-bound and NAD+-free
structures mediated by Asn-171 (Asn-170 in BphJ) in DmpF.
Asn-171 undergoes a 166° rotation in the presence of NAD+
relative to the apo structure, allowing the carboxamide of Asn-
171 to form hydrogen bonds with the ribosyl hydroxyls of
NAD+. This movement results in changes to the Cα backbone,
which subsequently affects the rotameric configuration of Ile-
172 and Ile-196 (Figure 3A). In the apo form, Ile-172 is seen in
multiple conformations, one of which is more predominant as
visualized in the electron density, obtained from the Electron
Density Server (Figure 3B).37 The distance between the Cδ
atom of Ile-172 and the Cγ2 atom of Ile-196 in the apo
structure (observed in two conformations) ranges from 3.4 to
3.7 Å. In the NAD+-bound form, the distance between these
[acyl‐CoA produced]
[4‐hydroxy‐2‐oxoacid utilized]
channeling efficiency (%) =
× 100%
(2)
Dissociation Constant of NAD+. The dissociation
constant of NAD+ was assessed by tryptophan fluorescence
quenching upon NAD+ binding. Experiments were completed
using a PTI QuantaMaster C-61 steady-state fluorimeter
(Photon Technology International, London, ON), with a 1
nm bandwidth for excitation (λex = 290 nm) and a 2 mm
bandwidth for emission (λem = 330 nm). Titrations were
completed in 500 μL of 20 mM HEPES (pH 8.5) at 25 °C with
100 μg of enzyme. NAD+ was added stepwise to the enzyme
mixture. The fluorescence intensity was corrected for dilution
factors, and the dissociation constant was calculated by fitting
to eq 3, which describes binding to one site:
ΔFmax
× 100 × [S]
(
)
F
ΔF
o
o
× 100 =
F
Kd + [S]
(3)
where ΔF/Fo × 100 is the percent fluorescence quenching
(percent change in fluorescence relative to the initial value, Fo)
following addition of a substrate at concentration [S]. Fitting
was conducted using nonlinear regression using SigmaPlot 11.0.
RESULTS
■
Structural Analysis of the Dehydrogenase. The crystal
structure of DmpF, an ortholog of BphJ, is composed of two
domains, an N-terminal Rossmann fold spanning residues 6−
119 and a C-terminal dimerization domain composed of
residues 127−273 that is responsible for interacting with the
aldolase.32 Analysis using the DALI server33 indicated that
DmpF structurally resembles aspartate-β-semialdehyde dehy-
drogenase (PDB entry 2R00)34 and glyceraldehyde-3-phos-
phate dehydrogenase (PDB entry 1CF2)35 (Figure 1A,B), with
4561
dx.doi.org/10.1021/bi300407y | Biochemistry 2012, 51, 4558−4567

Biochemistry
Article
Table 1. Steady-State Kinetic Parameters of BphJ with
a
Acetaldehyde as the Substrate
enzyme
Km (mM)
kcat (s−1
)
kcat/Km,app (M−1 s−1
)
WT
23.6 1.8
NA
17.2 0.5
NA
730 60
NA
C131A
C131S
D208A
NA
NA
NA
18.7 1.4
7.6 0.2
406 34
a
Assays were performed at 25 °C and contained 0.4 mM NAD+ and
0.1 mM coenzyme A in 100 mM HEPES buffer (pH 8.0) in a total
volume of 1 mL. NA means not available.
enzyme. The aldolase, BphI, could form a stable complex with
the cysteine variants of BphJ, and the associating aldolase had
kinetic parameters similar to those of wild-type BphI, further
supporting the premise that the aldolase−dehydrogenase
complex is properly folded (Table S2 of the Supporting
Information).
The pH dependence of the BphJ-catalyzed conversion of
acetaldehyde to acetyl-CoA was determined over a pH range of
6.0−9.0 and was fit to a one-pKa model with pKa values of 8.49
0.05 and 8.05
0.03 for the free enzyme and enzyme−
substrate complex, respectively (Figure 4). It was previously
proposed that the putative catalytic thiol Cys-132 is activated
via base attack from the bound water, which is activated by Asp-
209 in DmpF.23,36 Substitution of the equivalent Asp-208 in
BphJ with alanine (D208A) resulted in a <2-fold difference in
the steady-state catalytic efficiency (kcat/Km) for the dehydro-
genase reaction relative to that of the wild-type enzyme. In
addition, the pH dependency of the reaction was not
significantly altered relative to that of the wild-type enzyme
(pKa values of 8.57 0.08 and 8.08 0.03 for the free enzyme
and enzyme−substrate complex, respectively) (Figure 4). This
suggests that Asp-209 is not involved in thiol activation.
Dehydrogenase Substrate Specificity. The wild-type
enzyme exhibited similar catalytic efficiencies (kcat/Km) for
acetaldehyde and propionaldehyde with a ∼2-fold lower
catalytic efficiency for butyraldehyde compared to those for
the smaller aldehydes (Table 2). Specificity for pentaldehyde
was 10-fold lower than that for acetaldehyde primarily because
of the high apparent Km value for this substrate.
