Aldehyde dehydrogenase 2 activation and coevolution of its εPKC-mediated phosphorylation sites
Nene et al. Journal of Biomedical Science
Aldehyde dehydrogenase 2 activation and coevolution of its εPKC-mediated phosphorylation sites
Aishwarya Nene 0 1
Che-Hong Chen 0 1
Marie-Hélène Disatnik 1
Leslie Cruz 1
Daria Mochly-Rosen 1
0 Equal contributors Department of Chemical and Systems Biology, Stanford University, School of Medicine , Stanford, CA 94305-5174 , USA
1 Aishwarya Nene and Che-Hong Chen are 1st-coauthors of this publication
Background: Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a key enzyme for the metabolism of many toxic aldehydes such as acetaldehyde, derived from alcohol drinking, and 4HNE, an oxidative stress-derived lipid peroxidation aldehyde. Post-translational enhancement of ALDH2 activity can be achieved by serine/threonine phosphorylation by epsilon protein kinase C (εPKC). Elevated ALDH2 is beneficial in reducing injury following myocardial infarction, stroke and other oxidative stress and aldehyde toxicity-related diseases. We have previously identified three εPKC phosphorylation sites, threonine 185 (T185), serine 279 (S279) and threonine 412 (T412), on ALDH2. Here we further characterized the role and contribution of each phosphorylation site to the enhancement of enzymatic activity by εPKC. Methods: Each individual phosphorylation site was mutated to a negatively charged amino acid, glutamate, to mimic a phosphorylation, or to a non-phosphorylatable amino acid, alanine. ALDH2 enzyme activities and protection against 4HNE inactivation were measured in the presence or absence of εPKC phosphorylation in vitro. Coevolution of ALDH2 and its εPKC phosphorylation sites was delineated by multiple sequence alignments among a diverse range of species and within the ALDH multigene family. Results: We identified S279 as a critical εPKC phosphorylation site in the activation of ALDH2. The critical catalytic site, cysteine 302 (C302) of ALDH2 is susceptible to adduct formation by reactive aldehyde, 4HNE, which readily renders the enzyme inactive. We show that phosphomimetic mutations of T185E, S279E and T412E confer protection of ALDH2 against 4HNE-induced inactivation, indicating that phosphorylation on these three sites by εPKC likely also protects the enzyme against reactive aldehydes. Finally, we demonstrate that the three ALDH2 phosphorylation sites co-evolved with εPKC over a wide range of species. Alignment of 18 human ALDH isozymes, indicates that T185 and S279 are unique ALDH2, εPKC specific phosphorylation sites, while T412 is found in other ALDH isozymes. We further identified three highly conserved serine/threonine residues (T384, T433 and S471) in all 18 ALDH isozymes that may play an important phosphorylation-mediated regulatory role in this important family of detoxifying enzymes. Conclusion: εPKC phosphorylation and its coevolution with ALDH2 play an important role in the regulation and protection of ALDH2 enzyme activity.
ALDH2; Aldehyde dehydrogenase 2; εPKC; Phosphorylation; Coevolution; 4HNE
The mitochondrial aldehyde dehydrogenase 2, ALDH2,
is known for its role in ethanol metabolism, mediating
the rate-limiting step of metabolizing acetaldehyde to
acetic acid . However, this enzyme is also critical for
oxidation of fatty acid-derived aldehydes, such as
4hydrox-2-nonenal (4HNE) to non-electrophilic and
unreactive acids, 4-hydroxy-2-enoic acid (4HNA) [2, 3].
Therefore, ALDH2 plays a critical physiological role
both in the removal acetaldehyde derived from alcohol
drinking and the detoxification of lipid peroxidation
byproducts, 4HNE, under oxidative stress.
The functional ALDH2 is a homotetramer . In
human, a single point mutation in ALDH2 (E487K) greatly
reduces the enzyme’s activity [5–7]. This over-dominant
mutation, designated as ALDH2*2, is found in nearly
40% of East Asian populations, or approximately 560
million of the world population [8–10]. ALDH2*2
mutation leads to high levels of acetaldehydes accumulation
in the blood after ethanol consumption and causes the
well-known Asian Alcohol Flushing Syndrome [9, 11].
Because of the accumulation of acetaldehyde, a known
Group 1 carcinogen , the inactive variant of ALDH2*2
is associated with a much higher incidence of upper
aerodigestive track cancers as well as gastric, colorectal, lung,
and hepatocellular cancersc; a meta-analysis suggests up
to 80 fold higher incidence in heterozygotes who drink
more than 9 alcoholic beverages per week [9, 13–15].
