Oxidation of imidazole- and pyrazole-derived aldehydes by plant aldehyde dehydrogenases from the family 2 and 10

Article abstract of DOI:10.1016/j.cbi.2019.02.008

Plant cytosolic aldehyde dehydrogenases from family 2 (ALDH2s, EC 1.2.1.3) are non-specific enzymes and participate for example in the metabolism of acetaldehyde or biosynthesis of phenylpropanoids. Plant aminoaldehyde dehydrogenases (AMADHs, ALDH10 family, EC 1.2.1.19) are broadly specific and play an important role in polyamine degradation or production of osmoprotectants. We have tested imidazole and pyrazole carbaldehydes and their alkyl-, allyl-, benzyl-, phenyl-, pyrimidinyl- or thienyl-derivatives as possible substrates of plant ALDH2 and ALDH10 enzymes. Imidazole represents a building block of histidine, histamine as well as certain alkaloids. It also appears in synthetic pharmaceuticals such as imidazole antifungals. Biological compounds containing pyrazole are rare (e.g. pyrazole-1-alanine and pyrazofurin antibiotics) but the ring is often found as a constituent of many synthetic drugs and pesticides. The aim was to evaluate whether aldehyde compounds based on azole heterocycles are oxidized by the enzymes, which would further support their expected role as detoxifying aldehyde scavengers. The analyzed imidazole and pyrazole carbaldehydes were only slowly converted by ALDH10s but well oxidized by cytosolic maize ALDH2 isoforms (particularly by ALDH2C1). In the latter case, the respective K_m values were in the range of 10–2000 μmol l^?1; the k_cat values appeared mostly between 0.1 and 1.0 s^?1. The carbaldehyde group at the position 4 of imidazole was oxidized faster than that at the position 2. Such a difference was not observed for pyrazole carbaldehydes. Aldehydes with an aromatic substituent on their heterocyclic ring were oxidized faster than those with an aliphatic substituent. The most efficient of the tested substrates were comparable to benzaldehyde and p-anisaldehyde known as the best aromatic aldehyde substrates of plant cytosolic ALDH2s in vitro.

Full text of DOI:10.1016/j.cbi.2019.02.008

Accepted Manuscript
Oxidation of imidazole- and pyrazole-derived aldehydes by plant aldehyde
dehydrogenases from the family 2 and 10
Jan Frömmel, Radka Končitíková, David Kopečný, Miroslav Soural, Marek Šebela
PII:
S0009-2797(18)31226-2
https://doi.org/10.1016/j.cbi.2019.02.008
CBI 8541
DOI:
Reference:
To appear in: Chemico-Biological Interactions
Received Date: 14 September 2018
Revised Date: 20 December 2018
Accepted Date: 11 February 2019
Please cite this article as: J. Frömmel, R. Končitíková, D. Kopečný, M. Soural, M. Šebela, Oxidation of
imidazole- and pyrazole-derived aldehydes by plant aldehyde dehydrogenases from the family 2 and 10,
Chemico-Biological Interactions (2019), doi: https://doi.org/10.1016/j.cbi.2019.02.008.
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ACCEPTED MANUSCRIPT
Oxidation of imidazole- and pyrazole-derived aldehydes by plant aldehyde
dehydrogenases from the family 2 and 10
Jan Frömmel1), Radka Končitíková1), David Kopečný1) *, Miroslav Soural2) and Marek Šebela1)*
1) Department of Protein Biochemistry and Proteomics, Centre of the Region Haná for Biotechnological and
Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 27, 783 71 Olomouc, Czech Republic
2) Institute of Molecular and Translation Medicine, Faculty of Medicine and Dentistry, Palacký University,
Hněvotínská 5, 779 00 Olomouc, Czech Republic
*Corresponding authors: marek.sebela@upol.cz, kopecny_david@yahoo.co.uk
Tel.: +420 585634927 (M.S.), +420 585634840 (D.K.)
Keywords
Aldehyde dehydrogenase; aminoaldehyde dehydrogenase; imidazole; isoenzyme; pyrazole; substrate
Abbreviations
ALDH,
aldehyde
dehydrogenase;
AMADH,
aminoaldehyde
dehydrogenase;
APAL,
3
aminopropionaldehyde; abbreviations of heterocyclic aldehydes used in this study are completely listed
in Fig. 3 (ImCAL stands for imidazole carbaldehyde, PyrCAL for pyrazole carbaldehyde, Al- for allyl, Bn-
for benzyl, Bu- for Butyl, Et- for ethyl, Me- for methyl, Ph- for phenyl, Pm- for pyrimidinyl, Pr- for propyl,
and Th- for thienyl).
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Abstract
Plant cytosolic aldehyde dehydrogenases from family 2 (ALDH2s, EC 1.2.1.3) are non-specific enzymes
and participate for example in the metabolism of acetaldehyde or biosynthesis of phenylpropanoids.