Both I171A and I171F exhibited preferences for aldehydes
similar to that of the wild type (Table 2). In contrast, the I195A
variant exhibited a 5-fold reduction in the specificity constant
for acetaldehyde, because of the increase in the apparent Km
value. The specificity constants for butyraldehyde and
pentaldehyde in this variant increased by approximately 9-
and 20-fold, respectively, compared to that of the wild-type
enzyme. The increase in specificity constants for longer chain
aldehydes primarily resulted from lower Km values (<10 mM
for butyraldehyde and pentaldehyde). Significantly, the kcat/Km
of the I195A variant for pentaldehyde is 20-fold higher than
that of the wild-type enzyme, making it a more efficient enzyme
for utilization of this longer chain aldehyde (1584 156 M−1
s−1) compared to the wild-type enzyme for its natural substrate,
acetaldehyde (730 60 M−1 s−1). When Ile-195 was replaced
with a phenylalanine or tryptophan, kcat/Km values were 5−20-
fold lower than that of the wild type for aldehydes two to four
carbons in length. An increase in the Km for butyraldehyde in
both I195F and I195W variants is responsible for the reduction
in kcat/Km. Together, these results suggest that the alkyl chain of
the aldehyde orients toward Ile-195 and that this residue is
responsible for governing aldehyde substrate chain length
Figure 3. Structural changes upon NAD+ binding in DmpF (A)
Interaction of NAD+ with Asn-171 of DmpF. In the presence of
NAD+, Asn-171 rotates 166° and forms hydrogen bonds with the
hydroxyls of the nicotinamide ribose ring. This causes movement of
the adjacent isoleucine (Ile-172), one of three residues that line the
exit of the aldehyde channel leading to the dehydrogenase active site.
Carbon atoms in the apo structure are colored gray, and the NAD+-
bound structure is depicted as a cartoon with blue carbon atoms. (B)
Electron density of residues in the apo structure showing multiple
conformations of Ile-172 and Met-198.
residues increases to 4.0 Å. It was therefore suggested on the
basis of the crystal structure that Ile-172, Ile-196, and Met-198
in DmpF may gate the exit of channeled aldehydes from the
aldolase and the opening of the gate is mediated via Asn-171
upon NAD+ binding.23
To elucidate the function of these conserved residues in
BphJ, we replaced Ile-171 with alanine and phenylalanine
(I171A and I171F, respectively) and Ile-195 with the smaller
alanine (I195A) as well as the bulker phenylalanine and
tryptophan (I195F and I195W, respectively) residues. Asn-170
was replaced with alanine and aspartate (N170A and N170D,
respectively). Finally, to identify the catalytic thiol and the role
of Asp-208, we replaced Cys-131 with alanine and serine
(C131A and C131S, respectively) and Asp-208 with alanine
(D208A). All variants were expressed and purified in E. coli
BL21(λDE3) to homogeneity using the same protocol
developed for the wild-type enzyme.20
Analysis of Cys-131 and Asp-208. When Cys-131 was
replaced with alanine (C131A) or serine (C131S), no
detectable activity was observed (Table 1). Examination of
these cysteine variants (C131A and C131S) by circular
dichroism did not reveal significant differences in spectra
relative to that of the wild-type (WT) enzyme (Figure S1 of the
Supporting Information), suggesting that abrogation of catalysis
is not the result of drastic changes in the structure of the
enzyme. NAD+ was still able to bind to the enzyme; however,
the Kd was increased 20-fold compared to that of the wild-type
4562
dx.doi.org/10.1021/bi300407y | Biochemistry 2012, 51, 4558−4567

Biochemistry
Article
Figure 4. pH dependence of kcat and kcat/Km for WT BphJ and the D208A variant. Assays were conducted in a constant-ionic strength buffer
containing 0.1 M Tris, 0.05 M acetic acid, 0.05 M MES, and 1 mM MnCl2. (A) pH dependence of kcat of WT BphJ and the D208A variant (B) pH
●
○
dependence of kcat/Km of WT BphJ and the D208A variant. WT ( ) and D208A ( ) for both panels.