The α,β-unsaturated reactive 4HNE is well-known for
its genotoxicity and cytotoxicity, causing DNA damage
and proteins inactivation [16–18]. 4HNE is reactive and
readily forms Michael’s adducts on the nucleophilic
amino acids, cysteine, histidine and lysine [19, 20]. Many
protein targets of 4HNE have been identified, including
both serum and cellular components, such as albumin
and histones, and cytoprotective proteins, critical protein
quality control, such as HSP70, and the 20S proteasome
[21, 22]. Since 4HNE is a product of lipid peroxidation
and the mitochondrial respiratory electron transport
chain is the major source of ROS, it is likely that many
of the mitochondrial proteins are susceptible to 4HNE
modification. Indeed, a notably large proportion of the
4HNE modified proteins that have been identified reside
in the mitochondria . These include critical proteins
in respiratory chain and energy metabolism, such as
aconitase, ATP synthase, many dehydrogenases in the Krebs
cycle and, importantly, ALDH2 itself [23, 24]. 4HNE is a
substrate of ALDH2, but is also a potent inhibitor of
ALDH2, since it can readily inactivate this enzyme by
adducting to critical cysteine residue in the catalytic
active site, cysteine 302 (Cys 302) [24, 25].
Inactivation of ALDH2 by its own substrate, 4HNE, therefore
could lead to further accumulation of 4HNE, which
has been observed in many pathological conditions
including neurodegenerative, ischemic and
inflammatory diseases [26–29].
Enhancing the catalytic activity of ALDH2 and/or
protecting ALDH2 enzyme activity from 4HNE-induced
inactivation has recently emerged as new strategy for the
development of therapeutics [26, 27]. Our lab has
identified small molecules activators of ALDH2 (e.g., Alda-1)
that increase the catalytic activity of the enzyme directly
and also protect ALDH2 from 4HNE substrate-induced
inactivation . X-ray co-crystal structure of Alda-1
and ALDH2 showed that Alda-1 is bound at the
substrate tunnel of ALDH2, close to cysteine 302, thus likely
shielding and preventing the thiol-group of this amino
acid from interacting with 4HNE . In the absence of
Alda-1, we showed that ALDH2 was rapidly inactivated
by 4HNE within minutes. Whereas in the presence of
Alda-1, ALDH2 remained catalytically active for an
extended period of time .
Another way to enhance ALDH2 activity is by
posttranslational phosphorylation of the enzyme. We
previously found that activation of epsilon protein kinase C
(εPKC) at the mitochondria increases ALDH2 activity in
the heart by ~40%, thus protecting the heart from
ischemic injury [31, 32]; phosphorylation of ALDH2 by εPKC
increases metabolism of toxic aldehydes, including 4HNE.
However, the molecular basis for
phosphorylationinduced activation of the enzyme is not known. Using
liquid chromatography and mass spectrometry analysis we
identified previously three possible εPKC-mediated
phosphorylation sites on ALDH2 (Chen et al., 2008 supporting
online material and Fig. 1). These are serine 279 (S279),
which lies at N-terminal end of helix that immediately
precedes the catalytic residue Cys 302, threonine 185
(T185), which lies in the loop between end of the first
helix in the enzyme, and threonine 412 (T412), which
lies at the N-terminus of an α-helix . However,
the importance of these phosphorylation sites for the
enzymatic activity and the role (if any) of
phosphorylation at these sites in protecting ALDH2 against
4HNE inactivation are not known.
Using site-directed mutagenesis of the three possible
εPKC phosphorylation sites, we set out to determine their
role in enzyme activity, phosphorylation, folding, and
resistance to 4HNE inactivation. We also explored the
conservation of these sites with εPKC in evolution, as a means to
demonstrate their importance in regulating ALDH2.
Enzyme activity assay for aldehyde dehydrogenase
Enzymatic activity of ALDH2 was determined
spectrophotometrically, using purified recombinant protein to
measure the reductive reaction of NAD+ to NADH at
λ340 nm. All the assays were carried out in a 96-well plate
in triplicates at 30 °C in 50 mM sodium pyrophosphate
Fig. 1 Structure of ALDH2 enzyme. a ALDH2 monomer displaying the three phosphorylation sites identified by LC-MS-MS: Thr185, Ser279, and
Thr412 (blue). Also highlighted are the catalytic Cys302 (green) and the site of the ALDH2*2 or Asian mutation: Glu487 (red). b Tetramer of an
active ALDH2 enzyme form. Thr185, Ser279 and Thr412 are marked in subunits A as in (a). The distance of between the two Ser279s on subunits
A and D is also indicated
buffer, pH = 8.8, 2.5 mM NAD+ and 10 mM acetaldehyde
as the substrate, as described . ALDH2 activities were
expressed as μmole NADH/min/μg protein from the
linear range of the assay. The amount of mutant ALDH2
recombinant protein in each sample was determined by
Bradford assays and quantitative western blots, using
commercial bovine serum albumin and a highly purified
wild type ALDH2 as a standard. Where indicated, 4HNE
(50 μM) was added at the start of the kinetic assays
immediately following the addition of acetaldehyde. All kinetic
assays were measured for sixty minutes.