Plant aminoaldehyde dehydrogenases (AMADHs, ALDH10 family, EC 1.2.1.19) are broadly specific and
play an important role in polyamine degradation or production of osmoprotectants. We have tested
imidazole and pyrazole carbaldehydes and their alkyl-, allyl-, benzyl-, phenyl-, pyrimidinyl- or thienyl-
derivatives as possible substrates of plant ALDH2 and ALDH10 enzymes. Imidazole represents a building
block of histidine, histamine as well as certain alkaloids. It also appears in synthetic pharmaceuticals
such as imidazole antifungals. Biological compounds containing pyrazole are rare (e.g. pyrazole-1-
alanine and pyrazofurin antibiotics) but the ring is often found as a constituent of many synthetic drugs
and pesticides. The aim was to evaluate whether aldehyde compounds based on azole heterocycles are
oxidized by the enzymes, which would further support their expected role as detoxifying aldehyde
scavengers. The analyzed imidazole and pyrazole carbaldehydes were only slowly converted by ALDH10s
but well oxidized by cytosolic maize ALDH2 isoforms (particularly by ALDH2C1). In the latter case, the
respective Km values were in the range of 10-2000 μmol l-1; the kcat values appeared mostly between 0.1
and 1.0 s-1. The carbaldehyde group at the position 4 of imidazole was oxidized faster than that at the
position 2. Such a difference was not observed for pyrazole carbaldehydes. Aldehydes with an aromatic
substituent on their heterocyclic ring were oxidized faster than those with an aliphatic substituent. The
most efficient of the tested substrates were comparable to benzaldehyde and p-anisaldehyde known as
the best aromatic aldehyde substrates of plant cytosolic ALDH2s in vitro.
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1. Introduction
Aldehyde dehydrogenases (ALDHs) represent a large superfamily of NAD(P)+ dependent enzymes
oxidizing aldehydes to the respective carboxylic acids. Because many aldehydes are reactive compounds
with toxic effects in higher concentration, ALDHs play an important detoxification role by aldehyde
oxidation apart from their participation in physiological metabolic pathways. ALDHs have been purified
from different kingdoms [1-3] and their genes found in many sequenced genomes [4,5]. The subcellular
localization of ALDHs is broad: they appear in the cytosol or mitochondria, endoplasmic reticulum,
nucleus, chloroplasts or peroxisomes [6]. ALDHs are currently divided into 27 families according to their
sequence identity [5]. Each family contains enzymes sharing at least 40 % of sequence identity, whereas
enzymes with more than 60 % of identity form a subfamily [4]. Interestingly, mitochondrial ALDH2s
share a high identity with ALDH1A1 (68 %) and ALDH1B1 (75 %) [7-9] but for historical reasons they are
classified in a separate family. Plant ALDHs are currently divided into 13 families: ALDH2, ALDH3, ALDH5,
ALDH6, ALDH7, ALDH10, ALDH11, ALDH12, ALDH18, ALDH21-24 [6].
The family 2 of ALDHs (EC 1.2.1.3) contains related non-specific plant and mammalian enzymes
localized in the cytosol or mitochondria [6,7]. Plant cytosolic ALDH2 isoenzymes (subfamily ALDH2C)
oxidize both aliphatic and aromatic aldehydes. These enzymes participate in ethanol detoxification
(conversion of acetaldehyde to acetate), pyruvate metabolism, and phenylalanine degradation as well as
in the production of phenylpropanoids (Fig. 1) [6, 10, 11]. Plant ALDH2 enzymes were originally
identified based on the genes that restored fertility in plants and grouped with other fertility-restorer
(Rf) genes. Hence maize ALDH2 family members have been described as RF2A, RF2B (both
mitochondrial), RF2C, RF2D, RF2E, RF2F (all four cytosolic) [11-13]. Beside many other aldehydes, maize
cytosolic ALDH2s used in this study oxidize also aldehydes containing a carbaldehyde group such as
benzaldehyde, hydroxybenzaldehydes, or anisaldehydes [11]. So far, no report has been published on
the oxidation of imidazole- or pyrazole-based aldehydes by plant cytosolic ALDH2s.
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Plant ALDH10s are known as aminoaldehyde dehydrogenases (AMADHs, EC 1.2.1.19), γ-
guanidinobutyraldehyde dehydrogenases (EC 1.2.1.54) and betaine aldehyde dehydrogenases (BADHs,
EC 1.2.1.8) due to their ability to oxidize ω-aminoaldehydes (especially 3-aminopropionaldehyde i.e.
APAL or 4-aminobutyraldehyde, γ-guanidinobutyraldehyde, and betaine aldehyde, respectively)[3, 14-
16]. This nomenclature is based on the most preferred substrate. Many AMADH sequences contain a
peroxisomal targeting signal at the C-terminus [15, 17]. AMADHs have a broad substrate specificity.
