Direct comparison of the enzymatic characteristics and superoxide production of the four aldehyde oxidase enzymes present in mouse

Article abstract of DOI:10.1124/dmd.117.075937

Aldehyde oxidases (AOXs) are molybdoflavoenzymes with an important role in the metabolism and detoxification of heterocyclic compounds and aliphatic as well as aromatic aldehydes. The enzymes use oxygen as the terminal electron acceptor and produce reduced oxygen species during turnover. Four different enzymes, mAOX1, mAOX3, mAOX4, and mAOX2, which are the products of distinct genes, are present in the mouse. A direct and simultaneous comparison of the enzymatic properties and characteristics of the four enzymes has never been performed. In this report, the four catalytically active mAOX enzymes were purified after heterologous expression in Escherichia coli. The kinetic parameters of the four mouse AOX enzymes were determined and compared with the use of six predicted substrates of physiologic and toxicological interest, i.e., retinaldehyde, N¹-methylnicotinamide, pyridoxal, vanillin, 4-(dimethylamino)cinnamaldehyde (p-DMAC), and salicylaldehyde. While retinaldehyde, vanillin, p-DMAC, and salycilaldehyde are efficient substrates for the four mouse AOX enzymes, N¹-methylnicotinamide is not a substrate of mAOX1 or mAOX4, and pyridoxal is not metabolized by any of the purified enzymes. Overall, mAOX1, mAOX2, mAOX3, and mAOX4 are characterized by significantly different K_M and k_cat values for the active substrates. The four mouse AOXs are also characterized by quantitative differences in their ability to produce superoxide radicals. With respect to this last point, mAOX2 is the enzyme generating the largest rate of superoxide radicals of around 40% in relation to moles of substrate converted, and mAOX1, the homolog to the human enzyme, produces a rate of approximately 30% of superoxide radicals with the same substrate.