Site-directed mutagenesis and purification of human
recombinant enzymes of ALDH2 wild type, ALDH2*2 and
T185, S279, T412 phosphorylation site mutants
Human recombinant ALDH2 wild type and ALDH2*2
mutant enzymes were expressed in bacteria as previously
described . The PKC-mediated three
phosphorylation sites identified previously by LC/MS/MS, Thr185,
Ser279 and Thr412 were mutated to glutamic acid, to
mimic phosphorylation  or to alanine, as a control.
For sited-directed mutagenesis, primers were designed
and mutations were introduced by AccuPrime™ Pfx
DNA polymerase kit for cloning and mutagenesis
according to manufacture protocol (Life Technologies;
catalog number 12344–024). ALDH2 wild type clone
was used as the PCR template. Primer sets used for each
site-directed mutagenesis are as following: T185A
GC), T185E (Forward: GCTGGGCCCAGCCTTGGCA
GAGGGAAACGTGGTTGTG; Reverse: CACAACCAC
TATGGAT; Reverse: ATCCATATCGGCATCTGCCAT
GATGATGTTGGGGC), S279E (Forward: AAGAGCC
GC; Reverse: GCCCAATCCATATCGGCATCCTCCAT
GATGATGTTGGGGCTCTT), T412A (Forward: CAGA
AGGATCTG), T412E (Forward: ATGCAGATCCT
CTTCAGGATCTGCAT). All the constructed human
ALDH2 wild type and mutants were designed to express a
recombinant protein with the His-tag at the N-terminus of
the protein using E. coli BL21 host cells and purified by His
GraviTrap nickel-affinity column (GE Healthcare Life
Sciences) as described previously .
Phosphorylation of ALDH2 recombinant proteins by εPKC
For in vitro kinase reaction, recombinant εPKC (100 ng,
Life Technologies, Grand Island, NY, USA) and each
ALDH2 protein (8 μg) were incubated in the presence of
20 mM Tris–HCl pH 7.5, 200 μM ATP, 20 mM MgCl2
with 0.24 mg/ml phosphatidylserine (Avanti, AL, USA),
0.04 mg/ml 1,3-s-n-dioleylglycerol (Avanti, Alabaster,
AL) at 37 °C for 30 min as described in Chen et al. .
Protein sequence, structural alignment and analysis
Sequences for members of the ALDH family and the
ALDH2 protein from multiple species were found
through the NCBI protein database (see Additional file 1).
The sequence alignment of ALDH2 proteins from
multiple species was determined by using the NCBI
Constraint-based Multiple Protein Alignment Tool
(COBALT). Structures of the different ALDH2 mutants were
modeled using UCSF Chimera by running a sequence
alignment to reduce the Root Mean Square Deviation.
Structural analyses were carried out to determine whether
the phosphomimetic mutations (T185E, S279E and
T412E) affect the protein structure. Each mutation was
introduced using the MOE (Molecular Operating
Environment) program. Following energy minimization, the protein
model was searched for areas where the mutated residue
would clash with other surrounding residues using the
UCSF Chimera program which searches for atoms that
have a Van der Waals radius overlap of 0.6 angstroms and
ignores contacts of pairs that are 2 or fewer bonds apart.
Amino acid sequence alignment of 18 human ALDH
19 different, functional ALDH genes are known in the
human genome . Since ALDH18A1 showed very low
degree of homology with the rest of the 18 ALDH
isozyme and has no conservation of T185, S279 and T412
at the equivalent positions, it was omitted from our
sequence alignment. Multiple sequence alignment was
conducted using online software ClustalW (http://
embnet.vital-it.ch/software/ClustalW.html) and ALDH
sequences with the following GenBank Accession
numbers: ALDH2 (GI: 48146099), ALDH1A1 (GI: 16306661),
ALDH1A2 (GI: 119597936), ALDH1A3 (GI: 153266822),
ALDH1B1 (GI: 119578656), ALDH1L1 (GI: 393195306),
ALDH1L2 (GI:166198355), ALDH3A1 (GI: 206597441),
ALDH3A2 (GI:73466520), ALDH3B1 (GI:125950429),
ALDH3B2 (GI: 73695881), ALDH4A1 (GI: 23271000),
ALDH5A1 (GI: 21708023), ALDH6A1 (GI: 119601566),
ALDH7A1 (GI: 49117277), ALDH8A1 (GI: 88683005),
ALDH9A1 (GI: 119611164), ALDH16A1 (GI: 223972651).
For longer sequences of ALDH isozymes, both N- and
Cterminal sequences were truncated and small sequence
gaps were introduced to obtain the best fitted alignment
against the published ALDH2 protein sequence.