They are involved in many metabolic processes such as the catabolism of polyamines, amino acids or
choline. This is connected e.g. to the production of γ-aminobutyric acid, carnitine and osmoprotective
compounds such as β-alanine betaine, glycine betaine or dimethylsulfoniopropionate (Fig. 2) [18, 19].
Plant AMADHs also oxidize synthetic N-acyl-ω-aminoaldehydes, bromobenzaldehydes,
pyridinecarbaldehydes plus their halogen derivatives and other aldehydes containing a pyridine,
pyrimidine or purine ring [20-22]. On the other hand, their specificity towards carbaldehydes of azole
heterocycles has not yet been analyzed.
Imidazole is commonly present as a part of the histidine side chain, in its metabolite histamine or as a
constituent of plant alkaloids such as lepidilines from the garden cress (Lepidium sativum) and maca
(Lepidium meyenii) or pilocarpine from jaborandi (the Pilocarpus genus) leaves [23-25]. Also many
antifungal and antibacterial drugs such as miconazole, ketoconazole or ornidazole contain imidazole in
their structure [26-27].
Pyrazole is very rare in biological compounds. Pyrazole-1-alanine was isolated from water melon in
1960 [28]. Similarly to imidazole, also pyrazole is incorporated in the structure of many drugs with an
antibacterial, antitubercular, antifungal, antiviral, anticancer or neuroprotective activity such as 3-aryl-1-
methylpyrazoles, 1H-pyrazole-5-carbohydrazide hydrazones, sulfaphenazol or penthiopyrad [27, 29].
Substituted pyrazoles inhibit alcohol dehydrogenases by forming a complex with NAD+ and the zinc
atom at the active site [30].
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In this study, we tested the ability of plant ALDH2 and ALDH10 enzymes to oxidize imidazole and
pyrazole carbaldehydes and their derivatives with aliphatic and aromatic substituents. The activity assay
was based on a spectrophotometric monitoring of NADH production at 340 nm and the conversion of
the substrates was verified by liquid chromatography coupled to mass spectrometry (LC-MS).
2. Materials and methods.
2.1. Aldehydes
All fourteen tested aldehydes containing imidazole or pyrazole ring (Fig. 3) plus hexanal and APAL (in
the form of a diethylacetal) as reference substrates were purchased from Sigma-Aldrich. Most of the
compounds were solid, the others were liquid (1-Et-2-ImCAL, 1-Pr-2-ImCAL, 1-Al-3-Me-4-PyrCAL, 1-Al-3-
Me-4-PyrCAL and 1-Bn-4-PyrCAL). To make stock solutions during experiments, they were dissolved in 5-
20% acetonitrile to a concentration of 100 mmol l-1. The diethylacetal was converted to free APAL by
adding a calculated amount of 0.4 M HCl and incubating at 100 °C for 10 min freshly before the
measurement.
2.2. Enzymes
This study utilized four cytosolic ALDH2s from maize (Zea mays), namely RF2C, RF2D, RF2E and RF2F,
and five plant ALDH10s: three maize AMADHs (ZmAMADH1a, ZmAMADH1b, ZmAMADH2), one pea
(Pisum sativum) AMADH (PsAMADH2) and one tomato (Solanum lycopersicum) AMADH (SlAMADH1). All
nine enzymes were produced in Eschericha coli as recombinant proteins and purified as described
previously [11, 20, 21, 31].
In brief, transformed bacterial cells were grown in LB-medium (25 g l-1) containing streptomycin
(50 mg l-1) till OD600 ≈ 1.0. The expression was induced by adding isopropyl-β-D-1-thiogalactoside to a
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final concentration of 0.5 mmol l-1 and the culture was incubated at 20 °C overnight. ALDHs were
purified from cell lysates on HisPur Cobalt Spin Columns (Thermo Fisher Scientific) equilibrated with
20 mmol l-1 Tris-HCl buffer, pH 8.0, containing 300 mmol l-1 NaCl, 10 mmol l-1 imidazole and 5 % (v/v)
glycerol. A higher concentration of imidazole (250 mmol l-1) in the same buffer was used for elution. The
recombinant enzymes were concentrated and desalted using ultrafiltration with Amicon 30-kDa filters
(Merck). Enzyme concentration was determined by Bradford method [32]. Prior to storage at -80 °C, 5 %
glycerol was added as a stabilizer.