a
Table 2. Substrate Specificity of BphJ in the Dehydrogenase Reaction
acetaldehyde
propionaldehyde
(three carbons)
butyraldehyde
(four carbons)
pentaldehyde
enzyme
WT
kinetic parameter
(two carbons)
(five carbons)
picolinaldehyde
Km,app (mM)
23.6 1.8
17.2 0.5
730 60
17.0 1.4
37.9 1.2
2224 203
34.9 3.5
7.8 0.3
223 23
133 12
19.7 0.9
147 15
33.7 3.2
15.4 0.6
457 48
45.6 4.0
6.9 0.3
152 14
23.1 1.7
16.3 0.5
700 50
20.4 2.1
24.9 1.2
1219 141
27.2 2.4
6.0 0.2
221 21
31.5 2.7
12.1 0.6
384 35
45.7 4.1
7.6 0.3
167 16
79.5 6.9
3.4 0.2
42.5 4.1
31.7 2.2
9.5 0.2
300 20
12.6 1.2
16.7 0.9
1323 147
19.3 1.3
7.1 0.2
370 29
4.6 0.3
12.6 0.3
2763 181
−
−
−
18.2 1.3
2.8 0.1
154 12
3.2 0.3
1.9 0.1
597 53
12.0 1.0
2.4 0.1
202 17
6.9 0.5
3.2 0.1
468 34
−
kcat,app (s−1
)
b
k
cat/Km,app (M−1 s−1
Km,app (mM)
kcat,app (s−1
cat/Km,app (M−1 s−1
Km,app (mM)
kcat,app (s−1
cat/Km,app (M−1 s−1
Km,app (mM)
kcat,app (s−1
cat/Km,app (M−1 s−1
Km,app (mM)
kcat,app (s−1
cat/Km,app (M−1 s−1
Km,app (mM)
kcat,app (s−1
cat/Km,app (M−1 s−1
)
)
)
)
)
)
76 3.1
I171A
I171F
I195A
−
−
)
b
k
72.8 4.9
−
−
)
b
k
38.7 1.3
8.2 0.8
13.0 0.4
1584 156
−
)
k
I195F
)
−
−
−
b
b
b
k
43.3 2.3
34.6 2.3
0.76 0.03
−
−
−
−
−
−
I195W
)
b
b
b
k
45.8 1.4
26.5 1.1
0.11 0.01
a
Assays were performed at 25 °C and contained 5 μg of BphJ variants, 1 mM MnCl2, 100 μM CoA, and 400 μM NAD+ with varying concentrations
of aldehydes in 100 mM HEPES buffer (pH 8.0). Assays were performed at 25 °C and contained 5 μg of BphJ variants, 1 mM MnCl2, 200 μM CoA,
b
and 800 μM NAD+ with concentrations of picolinaldehyde varying between 0.1Km and at least 5Km in 100 mM HEPES buffer (pH 8.0). Because of
the low substrate solubility or high apparent Km for the substrate, the specificity constant can be estimated from the linear gradient of specific activity
vs substrate concentration.
specificity. The apparent Km values of NAD+ in I171F and I195
Table 3. Thiol Specificity of Wild-Type BphJ in the
a
variants were not significantly perturbed (≤3-fold) relative to
that of the wild-type enzyme.
Dehydrogenase Reaction
thiol
Km,app (μM)
kcat,app (s−1
)
kcat/Km,app (×104 M−1 s−1
46
)
The kcat for picolinaldehyde in wild-type BphJ was
approximately 5-fold lower compared to that for the natural
substrate, acetaldehyde. The I171A and I195A variants
exhibited the highest catalytic efficiencies for picolinaldehyde,
while the incorporation of an aromatic residue at position 195
(I195F and I195W) resulted in >200-fold reductions in kcat/Km,
possibly because of steric hindrance with the large picolinalde-
hyde side chain (Table 2).
Cofactor Binding and Specificity. BphJ was tested for its
ability to use various thiols during the deacylation step of the
reaction. The enzyme could utilize dithiothreitol (DTT) in
place of coenzyme A, albeit 300-fold less efficiently (Table 3).
coenzyme A
DTT
30.2 2.7
6609 525
NA
13.9 0.4
10.1 0.3
NA
4
0.15 0.01
NA
cysteine
glutathione
NA
NA
NA
a
Assays were performed at 25 °C and contained 5 μg of BphJ variants,
1 mM MnCl2, 5Km acetaldehyde, and 400 μM NAD+ with
concentrations of thiols varying between 0.1Km and 5Km in 100 mM
HEPES buffer (pH 8.0). NA means not available.
This difference was the direct result of the higher Km value for
DTT relative to that for coenzyme A. Free cysteine and
4563
dx.doi.org/10.1021/bi300407y | Biochemistry 2012, 51, 4558−4567

Biochemistry
Article
glutathione were not observed to act in lieu of coenzyme A in
the dehydrogenase reaction. BphJ had no detectable activity
(<0.0001 s−1) in the absence of coenzyme A and DTT,
indicating that water cannot replace the thiols in the
deacylation step under standard assay conditions.
Allosteric Communication. In the wild-type aldolase−
dehydrogenase complex, the aldol cleavage activity of BphI is
activated in the presence of the BphJ cofactor, NADH, by ∼5-
fold.19,20 When both enzymes are undergoing turnover, this
level of activation is increased to 16-fold over that of the
nonactivated aldolase. The level of allosteric activation of the
aldol cleavage reaction in the presence of the NADH in I171A,
N170A, and N170D was observed to be less than 2-fold (Table
6). When BphJ is undergoing turnover, BphI activities when in
The wild-type enzyme exhibited a dissociation constant (Kd)
for NAD+ of 1.8 0.2 μM (Table 4). The Kd for NAD+ in the
Table 4. Dissociation Constants of NAD+ for the Wild Type
a
and Variants
Table 6. Effects of NADH on Steady-State Kinetic
Parameters for the Aldol Cleavage of HOPA by BphI
enzyme
Kd (μM)
a
WT
1.8 0.2
BphJ in the
complex
Km,app
kcat,app
x-fold activation
N170A
N170D
I171A
C131A
C131S
8
1
b
(μM)
(×10−2 s−1
)
by NADH
3.2 0.4
WT
− NADH
+ NADH
− NADH
+ NADH
− NADH
+ NADH
− NADH
+ NADH
158 20
79
407
68
6
7
3
3
1
2
1
1
−
36
48
43
4
6
7
89
152 22
28
110 12
32
192 18
34
8
5.1 0.4
I171A
N170A
N170D
−
5
96
1.4 0.1
−
1.2 0.1
a
Dissociation constants were determined by monitoring tryptophan
44
fluorescence quenching upon NAD+ binding (excitation wavelength of
295 nm and emission wavelength of 330 nm). The titrations were
conducted in 500 μL of 20 mM HEPES (pH 8.5) at 25 °C with 100 μg
of enzyme, with stepwise addition of NAD+ aliquots. Mixtures
containing no NAD+ were used as a control.