The common East Asian ALDH2*2 single point
mutation (E487K), which is away from the catalytic site,
causes a >95% loss of activity in ALDH2 due to
structural changes that affect both the dimerization of the
enzyme and binding of the cofactor, NAD+ . To
determine whether phosphorylation causes a global
change in ALDH2 structure, in silico analysis of
structural models was carried out (Fig. 1a). Ser 279 lies on
the surface of the catalytic domain, near the
dimerdimer interface, between the A/B dimer and C/D dimer,
such that the residue is ~19 Å from its subunit related
Ser [A subunit and D subunit] (Fig. 1b). Ser 279 lies at
the N-terminal end of the helix that immediately
precedes the catalytic Cys (302) and is 27 Å from
Cys302. [For comparison, Glu487, which is mutated to
Lys in ALDH2*2, is 17 Å from Cys302.] Thr412, located
at the N-terminus of an α-helix, is only 10 Å from
Ser279 on the surface of the catalytic domain, though it
is further from the subunit interface. Finally, Thr185
residue is in the loop between the end of the first helix
and the beginning of the second strand in the Rossmann
coenzyme-binding fold [34, 35]. Thr185 is 9 Å from
Glu487, the mutated amino acid in ALDH2*2.
Therefore, Thr185 is adjacent to an area of the enzyme that is
known to affect activity and catalysis. Although it
appears buried, it is accessible to solvent if the C-terminal
residues contributed by a subunit in the opposing dimer
of the tetramer are displaced. Phosphorylation of Thr185
is predicted to preclude the binding of the C-terminal
carboxylate through electrostatic repulsion (Fig. 1).
We have reported previously that in vitro
phosphorylation of wild type ALDH2 recombinant protein increases
its enzymatic activity . We observed here an increase
of 70% the ALDH2 activity following phosphorylation by
recombinant εPKC (Fig. 2a). The effect of εPKC
phosphorylation on ALDH2*2 mutant enzyme was even
more pronounced, even though the ALDH2*2 mutant
enzyme had a much lower basal activity due to the
Glu487 substitution by Lys. As shown in Fig. 2a, we
observed that the enzymatic activity of the phosphorylated
ALDH2*2 is 270% of the non-phosphorylated ALDH2*2.
We set out to determine which of the phosphorylation
sites contributes to εPKC-mediated activation of ALDH2
enzymatic activity. Site-directed mutagenesis was carried
out for each of the putative εPKC phosphorylation sites,
Thr185, Ser279 and Thr412 on ALDH2. Since
phosphomimetic of an amino acid is a good estimation for the
function of phosphorylation, we first mutated the three
phosphorylation sites individually to a charged amino
acid residue, glutamate, to mimic the function of the
negatively charged phosphate group . We found that
all the single phosphomimetic ALDH2 mutants were less
active than the wild type ALDH, especially T185E.
Compared to the non-phosphorylated wild type ALDH2, the
T185E, S279E and T412E had only 14%, 68%, and 24%
of the wild type activity, respectively (Fig. 2b). A
structural model of the T185E mutant suggests that a
mutation to glutamate at position 185 will likely cause a
conformational change (Fig. 2c), as the glutamate
residue at that position appears to clash with the
surrounding amino acids, proline 181 and threonine 486. This
prediction is supported by replacing the glutamate
residue to an alanine residue. When the T185
phosphorylation site was mutated to alanine to serve as a
nonphosphorylatable control, the enzymatic activity of the
mutant enzyme was not affected as much as compared
with the T185E mutant. In this case, T185A retains 87%
Fig. 2 εPKC phosphorylation on wild type ALDH2, ALDH2*2 and Thr185, Ser279 and Thr412 mutant enzymes. a Increased activity for wild type
ALDH2 and ALDH2*2 mutant enzymes by εPKC phosphorylation. ALDH2 wild type (WT) and ALDH2*2 mutant enzyme activities were measured in the
absence or presence of εPKC. Enzyme activity was expressed in μmole NADH/min/μg recombinant protein (n = 3, **p < 0.001; bars represent the
mean ± SD). b Enzymatic active of the phosphomimetic ALDH2 site-directed mutants, T185E, S279E and T412E. Enzyme activity was expressed in
μmole NADH/min/μg recombinant protein (n = 3, *p < 0.05, **p < 0.001 vs. WT; bars represent the mean ± SD). c A structural analysis of the T185E
mutation reveals that a glutamate in the position of T185 would clash with the surrounding amino acids, proline 181 and threonine 486. d The effect
of εPKC phosphorylation on the phosphomimetic and non-phosphorylatable mutants of ALDH2. The graph displays the enzyme activity of T185, S279
and T412 mutants with or without the phosphorylation of εPKC (n = 3, *p < 0.05, **p < 0.001; bars represent the mean ± SD)
of the wild type ALDH2 activity (Fig. 2b). In contrast,
the S279A and T412A mutants showed a loss ~50%
(49% for S279A and 45% for T412A) of the wild type
activity (Fig. 2b). Interestingly, among the three
phosphomimetics, the S279E phosphomimetic was the only
mutant that had approximately 40% higher activity
relative to its S279A non-phosphorylatable mutant,
suggesting that S279 is probably a true allosteric site, capable of
increasing the catalytic activity of ALDH2 upon
phosphorylation. Similar to T185, either alanine or glutamate
substitution for T412 decreased the catalytic activity of
ALDH2. However, our structural modeling did not
indicate any clashes with surrounding amino acids for the
T412E substitution (Fig. 2c).