2.3. Activity assay
Activity measurements were based on spectrophotometric monitoring of NADH formation
(ε340 = 6,620 M-1 cm-1) at 30 °C in a UV-Vis spectrophotometer 8453 (Agilent) or detection of NADH
fluorescence emission at 460 nm upon excitation at 365 nm in a FluorolLog-3 spectrofluorometer
(Horiba). Fluorometric measurements were performed for aldehydes with absorbance maxima near
340 nm (2-Ph-4-ImCAL and 3-Th-4-PyrCAL). The reaction mixture contained 125 mmol l-1 Tris-HCl buffer,
pH 7.5, for RF2C, RF2D, RF2E and RF2F (or pH 9.0 for AMADHs), 1.0 mmol l-1 coenzyme NAD+ and an
appropriate amount of the enzyme. The reaction was started by the addition of aldehyde to a required
final concentration. A uniform aldehyde concentration of 1.0 mmol l-1 was used for substrate specificity
determinations by measuring relative reaction rates. Hexanal was a reference substrate for ALDH2
whereas APAL was a reference substrate for AMADH. For Km and V determinations, the substrate
concentration varied according to the needs. Data obtained from kinetic measurements were evaluated
using GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA, USA); substrate inhibition was
analyzed using the Haldane equation v = V·[S]/(Km+[S]·(1+[S]/Kss)), where Kss is the substrate inhibition
constant, according to previous examples of application [11,22].
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2.4. LC-MS analyses of reaction products
The reaction mixture for LC-MS analysis contained 125 mmol l-1 Tris-HCl buffer (pH 7.5 for ALDH2s
and pH 9.0 for ALDH10s), 1.5 mmol l-1 NAD+, 1.0 mmol l-1 aldehyde and an appropriate amount of the
enzyme. It was incubated at laboratory temperature for 24 h. After incubation, the enzyme was
removed by ultrafiltration using Amicon 30-kDa filters (Merck) and the filtrate analyzed.
All LC-MS analyses were carried out on a UHPLC chromatograph Acquity with photodiode array
detector coupled to a single quadrupole mass spectrometer (SQ Detector 2, Waters, Milford, MA, USA).
The separations were run on an X-Select CSH C18 column thermostated at 30 °C using a flow rate of 0.6
ml min-1. The mobile phases A and B were 10 mmol l-1 ammonium acetate and acetonitrile, respectively.
The gradient was linear from 20% to 80% B over 2.5 min, followed by 80% B for 1.5 min. The column was
re-equilibrated with 20% B for 1 min. The electrospray source operated at a discharge current of 5 μA,
vaporizer temperature of 350 °C and capillary temperature of 200 ºC.
3. Results and discussion
3.1. Substrate specificity
Table 1 provides a list of the enzymes, which were used in this study to perform enzyme kinetics
analyses with the respective nomenclature information, protein and gene accession numbers (in the
UniProt and GenBank databases, respectively), and measured specific activity values with reference
substrates. Although carbaldehydes of six-membered nitrogen-containing heterocycles are oxidized by
PsAMADH2 and SlAMADH1 at reasonable rates [20, 21], imidazole carbaldehydes, pyrazole
carbaldehydes and their derivatives are only weak substrates of all five AMADHs included in this study.
In a relative comparison at 1.0 mmol l-1 concentrations, the oxidation rate did not exceed 2% of that for
APAL, which is the best substrate of many AMADHs including PsAMADH2. On the other hand, cytosolic
ZmALDH2 (ALDH2C) isoforms oxidized the studied compounds much better and especially 1-Pm-4-
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ImCAL, 3-PyrCAL, 5-PyrCAL and 1-Bn-4-PyrCAL were oxidized at reaction rates above 10% of the rate for
the reference hexanal oxidation (Fig. 4). Moreover, this rate is comparable to those of benzaldehyde
and p-anisaldehyde known as the best aromatic aldehyde substrates of maize ALDH2C enzymes in vitro
[11]. The observed difference between ALDH2C isoforms and AMADHs probably resides in the absence
of certain aromatic and non-polar residues in the substrate cavity of ALDH10 family members. The F460
residue in RF2C (PDB ID: 4PXL) is replaced by C455 in ZmAMADH1a (PDB ID: 4I8P). This cysteine is almost
conserved across plant ALDH10. Exceptionally, serine (pea PsAMADH1) or threonine (tomato
SlAMADH1, barley HvAMADH1) residue can be found in a few AMADHs. There is a π-electron stacking
interaction possible in RF2C between the conserved aromatic residues F460 and Y171 and a nonpolar
substrate. In the case of ALDH10, the electronegative thiol group of C455 repels π electrons of substrate
aromatic ring (Fig. 5). In addition, there are two more polar residues, namely S297 and Q453 (in
ZmAMADH1), deeper in the cavity and below this cysteine that may interfere with aromatic or nonpolar
substrate moieties while forming the thiohemiacetal intermediate as deduced from the structure (PDB
ID: 4I9B) [11, 20, 31]. ALDH2 family members including RF2C and RF2F carry nonpolar valine and
phenylalanine residues, respectively, at the equivalent positions S297 and Q453, which make the
binding cavity highly nonpolar.