5
51
22
−
5
36
1.6 0.1
a
Assays contained 1 mM MnCl2 and HOPA concentrations that varied
b
from 0.1Km to 10Km in 100 mM HEPES buffer (pH 8.0). Ratio of kcat
(s−1) of BphI in the presence of NADH (final concentration of 0.4
mM) and in the absence of NADH (no NADH added to the assays).
N170D variant increased by only 1.7-fold, while substitution
with alanine, which is not capable of direct hydrogen bonding
to the C2 and C3 hydroxyls of the nicotinamide ribose, led to
an increase in Kd of ∼4-fold. In comparison, substitution of the
adjacent residue, Ile-171 (located ∼7 Å from the nicotinamide
moiety of NAD+), with alanine led to a 20-fold increase in Kd
for NAD+. The Km values for all variants were determined to
ensure all cofactors were present under saturating conditions
(Tables S2 and S3 of the Supporting Information). It was
observed that the I171A variant had ∼15-fold higher Km values
(>400 μM) for NAD+ relative to that of the wild-type enzyme.
Acetaldehyde Substrate Channeling. The acetaldehyde
channeling efficiency in N170A was observed to be 83 2%
(Table 5). Similar channeling efficiencies were observed in
N170D. The I195A variant exhibited a channeling efficiency
similar to those of other variants tested (∼80%), while I195W
displayed a more dramatic decrease in acetaldehyde channeling
efficiency (59 1%). Substitution of Ile-171 with either alanine
or phenylalanine resulted in a 20−30% decrease in channeling
efficiency.
complex with the I171A, N170A, and N170D BphJ variants
were 2.45, 0.91, and 0.54 s−1, respectively, representing 3.6-,
2.1-, and 2.5-fold increases in the catalytic rate, respectively.
Thus, these variants result in a significant reduction in the level
of activation of the aldol cleavage reaction by BphI.
DISCUSSION
■
BphJ is a nonphosphorylating acylating aldehyde dehydrogen-
ase that catalyzes the acylation of acetaldehyde to acetyl-CoA in
the presence of NAD+ and CoASH. Interestingly, BphJ and
related enzymes from bacterial aromatic hydrocarbon degrada-
tion pathways share no structural or sequence identity with the
only other acylating aldehyde dehydrogenase with known
structure, methylmalonate-semialdehyde dehydrogenase from
B. subtilis.18 Instead, BphJ and orthologs are evolutionarily
related, on the basis of structure, to phosphorylating ALDHs,
aspartate-β-semialdehyde dehydrogenase (ASADH) and glyc-
eraldehyde-3-phosphate dehydrogenase (GAPDH).
Replacement of Cys-131 in BphJ with alanine and serine led
to the abolishment of BphJ activity. Cys-132 in DmpF is in the
same relative position with respect to catalytic thiols of ASADH
and GAPDH. Unlike structurally similar GAPDH and ASADH,
BphJ and its homologues lack an invariant histidine located
proximal to the cysteine residue that is proposed to activate the
catalytic thiol38−41 or to stabilize a tetrahedral transition
state.15,42 It was previously proposed that the conserved Asp-
209 in DmpF (Asp-208 in BphJ) is required to activate the Cys-
132 via a bound water molecule observed in the apo
structure.23,36 However, the BphJ D208A variant was not
significantly affected either in terms of its steady-state kinetic
parameters or in its pH profile, discounting the involvement of
this residue in the catalytic mechanism. A single ionizable group
was observed in both free and bound forms of the BphJ.
However, in the substrate-bound form, the pKa of this group
was ∼0.5 pH unit lower than that of the free enzyme. A similar
Table 5. Acetaldehyde Channeling Efficiencies in BphI-BphJ
Variant Complexes
a
BphJ
WT
channeling efficiency (%)
95
83
83
84
77
59
84
5
2
2
3
6
1
1
N170A
N170D
I171A
I171F
I195W
I195A
a
Assays contained NAD+ at 5Km concentrations, coenzyme A at at
least 3Km concentrations, 1 mM MnCl2, 5 μg of enzyme complex, and
20 units of aldehyde dehydrogenase (ALDH), which converts
acetaldehyde to acetic acids. Reactions were quenched after 5 min
with 24 μL of 3 N HCl. Acetyl-CoA produced was detected at 254 nm
by HPLC.
4564
dx.doi.org/10.1021/bi300407y | Biochemistry 2012, 51, 4558−4567

Biochemistry
Article
shift was observed in methylmalonate-semialdehyde dehydro-
genase whereby the enzyme exhibits pKa values of 8.7 and 8.0
for the free enzyme and the enzyme−substrate−NAD+ binary
complex, respectively.18,43 These pKa values were assigned to
the catalytic cysteine with augmentation of the pKa in the
binary complex attributed to NAD+. Cofactor binding has also
been implicated in thiol activation in nonphosphorylating
GAPDH where binding of NADP+ and NAD+ increases the
reactivity of the catalytic thiol.44,45 Structurally, the nicotina-
mide ring is ∼4 Å from the thiol group of Cys-132 in the
NAD+-bound structure of DmpF, which supports its role in
activating the catalytic thiol.