Next, we determined whether further activation of the
enzymatic activity could be achieved by εPKC
phosphorylation of each of the single phosphomimetic or
nonphosphorylatable alanine substitution mutants. We
reasoned that since further activation by εPKC
phosphorylation on the specific phosphomimetic or
nonphosphorylatable alanine substitution was no longer
possible, such experiments will help to identify the true
phosphorylation site(s) contributing the enhanced ALDH2
enzyme activity by εPKC. We found that five out of six
possible amino acid substitutions, T185A/E and T412A/E
and S279A mutants, were significantly activated by
εPKCmediated phosphorylation, resulting in an increase of
50–150% above their basal activity (Fig. 2d). The
phosphomimetic S279E mutant was clearly the only
exception; it was insensitive to further activation by
εPKCmediated phosphorylation. These data are consistent with
the observation above that phosphomimetic substitution,
S279E, was the mutation that imparted the highest
increased in ALDH2 activity without phosphorylation and
that S279 phosphorylation is the critical event in
εPKCmediated activation of ALDH2.
Because 4HNE causes a fast inactivation of ALDH2 by
adduct formation with the critical catalytic Cys302
[24, 36], we also determined whether phosphorylation
mimetic mutations protect the enzyme and affect the
sensitivity of ALDH2 to 4HNE-induced inactivation.
We showed that wild type ALDH2 enzyme activity
decreases rapidly by ~65% immediately after addition
of 50 μM 4HNE (Fig. 3). Compared to the wild type
ALDH2, the non-phosphorylatable mutants, T185A or
S279A were more sensitive to 4HNE inactivation and
lost 79% and 85% of their activity, respectively.
Surprisingly, the T412A mutation only lost 24% activity and was
more resistant to 4HNE inactivation than the wild type.
Importantly, the phosphomimetic mutations, T185E and
S279E, increased the resistance to 4HNE-induced
inactivation. Compared to a 65% decrease in the wild type,
ALDH2 activity of T185E and S279E mutant enzymes
showed only a decrease of 47% and 49%, respectively
(Fig. 3). On the other hand, although the phosphomimetic
T412E mutant was not as resistant to 4HNE-induced
inactivation as the T412A mutant, it conferred some
protection to ALDH2 after incubation with 4HNE with a 55%
reduction of activity as compared to the loss 65% observed
from the wild type ALDH2 (Fig. 3). The simplest
explanation for these results is that phosphorylation on
ALDH2 may induce a conformational change in the
enzyme structure thus allosterically protect Cys302
adduction by 4HNE.
We also used multiple sequence alignments to
determine whether the three ALDH2 phosphorylation sites
were conserved among species and co-evolved with
εPKC. We reasoned that if εPKC-mediated
phosphorylation of ALDH2 is critical for the regulation of ALDH2
activity, the critical phosphorylation sites should
coevolve with εPKC. We aligned multiple sequences of
ALDH2 from a wide range of eukaryotic species that
express εPKC, and compared ALDH2 sequence
conservation with species that do not express this protein kinase
(Additional files 1 and 2). Focusing on T185, S279 and
T412-equivalent phosphorylation positions in ALDH2,
we compared the conservation of the regions
corresponding to the phosphorylated site in 10 species that
express εPKC and 10 species that lack εPKC (Fig. 4,
Additional files 1 and 2). Remarkably, in the 10 species
that express εPKC, either a serine or a threonine was
invariably found at the three putative phosphorylation
sites in ALDH2 (Fig. 4, left columns). In contrast, in
the 10 species that lack εPKC, conservation of
phosphorylatable amino acids, T185, S279, T412 was
minimal (Fig. 4, right columns).