Interestingly, even if benzaldehyde, m-anisaldehyde and p-anisaldehyde are oxidized less efficiently
by RF2C than by the three other ZmALDH2 enzymes [11], it displays the broadest substrate specificity
for the five-membered heterocyclic aldehydes. RF2C converted 1-Pm-4-ImCAL and 3-PyrCAL at relative
reaction rates of 58% and 55%, respectively, compared with that for hexanal. Also 2-ImCAL, 4-ImCAL, 5-
PyrCAL and 1-Bn-4-PyrCAL were oxidized well by RF2C. The highest relative rate for oxidation by RF2D
was observed in the case of 4-ImCAL (11%) and by RF2E and RF2F for 1-Bn-4-PyrCAL (16% and 18%,
respectively) (Fig. 4).
According to the relative rates, 4-ImCAL was oxidized better than its isomer 2-ImCAL by all studied
enzymes. Both derivatives of 2-ImCAL (1-Et-2-ImCAL and 1-Pr-2-ImCAL) were oxidized more slowly than
the corresponding derivatives of 4-ImCAL (2-Me-4-ImCAL and 2-Bu-4-ImCAL). In a mutual comparison,
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very similar results were obtained for 3-PyrCAL and 5-PyrCAL in accordance with the existing
tautomerism of 3(5)-substituted pyrazoles [33]. The aldehydes with an aromatic substituent on the
imidazole or pyrazole ring were usually oxidized better than those with an aliphatic substituent. The
only exception was the sulfur-containing 3-Th-4-PyrCAL, which was a weak substrate of all enzymes.
3.2. Enzyme kinetics
For selected efficient substrates, we measured basic kinetic parameters of their oxidation (Km, V and,
if applicable, Kss from substrate inhibition curves) by the cytosolic ZmALDH2 isoforms (Fig. 6). The Km
values were in the range of 10-2000 μmol l-1 (Table 2) with only four values higher than 1000 μmol l-1:
for the oxidation of 1-Bn-4-PyrCAL by RF2E and 1-Al-3-Me-4-PyrCAL by RF2E and RF2F. The experimental
V values did not exceed 150 nmol s-1 mg-1 and were higher than 50 nmol s-1 mg-1 in a few cases e.g. for 1-
Pm-4-ImCAL, 3-PyrCAL, 5-PyrCAL and 1-Bn-4-PyrCAL in RF2C reactions. In Table 2, the respective
calculated kcat values are provided instead of V values to allow a better comparison with previous papers
on plant ALDH2Cs [11] and ALDH10s [22,31}. It is worth of mentioning that the specific activities
measured for hexanal in this study were 2-3 fold higher compared to those published by Končitíková et
al. [11]. The reason resides in using different enzyme preparations: freshly purified ALDH2C enzymes
(without freezing for storage) were first checked with hexanal as a reference substrate. In consequence,
the measured V (kcat) values were significantly higher and also the subtracted Km values were influenced
because of fitting slightly different experimental curves.
1-Pm-4-ImCAL was a better substrate of RF2C than the reference hexanal based on the kcat/Km ratio
of 1.38 (the value for hexanal was arbitrarily taken as 1.00) due to the low Km value (25.4 μmol l-1), even
though the respective kcat (7.8 s-1) was significantly lower compared to that for hexanal (12.6 s-1). The
kcat/Km ratio was high enough also for the oxidation of 2-Ph-4-ImCAL, 3-PyrCAL, 5-PyrCAL, 1-Al-3-Me-4-
PyrCAL and 1-Bn-4-PyrCAL by RF2C. The best substrate for RF2D among the tested compounds was 1-
Pm-4-ImCAL with a relative kcat/Km ratio of 0.23. Also 3-PyrCAL is an efficient RF2D substrate (relative
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kcat/Km ratio of 0.19). RF2E and RF2F oxidized 2-Ph-4-ImCAL with much higher relative kcat/Km ratios than
it was observed for any other tested heterocyclic aldehyde (0.79 and 0.46 respectively) followed by 1-
Bn-4-PyrCAL and 1-Pm-4-ImCAL. Our enzyme kinetics data clearly document that the substrates with an
aromatic substituent on the azole ring are better than those with an aliphatic substituent. Compared to
RF2C, both RF2E and RF2F displayed higher Km values (and likely lower affinity) for the tested
heterocyclic aldehydes although the previously described Km values for benzaldehyde and two
anisaldehydes with RF2E were lower than those of RF2C [11]. For example, the Km value for 1-Al-3-Me-4-
PyrCAL with RF2C to is more than 60 times lower than those with RF2E and RF2F for this substrate (26,
1870 and 1590 μmol l-1, respectively). In general, RF2C has lower Km values than RF2F for the same
substrates. This is probably caused by the replacement of the aromatic F460 in RF2C by M477 in RF2F
(PDB ID: 4PZ2) and loss of the aromatic ring of F178 (RF2C) and its replacement by V192 (RF2F) above
the catalytic cysteine. An additional shift aside of M477 in RF2F makes the substrate binding site much
wider and eliminates in this way a stacking interaction with heterocyclic aldehydes. Moreover, the
binding site in RF2F is slightly rearranged due to the absence of an aromatic residue at the position of
V192 as deduced from the positioning of solvent molecules in both crystal structures (Fig. 5). The Km
values of RF2C for 3-PyrCAL and 5-PyrCAL (97 and 131 μmol l-1) were found similar to that for
benzaldehyde (83 μmol l-1) [11] and lower than those for m- and p-anisaldehyde (203 and 299 μmol l-1)
[11]. Interestingly, the value for2-ImCAL was significantly higher (821 μmol l-1). Both RF2D and RF2E
displayed higher Km values for 3-PyrCAL and 5-PyrCAL (Table 2) than for benzaldehyde or both
anisaldehydes provided in ref. [11]. The Km values of RF2F for 3-PyrCAL (231 μmol l-1) and 5-PyrCAL
(371 μmol l-1) were found higher than that for benzaldehyde (146 μmol l-1) [11] and similar to that for m-
anisaldehyde (260 μmol l-1) [11].