Replacement of Ile-195 with alanine (I195A) increased the
size of the active site, allowing for the accommodation of longer
and bulkier aldehydes and hence increasing the specificity
constants for these substrates. The specificity constant for
acetaldehyde was lowered in this variant, possibly because of
the loss of specific interactions between the enzyme and the
shorter substrate leading to nonproductive binding. Conversely,
incorporation of bulky aromatic residues at position 195 in
I195F and I195W perturbed the binding of longer alkyl chain
aldehydes. These data suggest that the aldehyde side chain
extends toward Ile-195.
MOLE,19 Ile-171, Ile-195, and Leu-197 in BphJ form the exit of
the substrate channel connecting the active sites of the aldolase
and dehydrogenase. This was confirmed by reduced aldehyde
channeling efficiencies when Ile-195 was replaced with bulky
residues phenylalanine and tryptophan. A BphJ I195F variant
was previously demonstrated to have a channeling efficiency of
80 1%,19 while I195W exhibited a channeling efficiency of 59
1%. Aldehyde channeling in the BphI-BphJ system is still
efficient (83 2%) when Asn-170 was replaced with alanine,
which cannot form hydrogen bond interactions with the ribosyl
hydroxyls of the nicotinamide moiety of NAD+. Therefore, the
previous proposal,23 that substrate channeling is dependent on
hydrogen bonding interaction between the side chain of Asn-
171 (Asn-170 in BphJ) and NAD+, which alters the Cα
backbone and rotameric configuration of Ile-172, Ile-196, and
Met-198 to allow the exit of the channeled aldehyde, is not
supported by the experimental evidence. In comparison, Tyr-
290 in BphI partially blocks the entrance of the aldehyde
channel. Previous replacement of Tyr-290 compromised
channeling efficiency (up to 85% reduction in channeling
efficiency). Therefore, Tyr-290 in the aldolase appears to be the
important gating residue for aldehyde channeling in BphI-
BphJ.19
Invariant residues Arg-245 and Glu-220 in ASADH from S.
pneumoniae (PDB entry 2GZ3) are spatially equivalent to Met-
198 and Ile-196 in DmpF, respectively.46,47 Kinetic studies48
and structures of native enzyme complexes containing a
hemithioacetal intermediate39,47,49 or the inactivator S-methyl-
L-cysteine sulfoxide50 have further established that Arg-245
forms bidenate interactions with the α-carboxyl group of the
substrate L-aspartate-β-semialdehyde. The role of Glu-220 in
ASADHs has not been definitively established,46 but the side
chain carboxyl group may interact with the α-amino group of L-
aspartate-β-semialdehyde.47,49 Residues Arg-231 and Arg-195 in
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of B.
stearothermophilus (PDB entry 1NQA) are similar in position to
Ile-196 in DmpF and Ile-172, respectively. These residues have
been implicated in binding the phosphate group of the
substrate.51 Thus, divergent substrate specificities of these
evolutionarily related enzymes may be governed by the side
chains of the two residues at the back of the active sites of these
enzymes (Figure 1C).
Replacement of Ile-171 with alanine led to a 20-fold increase
in the NAD+ dissociation constant for BphJ. In DmpG, the
equivalent residue Ile-172, is located on the N-terminus on an
α-helix and forms hydrophobic interactions with Ile-196 and
Met-198 on an opposing β-sheet. It is envisaged that shortening
the side chain in the BphJ I171A variant may disrupt this
interaction, thereby altering the Cα backbone and affecting the
tertiary structure of the NAD+-binding domain (Figure 3A).
Dissociation constants for the Asn-170 variants of BphJ were
also increased, but by <10-fold, indicating that although Asn-
170 interacts with NAD+, it is not critical for cofactor binding.
Thus, when Asn-170 is replaced with aspartate or shortened to
alanine, hydrogen bonding with NAD+ can still occur between
the carboylate oxygen and ribosyl hydroxyls in N170D, or space
may be created for water molecules to interact with NAD+ in
N170A. Indeed, in the structurally similar GAPDH and
ASADH, NAD+ binds in a position similar to that of DmpF
(Figure 1C), but ordered water molecules form hydrogen bond
interactions with the hydroxyls of the ribose of NAD+ instead.
Substrate Channeling and Allostery. By inference from
the DmpF structure23 and previous channel identification using
Interestingly, Asn-170 and Ile-171 appear to be important in
mediating allosteric activation of the aldol cleavage reaction of
BphI upon NAD+ binding in BphJ. Replacement of these
residues led to attenuation of BphI activation. This allostery
likely ensures that aldehydes released to the solvent in the
absence of cofactors are kept to a minimum because the aldol
cleavage reaction always occurs in the presence of 4-hydroxy-2-
oxoacids. The equivalent Asn-171 and Ile-172 are located in the
α7 and α8 secondary structures of DmpF that interact with the
α5 secondary structure of the HMGL-like domain in the
aldolase, DmpG. It is possible that the allosteric activation is
mediated through these intersubunit contacts. However, the
precise molecular mechanism of this activation is currently
unclear because no obvious structural changes in the aldolase
can be observed between the crystal structures of the apo and
NAD+-bound forms of the DmpF-DmpG complex.