It is expected that in the absence of a kinase, if a
phosphorylation site is important for an enzymatic activity or
biological function, that position will be substituted for
by a negative amino acid (glutamate or aspartate) to
mimic phosphorylation . We found that, for ALDH2
T185, among the 10 species that did not express εPKC
Fig. 3 Sensitivity of the non-phosphorylatable and phosphomimetic ALDH2 mutants to 4HNE inactivation. Enzyme activity of each of the T185,
S279 and T412 single phosphorylation mutant (A or E) with or without incubation with 50 μM of 4HNE. All enzyme activities are presented as a
percentage of the no 4HNE treatment for each of the mutant
Fig. 4 Co-evolution of εPKC and phosphorylation residues in ALDH2. Shown is the amino acid (one-letter code) at the indicated position in
ALDH2 from 20 different evolutionarily diverged species. Each cell represents one species. The left column depicts the amino acids at that site for
the ten species that have εPKC. The right hand column depicts the amino acids at that corresponding site (determined by alignment of the
whole sequence) for the ten species that do not have εPKC. In both columns, the size of the amino acid represents the frequency of the given
amino acid at that site. Residues that can be phosphorylated by εPKC, serine and threonine, are colored in blue. Residues colored in red are
negative amino acids, thus mimicking the phosphorylated serine and threonine. Other amino acids are colored in black. For a list of the 20
species, their phylogenetic tree and their respective amino acid residues at corresponding T187, S279 and T412, see Additional files 1 and 2
only 1 of the 10 species had a negative amino acid. For
both S279 and T412, out of the 10 species that lacked
εPKC, half had a negative amino acid in the place of the
phosphorylation site. These data are consistent with the
idea that evolutionary conservation of a negatively charged
amino acid for a phosphorylatable serine/threonine in that
position indicates a functionally important residue for
activity. In addition, we also found that in several species
that did not express εPKC, serine or threonine was still
conserved. 2 of the 10 species that did not have εPKC
retained threonine at position T185, 1 of 10 species
retained serine at position S279 and 2 of 10 species
had a serine substitution at the equivalent for T412
position. These data suggest that in the absence of
εPKC, another serine/threonine protein kinase may
phosphorylate ALDH2 in these species.
We also aligned and compared amino acid sequences
of all 19 identified and functional ALDH isozymes
within the human genome and determined how the
positions equivalent to T185, S279 and T412 are conserved
among the human ALDH supergene family (Fig. 5). We
reasoned that such a comparison will reveal whether other
ALDH isozymes may also be regulated by phosphorylation
(perhaps even by εPKC-mediated phosphorylation) in a
similar fashion. Because ALDH18A1 showed very low
degree of homology with the rest of the 18 ALDH isozyme
and no conservation of an equivalent to T185, S279 and
T412 was found, it was omitted from this comparison.
Figure 5 depicts the best alignment of the remaining 18
human ALDH isozymes. We found that the equivalents of
either T185 or S279 of ALDH2 were preserved in only
one other ALDH isozyme each; ALDH1B1 has a
threonine at the equivalent position T185 and ALDH9A1 has a
serine at the equivalent position at S279. It is also
interesting to note that in 6 of the remaining 17 ALDHs, the S279
is substituted for with E or D, but none of the equivalent
of T185 substitution are negatively charged amino acid
mimetics. On the other hand, T412 had much higher
conservation in that 12 out of the 18 ALDH isozymes had
either a threonine or serine, and 2 members of the ALDH
family had a negatively charged amino acid, Asp, at the
equivalent position of T412
Finally, in contrast to the low degree of conservation
of T185 and S279, we found three other
serine/threonine sites that were highly conserved among all the 18
ALDH isozymes: T384 was conserved in 16 of the 18
ALDH isozymes, and T433 and S471 were conserved in
all 18 ALDH isozymes. These data suggest that these
three sites may be universal serine/threonine
phosphorylation sites for the ALDH super gene family. Note that
as a reference point for the accuracy of alignment, the
critical catalytic site, Cys 302, was found at the
equivalent position in 17 of the 18 ALDH isozymes, except for
the more divergent member of ALDH16A1.
It is well-established that post-translational modification
of proteins and enzyme can modulate the activity of
many enzymes, thus playing an important role in cellular
functions. Phosphorylation affects the activity of many
enzymes through increased interaction with a partner
protein [38, 39], inhibition of intramolecular interaction
[40, 41], decreased ability to be modified by
ubiquitination and subsequent degradation [42, 43] and/or through
altered access to the substrate [44, 45]. We previously
showed that ALDH2 is a substrate of εPKC and that
εPKC-mediated phosphorylation of ALDH2 leads to
enhanced catalytic activity towards oxidation of toxic
aldehydic substrate and confers cardioprotection
against ischemia-reperfusion injury . However,
nonenzymatic modification of ALDH2, in particular, on the
critical catalytic cysteine 302 residue also occurs by its
electrophilic and reactive aldehyde substrate, 4HNE .
In our previous studies, we demonstrated that a small
Fig. 5 (See legend on next page.)
(See figure on previous page.)
Fig. 5 Alignment of amino acid sequences of the 18 human ALDH isozymes. Amino acid sequences of 18 human ALDH isozymes were aligned
based on their sequence homology. For longer ALDH isozymes, both N- and C-terminal sequences were truncated to obtain the best fitted alignment
against the ALDH2 protein sequence from its amino acid residues 76 to 500 as marked (without the 17 a.a. N-terminal mitochondria targeting sequence).