An excess substrate inhibition of RF2C was observed for all tested aldehydes including hexanal, the
Kss values exceeded 1.0 mmol l-1 (Table 3). The other ALDH2C isoenzymes were inhibited in this way only
by specific aldehydes. In the case of RF2D, such an inhibition was observed for hexanal, 1-Pm-4-ImCAL
and 3-PyrCAL oxidation, but not for 4-ImCAL and 5-PyrCAL oxidation. Hexanal as well as 3-PyrCAL and 5-
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PyrCAL did not cause any substrate inhibition of RF2E or RF2F. On the other hand, RF2E and RF2F were
inhibited by high concentrations of 2-Ph-4-ImCAL, 1-Al-3-Me-4-PyrCAL and 1-Bn-4-PyrCAL. For 1-Pm-4-
ImCAL oxidation, excess substrate inhibition was observed with RF2E but not RF2F (Table 3).
3.3 LC-MS detection of carboxylic acids
The above enzyme kinetics data were based on the detection of NADH produced during the
enzymatic reaction, which employed the classical Warburg`s optical test [34] or a fluorometric detection
of NADH emission (λ = 460 nm) [35] upon excitation at 365 nm in the cases of 2-Ph-4-ImCAL and 3-Th-4-
PyrCAL. In order to verify the results of the spectroscopic assays, LC-MS analyses of the complete
reaction mixtures were performed after their incubating for 24 h.
As an example for illustration, the LC-MS output data for the reaction mixture of 2-Me-4-ImCAL and
RF2C are shown in Fig. 7. 2-Methyl-1H-imidazole-4-carboxylic acid (m/z 127) was produced and eluted at
1.07 min. The remaining aldehyde (m/z 111) was eluted at 0.86 min (not shown). Similarly, 1H-pyrazole-
3-carboxylic acid, 1H-pyrazole-5-carboxylic acid and 1H-imidazole-4-carboxylic acid (m/z 113 for all three
acids) as the oxidation products of 3-PyrCAL, 5-PyrCAL and 4-ImCAL conversion, respectively, were
detected.
3.4 The role of ALDH2s and ALDH10s in detoxification
In humans, the mitochondrial ALDH2 is ubiquitous. It is most abundant in the liver and also in organs
with a high level of oxidative phosphorylation (heart, brain) [36]. The enzyme is crucial for ethanol
metabolism as it efficiently converts acetaldehyde to acetate. It oxidizes short-chain aliphatic aldehydes
and aromatic aldehydes. Additionally, it has been shown to neutralize cytotoxic products of lipid
peroxidation, trans-4-hydroxy-2-nonenal or malondialdehyde, as well as endogenous/exogenous
acrolein [36,37]. Medium- and long-chain aliphatic aldehydes (including trans-4-hydroxy-2-nonenal) and
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aromatic aldehydes are oxidized also by ALDH3 [8,38]. Such a cytoprotective role has been anticipated
for the maize cytosolic ALDH2C isoforms based on their substrate specificity including aliphatic and
aromatic aldehydes, especially for ALDH2C1 (RF2C), which is highly expressed in all tissues for both
seedlings and developed plants [11]. This work is in line with the previous observations and provides
additional clues supporting a general detoxification and protective role of ALDH2. We show that the
enzymes might efficiently oxidize heterocycle-based aldehydes originating from endogenous
metabolism or conversion of xenobiotics.
Recent reports on numerous aromatic and heterocyclic aldehydes as substrates of plant ALDH10
enzymes [20,21] has now been extended by demonstrating their ability to oxidize azole-containing
aldehydes. As plant ALDH10s are localized in peroxisomes or in the cytosol (some isoforms lack the
presence of a peroxisomal targeting signal) [31,39], they could represent another protective shield in
the plant cell complementing the more dedicated and detoxifying ALDH2s.