CONCLUSION
■
In summary, BphJ utilizes Cys-131 as the catalytic thiol;
however, this thiol is not activated by Asp-208 as previously
proposed.23,36 Ile-195 is involved in substrate binding, and
residues in equivalent positions in phosphorylating ALDHs also
appear to interact with their respective substrates. Interaction of
Asn-170 with the nicotinamide cofactor does not appear to be
important for substrate channeling. However, Asn-170 and Ile-
171 are important for allosteric activation of BphI. The results
presented herein complement our previous work and shed
further light on the mechanism of substrate channeling in this
enzyme complex.19−22,32,52
ASSOCIATED CONTENT
■
S
* Supporting Information
Circular dichroism spectra of wild-type BphJ and its C131A and
C131S variants (Figure S1), primers used in polymerase chain
reaction site-directed mutagenesis (Table S1), steady-state
kinetic parameters of BphJ variants (Table S2), and steady-state
kinetic parameters of BphJ cofactors (Table S3). This material
is available free of charge via the Internet at http://pubs.acs.org.
4565
dx.doi.org/10.1021/bi300407y | Biochemistry 2012, 51, 4558−4567

Biochemistry
Article
dehydrogenase as probed by site-directed mutagenesis. Biochemistry
34, 237−243.
AUTHOR INFORMATION
Corresponding Author
*Department of Molecular and Cellular Biology, University of
Guelph, Guelph, Ontario, Canada N1G 2W1. E-mail: sseah@
uoguelph.ca. Phone: (519) 824-4120, ext. 56750. Fax: (519)
837-1802.
■
(13) Vedadi, M., and Meighen, E. (1997) Critical glutamic acid
residues affecting the mechanism and nucleotide specificity of Vibrio
harveyi aldehyde dehydrogenase. Eur. J. Biochem. 246, 698−704.
(14) Cobessi, D., Tete-Favier, F., Marchal, S., Branlant, G., and
Aubry, A. (2000) Structural and biochemical investigations of the
catalytic mechanism of an NADP-dependent aldehyde dehydrogenase
from Streptococcus mutans. J. Mol. Biol. 300, 141−152.
Author Contributions
P.B. and J.C. contributed equally to this work.
(15) Soukri, A., Mougin, A., Corbier, C., Wonacott, A., Branlant, C.,
and Branlant, G. (1989) Role of the histidine 176 residue in
glyceraldehyde-3-phosphate dehydrogenase as probed by site-directed
mutagenesis. Biochemistry 28, 2586−2592.
(16) D’Ambrosio, K., Pailot, A., Talfournier, F., Didierjean, C.,
Benedetti, E., Aubry, A., Branlant, G., and Corbier, C. (2006) The first
crystal structure of a thioacylenzyme intermediate in the ALDH family:
New coenzyme conformation and relevance to catalysis. Biochemistry
45, 2978−2986.
Funding
This research is supported by the National Science and
Engineering Research Council of Canada (NSERC), Grant
238284 (to S.Y.K.S.). P.B. is a recipient of a postgraduate
research scholarship from NSERC.
Notes
The authors declare no competing financial interest.
(17) Talfournier, F., Pailot, A., Stines-Chaumeil, C., and Branlant, G.
(2009) Stabilization and conformational isomerization of the cofactor
during the catalysis in hydrolytic ALDHs. Chem.-Biol. Interact. 178,
79−83.
(18) Talfournier, F., Stines-Chaumeil, C., and Branlant, G. (2011)
Methylmalonate-semialdehyde dehydrogenase from Bacillus subtilis:
Substrate specificity and coenzyme A binding. J. Biol. Chem. 286,
21971−21981.
(19) Carere, J., Baker, P., and Seah, S. Y. (2011) Investigating the
Molecular Determinants for Substrate Channeling in BphI-BphJ, an
Aldolase-Dehydrogenase Complex from the Polychlorinated Biphenyls
Degradation Pathway. Biochemistry 50, 8407−8416.
(20) Baker, P., Pan, D., Carere, J., Rossi, A., Wang, W., and Seah, S. Y.
(2009) Characterization of an aldolase-dehydrogenase complex that
exhibits substrate channeling in the polychlorinated biphenyls
degradation pathway. Biochemistry 48, 6551−6558.
(21) Wang, W., Baker, P., and Seah, S. Y. (2010) Comparison of two
metal-dependent pyruvate aldolases related by convergent evolution:
Substrate specificity, kinetic mechanism, and substrate channeling.
Biochemistry 49, 3774−3782.
(22) Baker, P., Carere, J., and Seah, S. Y. (2011) Probing the
Molecular Basis of Substrate Specificity, Stereospecificity, and Catalysis
in the Class II Pyruvate Aldolase, BphI. Biochemistry 50, 3559−3569.
(23) Manjasetty, B. A., Powlowski, J., and Vrielink, A. (2003) Crystal
structure of a bifunctional aldolase-dehydrogenase: Sequestering a
reactive and volatile intermediate. Proc. Natl. Acad. Sci. U.S.A. 100,
6992−6997.
(24) Dubourg, H., Stines-Chaumeil, C., Didierjean, C., Talfournier,
F., Rahuel-Clermont, S., Branlant, G., and Aubry, A. (2004)
Expression, purification, crystallization and preliminary X-ray
diffraction data of methylmalonate-semialdehyde dehydrogenase
from Bacillus subtilis. Acta Crystallogr. D60, 1435−1437.