Serine and threonine at positions T187, S279, T384, T412, T433 and S471 the conserved are denoted in red letters. The negatively charged amino acids, D
and E, are in blue. The conserved catalytic site, Cysteine 302 (C302) residues, are marked in green. For GenBank accession numbers of all ALDH isozymes,
molecular agonist of ALDH2, Alda-1, positioned at the
substrate tunnel near Cys302 could protect ALDH2 from
4HNE inactivation. Here, we determined whether εPKC
phosphorylation or phosphor-memetic of three serine/
threonine residues of ALDH2 (T185, S279 and T412)
mediate activation of the enzyme and/or protect ALDH2
from 4HNE inactivation.
Mutation of T185 to A did not affect ALDH2 activity
(Fig. 2b), and T185E (phosphomimetic mutation) resulted
in lower ALDH2 activity relative to wild type or T185A
mutant, suggesting a structural role of this residue, and/or
that T185 is a site that mediates phosphorylation-induced
inactivation of ALDH2 (Fig. 2b). T185A and T185E
mutants were also sensitive to 4HNE inactivation (Fig. 3), but
T185E may have a lower sensitivity, relative to T185A
(Fig. 3). Together, these data indicate that although T185
is relatively close to the catalytic site and may protect from
4HNE inactivation when negatively charged,
phosphorylation of T185 by εPKC is unlikely to mediate activation of
ALDH2. Furthermore, we find that T185 is conserved in
species that have εPKC and there is threonine in that
position in two species that lack εPKC, further supporting a
role of this amino acid in ALDH2 activity. However, its
role is not the same for other ALDH isozymes; S or T is
not found in any other 18 ALDH isozymes in humans in
the equivalent position of T185 (except for ALDH1B1),
and only one of the ten species that lack εPKC have
the expected phosphomimetic amino acid substitution,
which is predicted to make up for the lack of the kinase
(Figs. 4 and 5). Together, we conclude that if T185
phosphorylation in ALDH2 is mediated by εPKC, it does not
affect ALDH2 catalysis, but it may contribute to
protection of ALDH2 from 4HNE-induced inactivation.
Mutation of S279A and T412A each resulted in
enzyme with only 50% activity relative to the wild type
enzyme (Fig. 2b). Whether the loss of activity reflects a
structural defect or role for these two amino acids in
catalysis, per se, cannot be determined based on our study.
However, whereas mutation to a phosphomimetic E
(T412E) resulted in an enzyme with even lower activity
relative to T412A, S279E is more active relative to
S279A. These data suggest that S279 is the
phosphorylation site that mediates the increase in ALDH2 activity
by εPKC; indeed, S279E mutant was completely
insensitive to further activation by εPKC-mediated
phosphorylation (Fig. 2d).
So, what is the role of T412 phosphorylation? T412A
is greatly activated by εPKC-mediated phosphorylation
(2.5-fold increase in ALDH2 activity relative to
nonphosphorylated enzyme; Fig. 2d) and T412A mutant is
completely insensitive to 4HNE-induced inhibition of
ALDH2 (Fig. 3). We also find that T412 is highly
conserved in evolution; even among the species that lack
εPKC, 3/10 have S at that position and 5/7 of the
remaining species have a phosphomimetic D in that
position (Fig. 4, right panel). Finally, in 12 of the 18 other
ALDH isoforms in humans, the equivalent of T412 is
conserved and 2 of the remaining 6 have a
phosphomimetic D at that position. Together, these data suggest an
important regulatory role for T412; its phophsorylation
may inhibit 4HNE inactivation. Importantly, because the
T412E mutant was also less sensitive to εPKC-mediated
increase in ALDH2 activity, we conclude that T412
likely also contribute to εPKC-mediated activation of
ALDH2. The physical proximity of S279 and T412 in
ALDH2 (Fig. 1b) may also contribute to the role of these
two putative phosphorylation sites by the same protein
kinase, εPKC. We suggest that T412/S279, the two
neighboring amino acids on enzyme surface in 3D, are
allosteric sites that are protecting ALDH2 from 4HNE
inactivation, possibly by altering the structure of the
catalytic tunnel and the access of 4HNE to the channel.