4. Conclusions
Plant AMADHs are well known for oxidizing ω-aminoaldehydes, which represent products of amino
acid metabolism in prokaryotic as well as eukaryotic organisms. Nevertheless, recent studies have
shown their ability to catalyze the oxidation of aromatic and nitrogenous heterocyclic aldehydes [20,
21]. Now we have demonstrated that plant AMADHs oxidize slowly aldehydes with nitrogen-containing
five-membered heterocycles, namely those derived from imidazole and pyrazole. Moreover, many of
these aldehydes are good substrates of non-specific plant cytosolic ALDH2s (subfamily ALDH2C).
It seems that if similar heterocyclic aldehydes would appear as products of a natural metabolic
conversion or enter the plant cell as exogenous compounds, they could be eliminated by ALDH2 family
members. The broadest substrate specificity towards imidazole- and pyrazole-containing aldehydes was
observed for the maize RF2C isoenzyme. Especially 4-ImCAL, 2-Ph-4-ImCAL, 1-Pm-4-ImCAL, 3-PyrCAL/5-
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PyrCAL and 1-Bn-4-PyrCAL are oxidized well by maize ALDH2s. The aldehydes with an aromatic
substituent on the azole ring were usually oxidized better than those with an aliphatic substituent. The
only exception was the sulfur containing 3-Th-4-PyrCAL, which was a weak substrate of all enzymes.
Acknowledgements
This work was supported by the National Program of Sustainability I (LO1204) from the Ministry of
Education, Youth and Sports of the Czech Republic and by IGA_PrF_2017_016 (Palacký University,
Faculty of Science).
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Table 1. Nomenclature and database accession numbers of the studied ALDH2 and ALDH10 enzymes.
The nomenclature used is based on literature records [6,11,31]. The specific activity values were
measured with hexanal as a substrate for ALDH2s and APAL for ALDH10s.
Enzyme
(systematic
name)
Source
Alternative
names/aliases accession
number
UniProt
GenBank
accession
number
Specific activity
(nkat·mg-1)
ALDH2C1
ALDH2C2
ALDH2C4
ALDH2C5
ALDH10A8
ALDH10A9
ALDH10A5
ALDH10A8
Maize
(recombinant) RF2C
ZmALDH2-3,
W8T3P5
KJ004512
145
42
Maize
(recombinant) RF2D
ZmALDH2-4,
A0A0A0R8A4 KM225857
A0A0A0R570 KM225858
Maize
(recombinant) RF2E
ZmALDH2-5,
38
Maize
(recombinant) RF2F
ZmALDH2-6,
W8SZG1
KJ004513
GQ184593
JN635700
GQ184594
AJ315853
AY796114
15
Maize
(recombinant)
ZmAMADH1a C0P9J6
ZmAMADH1b G5DDC2
44
Maize
(recombinant)
33
Maize
(recombinant)
ZmAMADH2
PsAMADH2
SlAMADH1
C6KEM4
Q93YB2
Q56R04
69
Pea
(recombinant)
102
53
ALDH10A12 Tomato
(recombinant)
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Table 2. Basic kinetic parameters of the oxidation of imidazole- or pyrazole-containing aldehydes by
maize cytosolic ALDH2s. The measurements were performed in 125 mmol l-1 Tris-HCl buffer, pH 7.5,
containing 1.0 mmol l-1 NAD+. The symbol x denotes experiments in which the kinetic parameters could
not be measured due to a very slow aldehyde oxidation.
RF2C
RF2D
Aldehyde
Km
kcat
(s-1)
kcat/Km
Km
kcat
(s-1)
kcat/Km
(μmol l-1)
(rel.)
(μmol l-1)
(rel.)
Hexanal
56 ± 3
12.62 ± 0.30 1.000
1.05 ± 0.19 0.006
0.94 ± 0.06 0.012
0.28 ± 0.03 0.002
0.15 ± 0.02 0.002
0.12 ± 0.003 0.009
0.31 ± 0.01 0.039
7.77 ± 0.56 1.381
6.71 ± 0.28 0.307
3.39 ± 0.15 0.115
0.35 ± 0.01 0.060
0.26 ± 0.01 0.005
2.78 ± 0.12 0.537
RF2E
111 ± 7
2.86 ± 0.08 1.000
2-ImCAL
821 ± 73
354 ± 40
578 ± 103
415 ± 77
57 ± 4
x
x
x
4-ImCAL
212 ± 33
0.29 ± 0.01 0.053
2-Me-4-ImCAL
1-Et-2-ImCAL
2-Bu-4-ImCAL
2-Ph-4-ImCAL
1-Pm-4-ImCAL
3-PyrCAL
x
x
x
x
x
x
x
x
x
x
x
35 ± 3
x
25 ± 5
45 ± 4
0.27 ± 0.01 0.231
0.21 ± 0.01 0.192
0.15 ± 0.004 0.043
97 ± 8
42 ± 4
5-PyrCAL
131 ± 12
133 ± 13
1-Al-3-Me-4-PyrCAL 26 ±2
x
x
x
x
x
x
x
x
x
1-Al-5-Me-4-PyrCAL 236 ± 23
1-Bn-4-PyrCAL
23 ± 3
RF2F
Aldehyde
Km
kcat
(s-1)
kcat/Km
Km
kcat
kcat/Km
(rel.)