(25) Lei, Y., Pawelek, P. D., and Powlowski, J. (2008) A shared
binding site for NAD+ and coenzyme A in an acetaldehyde
dehydrogenase involved in bacterial degradation of aromatic
compounds. Biochemistry 47, 6870−6882.
(26) Ricagno, S., Jonsson, S., Richards, N., and Lindqvist, Y. (2003)
Crystallization and preliminary crystallographic analysis of formyl-CoA
tranferase from Oxalobacter formigenes. Acta Crystallogr. D59, 1276−
1277.
ABBREVIATIONS
■
ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogen-
ase; ASADH, aspartate-semialdehyde dehydrogenase; EDTA,
ethylenedinitrilotetraacetic acid; GAPDH, glyceraldehyde-3-
phosphate dehydrogenase; HEPES, 4-(2-hydroxyethyl)-1-pi-
perazinepropanesulfonic acid; HOPA, 4-hydroxy-2-oxopenta-
noate; IPTG, isopropyl β-D-thiogalactopyranoside; LDH, L-
lactate dehydrogenase; MES, 2-(N-morpholino)ethanesulfonic
acid.
REFERENCES
■
(1) Segal, H. L., and Boyer, P. D. (1953) The role of sulfhydryl
groups in the activity of D-glyceraldehyde 3-phosphate dehydrogenase.
J. Biol. Chem. 204, 265−281.
(2) Racker, E., and Krimsky, I. (1952) The mechanism of oxidation
of aldehydes by glyceraldehyde-3-phosphate dehydrogenase. J. Biol.
Chem. 198, 731−743.
(3) Feldman, R. I., and Weiner, H. (1972) Horse liver aldehyde
dehydrogenase. II. Kinetics and mechanistic implications of the
dehydrogenase and esterase activity. J. Biol. Chem. 247, 267−272.
(4) Bradbury, S. L., and Jakoby, W. B. (1971) Ordered binding of
substrates to yeast aldehyde dehydrogenase. J. Biol. Chem. 246, 1834−
1840.
(5) MacGibbon, A. K., Blackwell, L. F., and Buckley, P. D. (1977)
Pre-steady-state kinetic studies on cytoplasmic sheep liver aldehyde
dehydrogenase. Biochem. J. 167, 469−477.
(6) Vallari, R. C., and Pietruszko, R. (1981) Kinetic mechanism of
the human cytoplasmic aldehyde dehydrogenase E1. Arch. Biochem.
Biophys. 212, 9−19.
(7) Marchal, S., Rahuel-Clermont, S., and Branlant, G. (2000) Role of
glutamate-268 in the catalytic mechanism of nonphosphorylating
glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans.
Biochemistry 39, 3327−3335.
(8) Shone, C. C., and Fromm, H. J. (1981) Steady-state and pre-
steady-state kinetics of coenzyme A linked aldehyde dehydrogenase
from Escherichia coli. Biochemistry 20, 7494−7501.
(9) Sohling, B., and Gottschalk, G. (1993) Purification and
characterization of a coenzyme-A-dependent succinate-semialdehyde
dehydrogenase from Clostridium kluyveri. Eur. J. Biochem. 212, 121−
127.
(10) Yan, R. T., and Chen, J. S. (1990) Coenzyme A-acylating
aldehyde dehydrogenase from Clostridium beijerinckii NRRL B592.
Appl. Environ. Microbiol. 56, 2591−2599.
(11) Farres, J., Wang, T. T., Cunningham, S. J., and Weiner, H.
(1995) Investigation of the active site cysteine residue of rat liver
mitochondrial aldehyde dehydrogenase by site-directed mutagenesis.
Biochemistry 34, 2592−2598.
(12) Wang, X., and Weiner, H. (1995) Involvement of glutamate 268
in the active site of human liver mitochondrial (class 2) aldehyde
(27) Ricagno, S., Jonsson, S., Richards, N., and Lindqvist, Y. (2003)
Formyl-CoA transferase encloses the CoA binding site at the interface
of an interlocked dimer. EMBO J. 22, 3210−3219.
(28) Wolodko, W. T., Fraser, M. E., James, M. N., and Bridger, W. A.
(1994) The crystal structure of succinyl-CoA synthetase from
Escherichia coli at 2.5-Å resolution. J. Biol. Chem. 269, 10883−10890.
(29) Liu, H., and Naismith, J. H. (2008) An efficient one-step site-
directed deletion, insertion, single and multiple-site plasmid muta-
genesis protocol. BMC Biotechnol. 8, 91.
4566
dx.doi.org/10.1021/bi300407y | Biochemistry 2012, 51, 4558−4567

Biochemistry
Article
(30) Bradford, M. M. (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal. Biochem. 72, 248−254.
(50) Blanco, J., Moore, R. A., Kabaleeswaran, V., and Viola, R. E.
(2003) A structural basis for the mechanism of aspartate-β-
semialdehyde dehydrogenase from Vibrio cholerae. Protein Sci. 12,
27−33.
(51) Skarzynski, T., Moody, P. C., and Wonacott, A. J. (1987)
Structure of holo-glyceraldehyde-3-phosphate dehydrogenase from
Bacillus stearothermophilus at 1.8 Å resolution. J. Mol. Biol. 193, 171−
187.