The limitations of this in vitro study should be pointed
out. Since the first study by Thorsten and Koshland 
mutation of potential phosphorylation site to an amino
acid with a negative charge, to mimic phosphorylation,
has been used extensively. Furthermore, mutation of
amino acids to an alanine residue seems to be of
minimal structural consequences and is therefore often used
to identify the role of a particular amino acid; a loss of
function is taken to indicate that the particular amino
acid is required for that function. However, clearly, any
mutagenesis of proteins may have additional ‘gain of
function’ consequences due to problem in folding,
maturation and/or stability of the enzyme. Furthermore, as
all these proteins were expressed in bacteria, they were
missing additional co- and post-translational
modifications that may affect the activity of the enzyme. Relevant
to this point, we found that with one exceptions, all the
ALDH2 mutants had lower activity relative to wild type
enzyme and that, together with the work with
recombinant enzymes remain caveats of our study. Nevertheless,
we believe that this work provides the first evidence for
the role of particular sites in ALDH2 in responding to
εPKC-mediated phosphorylation and to 4HNE-induced
inhibition of the enzyme, through a mechanism termed
The co-evolution study strengthens our in vitro
observations. It was striking to observe that the three ALDH2
phosphorylation sites identified by εPKC appear to
coevolve well with this particular εPKC isozyme. Among
all the species that have εPKC, we found that all the
three phosphorylation sites were invariably conserved. It
implies that there was a strong selection to preserve
these three sites for εPKC phosphorylation. It is only in
the species where εPKC is absent or lost, these three
phosphorylation sites would begin to drift. This
coevolution was even more striking when we aligned all 19
known functional human ALDH isozymes to evaluate
the degree of conservation of these putative
phosphorylation sites within this supergene family. We found that
except for T412 position, which was conserved in 12/18
isozymes, T185 and S279 were unique to the ALDH2
isozyme and one addition isozyme each (ALDH1B1 for
T185 and ALDH9A1 for S279). This implies that the
coevolution relationship was uniquely maintained between
ALDH2 and εPKC and these three phosphorylation sites
may be preferentially regulated by εPKC. We also
identified three other serine/threonine residues, T384, T433
and S471 that were extremely well conserved across all
the ALDH gene family members. Based on the
alignment of 16 known ALDH sequences, Sheikh et al., also
identified T384 and S471 as critical conversed amino
acids . T384 is located close to the solvent surface
and binds to the carbonyl backbone of another
conserved amino acid Proline 383. Such interaction appears
to be critical for the stability of a local structure in all
ALDHs. S471, on the other hand, is located closer to the
catalytic tunnel and interacts with residues 269 and 270.
Site-directed mutagenesis indicated that mutation at this
position would affect the critical conversed general base,
Glu268, and dramatically reduced the enzyme activity.
Whether these three residues are preserved for ALDH
phosphorylation and or for structural effects remain to
Mitochondrial ALDH2 is a key detoxifying enzyme
guarding the integrity and health of this important
organelle . As most mammalian cells rely on oxidative
respiration for ATP production, mitochondrial lipid
bilayer is undoubtedly one of the major cellular sites
where lipid peroxidation-derived 4HNE is produced by
ROS generated from the electron transport chain .
The association between ALDH2, 4HNE accumulation
and human disease have been the subject of extensive
reviews in recent years [27, 48, 49]. The identification of
the sites that mediate εPKC -induced increase in ALDH2
activity to detoxify acetaldehyde, 4HNE and other toxic
aldehydes from food, environmental sources and normal
metabolism and protection from inactivation by its toxic
substrates, such as 4HNE, contributes to our
understanding how this mitochondrial enzyme is regulated by
signal transduction. We believe that improving
mitochondrial health via εPKC activation and its downstream
substrate, ALDH2, should be a viable strategy to confer
beneficial effects in a variety of human diseases . In
the context of human diseases that are associated with
ALDH2 activity or ALDH2 mutation, it will therefore be
worthwhile to explore in the future the role
εPKCmediated phosphorylation of ALDH2.
The role of three serine/threonine phosphorylation sites
by εPKC on ALDH2 were characterized. Site-directed
mutagenesis and in vitro phosphorylation revealed that
S279 was a critical εPKC phosphorylation site for the
activation of ALDH2. Whereas, phosphorylation of T185,
S279 and T412 conferred protection against reactive
aldehyde, 4HNE, inactivation of ALDH2. Alignment
across a wide range of diverse biological species and of
18 known human ALDH multigene family members
showed that the three phosphorylation sites co-evolved
tightly with species that expressed εPKC. Such alignment
also identified both unique and conserved
serine/threonine on ALDH2 and its isozymes. Our findings indicated
that εPKC phosphorylation and its coevolution with
ALDH2 played an important role in the regulation and
protection of ALDH2 enzyme activity.
Additional file 1: Phylogenetic Tree of the 20 species for ALDH2 and
εPKC coevolution comparison. Species in green letters are those with a
homology of εPKC. Species in red letters are those without a homology
of εPKC. (PDF 73 kb)
Additional file 2: The table shows the 10 species with εPKC (left panel),
the 10 species without εPKC (right panel) and their amino acid residues
at the three human ALDH2 phosphorylation sites, T185, S279 and T412.
(PDF 313 kb)
4HNE: 4-hydroxy-nonenal; ALDH2: Aldehyde dehydrogenase 2;
ALDH: Aldehyde sehydrogenase; C302: Cysteine 302; S279: Serine 279;
T185: Threonine 185; T412: Threonine 412; εPKC: Epsilon protein kinase C.
Availability of data and materials
All data generated or analyzed during this study are included in this
published article [and its Additional files 1 and 2].
AN, CHC, MHD, LC conducted original experiments and analyzed all the data
described in this manuscript. DMR conceived the original experimental
concepts and designed, participated in data analysis and discussion. AN, CHC
and DMR were responsible for the preparation of this manuscript. All authors
read and approved the final manuscript.
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