(μmol l-1)
276 ± 22
31 ± 3
(rel.)
(μmol l-1)
540 ± 56
73 ± 10
(s-1)
Hexanal
2.48 ± 0.06 1.000
0.22 ± 0.01 0.788
0.71 ± 0.09 0.085
0.10 ± 0.004 0.041
0.07 ± 0.002 0.035
0.62 ± 0.09 0.037
3.09 ± 1.58 0.267
1.27 ± 0.05
0.08 ± 0.01
1.000
0.457
2-Ph-4-ImCAL
1-Pm-4-ImCAL
3-PyrCAL
923 ± 81
269 ± 33
224 ± 19
351 ± 28
231 ± 21
371 ± 35
1594 ± 241
715 ± 124
0.10 ± 0.004 0.119
0.05 ± 0.001 0.090
0.07 ± 0.002 0.079
5-PyrCAL
1-Al-3-Me-4-PyrCAL 1866 ± 259
1-Bn-4-PyrCAL 1287 ± 244
0.16 ± 0.02
0.31 ± 0.04
0.042
0.181
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Table 3. Substrate inhibition of maize cytosolic ALDH2s reaction. The measurements were performed
in 125 mmol l-1 Tris-HCl buffer, pH 7.5, containing 1.0 mmol l-1 NAD+. The abbreviation n. i. indicates that
no substrate inhibition was observed; the symbol x denotes experiments in which the kinetic
parameters could not be measured due to a very slow aldehyde oxidation.
Kss (μmol l-1)
Aldehyde
RF2C
1889
1141
2936
2425
1013
6323
1332
1062
2666
6282
6860
6061
2552
RF2D
RF2E
n. i.
x
RF2F
n. i.
x
Hexanal
6385
2-ImCAL
x
4-ImCAL
n. i.
x
x
2-Me-4-ImCAL
1-Et-2-ImCAL
2-Bu-4-ImCAL
2-Ph-4-ImCAL
1-Pm-4-ImCAL
3-PyrCAL
x
x
x
x
x
x
x
x
x
x
1312
2674
n. i.
n. i.
2015
x
1457
n. i.
n. i.
n. i.
3678
x
1935
2606
n. i.
x
5-PyrCAL
1-Al-3-Me-4-PyrCAL
1-Al-5-Me-4-PyrCAL
1-Bn-4-PyrCAL
x
x
503
2709
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Figure legends
Fig. 1: Reactions catalyzed by plant ALDH2 enzymes.
Fig. 2: Reactions catalyzed by plant ALDH10 enzymes (AMADHs and BADHs).
Fig. 3: Pyrazole- and imidazole-containing aldehydes and the corresponding name abbreviations.
Chemical formulas were drawn using ChemSketch software.
Fig 4: Substrate specificity of ALDHs towards the tested heterocyclic aldehydes. The substrate and
coenzyme (NAD+) concentrations were 1.0 mmol l-1; the reaction rates are expressed as relative to those
for physiological substrates at the same concentration (hexanal for ALDH2s and APAL for AMADHs).
Fig 5: A comparison of the substrate cavity of ZmAMADH1a (gray), RF2C (blue) and RF2F (red).
Ethylene glycol is displayed at the bottom (EDO; in gray for ZmAMADH1a and RF2C, in red for RF2F). The
catalytic cysteine is not labeled for clarity: C303 (RF2C), C296 (ZmAMADH1), C317 (RF2F).
Fig 6: Saturation curves for activity determination of maize cytosolic ALDH2s. The measurements were
performed in 125 mmol l-1 Tris-HCl buffer, pH 7.5, containing 1.0 mmol l-1 NAD+.
Fig 7: LC-MS analysis of the reaction mixture of RF2C and 2-Me-4-ImCAL. Main panel: LC separation of
the reaction mixture (a photodiode array detector chromatogram). Inset: A mass spectrum referring to
the peak eluted at 1.07 min.
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Figure 1

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CHEMBIOINT_2018_1121
Oxidation of imidazole- and pyrazole-derived aldehydes by plant aldehyde dehydrogenases from the
family 2 and 10
by Jan Frömmel, Radka Končitíková, David Kopečný, Miroslav Soural and Marek Šebela
Highlights:
•
•
•
•
Plant aldehyde dehydrogenases (family 2 and 10) were analyzed by enzyme kinetics
Commercial imidazole- and pyrazole-derived aldehydes were tested as substrates
These substrates were well oxidized particularly by maize ALDH2C1 (RF2C)
Cytosolic ALDH2C enzymes have protective and detoxification role in the plant cell