(31) Cornish-Bowden, A. (1995) Analysis of enzyme kinetic data,
Oxford University Press, New York.
(32) Baker, P., Hillis, C., Carere, J., and Seah, S. Y. (2012) Protein-
protein interactions and substrate channeling in orthologous and
chimeric aldolase-dehydrogenase complexes. Biochemistry 51, 1942−
1952.
(33) Holm, L., and Rosenstrom, P. (2010) Dali server: Conservation
mapping in 3D. Nucleic Acids Res. 38, W545−W549.
(52) Baker, P., and Seah, S. Y. (2012) Rational design of
stereoselectivity in the class II pyruvate aldolase BphI. J. Am. Chem.
Soc. 134, 507−513.
(34) Viola, R. E., Liu, X., Ohren, J. F., and Faehnle, C. R. (2008) The
structure of a redundant enzyme: A second isoform of aspartate β-
semialdehyde dehydrogenase in Vibrio cholerae. Acta Crystallogr. D64,
321−330.
(35) Charron, C., Talfournier, F., Isupov, M. N., Littlechild, J. A.,
Branlant, G., Vitoux, B., and Aubry, A. (2000) The crystal structure of
D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermo-
philic archaeon Methanothermus fervidus in the presence of NADP+ at
2.1 Å resolution. J. Mol. Biol. 297, 481−500.
(36) Smith, N. E., Vrielink, A., Attwood, P. V., and Corry, B. (2012)
Biological Channeling of a Reactive Intermediate in the Bifunctional
Enzyme DmpFG. Biophys. J. 102, 868−877.
(37) Kleywegt, G. J., Harris, M. R., Zou, J. Y., Taylor, T. C., Wahlby,
A., and Jones, T. A. (2004) The Uppsala Electron-Density Server. Acta
Crystallogr. D60, 2240−2249.
(38) Moras, D., Olsen, K. W., Sabesan, M. N., Buehner, M., Ford, G.
C., and Rossmann, M. G. (1975) Studies of asymmetry in the three-
dimensional structure of lobster D-glyceraldehyde-3-phosphate de-
hydrogenase. J. Biol. Chem. 250, 9137−9162.
(39) Blanco, J., Moore, R. A., and Viola, R. E. (2003) Capture of an
intermediate in the catalytic cycle of L-aspartate-β-semialdehyde
dehydrogenase. Proc. Natl. Acad. Sci. U.S.A. 100, 12613−12617.
(40) Blanco, J., Moore, R. A., Faehnle, C. R., and Viola, R. E. (2004)
Critical catalytic functional groups in the mechanism of aspartate-β-
semialdehyde dehydrogenase. Acta Crystallogr. D60, 1808−1815.
(41) Zhang, L., Ahvazi, B., Szittner, R., Vrielink, A., and Meighen, E.
(2000) A histidine residue in the catalytic mechanism distinguishes
Vibrio harveyi aldehyde dehydrogenase from other members of the
aldehyde dehydrogenase superfamily. Biochemistry 39, 14409−14418.
(42) Talfournier, F., Colloc’h, N., Mornon, J. P., and Branlant, G.
(1998) Comparative study of the catalytic domain of phosphorylating
glyceraldehyde-3-phosphate dehydrogenases from bacteria and archaea
via essential cysteine probes and site-directed mutagenesis. Eur. J.
Biochem. 252, 447−457.
(43) Stines-Chaumeil, C., Talfournier, F., and Branlant, G. (2006)
Mechanistic characterization of the MSDH (methylmalonate semi-
aldehyde dehydrogenase) from Bacillus subtilis. Biochem. J. 395, 107−
115.
(44) Marchal, S., Cobessi, D., Rahuel-Clermont, S., Tete-Favier, F.,
Aubry, A., and Branlant, G. (2001) Chemical mechanism and substrate
binding sites of NADP-dependent aldehyde dehydrogenase from
Streptococcus mutans. Chem.-Biol. Interact. 130−132, 15−28.
(45) Harris, J. I., and Waters, M. (1976) The Enzymes, 3rd ed.,
Academic Press, San Diego.
(46) Blanco, J., Moore, R. A., Faehnle, C. R., Coe, D. M., and Viola,
R. E. (2004) The role of substrate-binding groups in the mechanism of
aspartate-β-semialdehyde dehydrogenase. Acta Crystallogr. D60, 1388−
1395.
(47) Faehnle, C. R., Ohren, J. F., and Viola, R. E. (2005) A new
branch in the family: Structure of aspartate-β-semialdehyde dehydro-
genase from Methanococcus jannaschii. J. Mol. Biol. 353, 1055−1068.
(48) Ouyang, J., and Viola, R. E. (1995) Use of structural
comparisons to select mutagenic targets in aspartate-β-semialdehyde
dehydrogenase. Biochemistry 34, 6394−6399.
(49) Faehnle, C. R., Le Coq, J., Liu, X., and Viola, R. E. (2006)
Examination of key intermediates in the catalytic cycle of aspartate-β-
semialdehyde dehydrogenase from a Gram-positive infectious bacteria.
J. Biol. Chem. 281, 31031−31040.
4567
dx.doi.org/10.1021/bi300407y | Biochemistry 2012, 51, 4558−4567