Full text of DOI:10.1124/dmd.117.075937

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Direct comparison of the enzymatic characteristics and superoxide
production of the four aldehyde oxidase enzymes present in mouse
Gökhan Kücükgöze, Mineko Terao, Enrico Garattini, and Silke Leimkühler
Institut für Biochemie and Biologie (G.K. and S.L.), Universität Potsdam, Potsdam,
Germany.
Laboratory of Molecular Biology (M.T and E.G.), Department of Biochemistry and
Molecular Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri, via La Masa 19,
I-20156 Milano, Italy.
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Running Title: Characterization of aldehyde oxidase enzymes from mouse
Corresponding author:
Silke Leimkühler, Tel: +49-331-977-5603, Fax: +49-331-977-5128,
E-mail: sleim@uni-potsdam.de
Text Pages (including references): 31
Tables: 3
Figures: 5
References: 40
Abstract: 249
Introduction: 761
Discussion: 1953
Abbreviations: AOX1-4: aldehyde oxidase 1-4; AO: aldehyde oxidase; XOR: xanthine
oxidoreductase; XO: xanthine oxidase; XDH: xanthine dehydrogenase; Moco: molybdopterin
cofactor; E. coli: Escherichia coli; DTT: dithiotreitol; 4-(dimethylamino)cinnamaldehyde: p-
DMAC.
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Abstract
Aldehyde oxidases (AOXs) are molybdoflavoenzymes with an important role in the
metabolism and detoxification of heterocyclic compounds and aliphatic as well as aromatic
aldehydes. The enzymes use oxygen as the terminal electron acceptor and produce reduced
oxygen species during turnover. Four different enzymes, mAOX1, mAOX3, mAOX4 and
mAOX2, which are the products of distinct genes, are present in the mouse. A direct and
simultaneous comparison of the enzymatic properties and characteristics of the four enzymes
has never been performed. In this report, the four catalytically active mAOX enzymes were
purified after heterologous expression in Escherichia coli. The kinetic parameters of the four
mouse AOX enzymes were determined and compared with the use of six predicted substrates
of physiological and toxicological interest, i.e. retinaldehyde, N1-methylnicotinamide,
pyridoxal, vanillin, 4-(dimethylamino)cinnamaldehyde (p-DMAC) and salicyladehyde. While
retinaldehyde, vanillin, p-DMAC and salycilaldehyde are efficient substrates for the four
mouse AOX enzymes, N1-methylnicotinamide is not a substrate of mAOX1 or mAOX4, and
pyridoxal is not metabolized by any of the purified enzymes. Overall, mAOX1, mAOX2,
mAOX3 and mAOX4 are characterized by significantly different Km and kcat values for the
active substrates. The four mouse AOXs are also characterized by quantitative differences in
their ability to produce superoxide radicals. With respect to this last point, mAOX2 is the
enzyme generating the largest rate of superoxide radicals of around 40% in relation to mol of
substrate converted, while mAOX1 the homologue to the human enzyme, produces a rate of
approximately 30% of superoxide radicals with the same substrate.
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INTRODUCTION
Aldehyde oxidases (AOX; EC 1.2.3.1) are molybdo-flavoenzymes present in the
cytosol of various tissues and animal species (Garattini et al., 2008). The number of active
AOX genes varies according to the animal species considered. While a single AOX protein is
known in humans, at least four AOX enzymes (AOX1, AOX3, AOX4 and AOX2, Figure 1)
encoded by distinct genes have been described for the mouse and other rodents including rats
(Garattini et al., 2009). The four mouse Aox genes cluster on chromosome 1 at a short
distance from one another (Figure 1). The human genome is characterized by an active gene
homologous to mouse Aox1 and two inactive pseudogenes (Garattini et al., 2008).
The biochemical and physiological functions of mammalian AOXs are still unclear
(Garattini et al., 2008). Nevertheless, human AOX1 is an enzyme of emerging relevance for
phase I drug metabolism (Garattini and Terao, 2012; Kitamura and Sugihara, 2014). Indeed,
the number of xenobiotics acting as human AOX1 substrates is increasing. In particular, the
current trend in drug development, focusing on the design and synthesis of molecules that are
not recognized and inactivated by cytochrome P-450 dependent monoxygenases, has resulted
in an enrichment of molecules which can be oxidized by AOXs (Pryde et al., 2010; Garattini
and Terao, 2012). Very little is known about the physiological substrates of human AOX1
and other mammalian AOXs. AOXs are deemed to be involved in the oxidation of all-trans
retinal into all-trans-retinoic acid, the active metabolite of vitamin A, and are believed to
participate in vitamin B6 (pyridoxal) metabolism (Stanulović and Chaykin, 1971; Terao et
al., 2009).
While the expression of human AOX1 is relatively ubiquitous, the four mouse AOX
enzymes are expressed in a tissue-specific manner (Garattini et al., 2003). The two enzymes,
mAOX1 and mAOX3, have largely overlapping expression profiles and the highest levels of
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the two proteins are observed in liver, lung, and testis (Vila et al., 2004). Significant amounts
of mAOX4 are detectable in the skin, the epithelium lining the oral cavity, the esophagus and
the Harderian gland, the major exocrine gland of the orbital cavity in many vertebrates.
Knock-out of the gene coding for mAOX4 causes perturbations in the circadian rhythm and
resistance to diet-induced obesity (Terao et al., 2016). The expression of mAOX2 is restricted
to the nasal cavity. High levels of the corresponding transcript and protein are detectable
especially in Bowman’s gland and olfactory mucosa (Terao et al., 2000). It has been
suggested that the different mouse aldehyde oxidase enzymes have different substrate
specificities. So far, mAOX3 is the only mouse AOX for which a crystal structure is available
(PDB ID: 3ZYV) (Coelho et al., 2012), while the crystal structure of human AOX1 has
recently been solved (Coelho et al., 2015). The structures were very similar in respect to their
active sites, which suggested an overlapping spectrum of substrates. Comparisons between
the crystal structures of hAOX1 and mAOX3 demonstrated differences between the two
enzymes in the position of one loop at the FAD site, the site of oxygen reduction. The loop
close to the FAD pocket (T1230RGPDQ1235) of hAOX1 is flipped by almost 180° relative to
the mAOX3 counterpart.
AOXs are cytosolic proteins and use molecular oxygen as the sole electron acceptor.
During substrate oxidation and molecular oxygen reduction, AOXs generate significant
.-
amounts of H2O2 in addition to toxic O2 radicals. This specific aspect of AOXs activity has
been largely overlooked and may be of patho-physiological interest. In fact, significant levels
.-
of AOX activity are present in different tissues and AOX-dependent O2 production may
contribute to damage by reactive oxygen species in vivo. Given these reasons and their
potential relevance for the human enzyme, it is interesting to evaluate whether there are
.-
differences in the generation of O2 among the four mouse AOX enzymes.
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In this study, we set up and optimized an efficient system for the heterologous
expression of the four mouse AOX enzymes in Escherichia coli. We used this system to
purify significant amounts of highly purified and active mAOX1, mAOX3, mAOX4 and
mAOX2. We compared the kinetic and spectroscopic properties of the four enzymes using a
selected number of potential substrates. In addition, we determined the levels of superoxide
anions generated by each enzyme. Our data reveal significant differences in the enzymatic
characteristics and substrate specificities of the mouse AOX enzymes. This is of relevance for
the use of mouse as an animal model in the context of drug discovery programs. The results
presented support the idea that mice and other rodents are unlikely to represent suitable
models for drug metabolism predictions in humans.
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MATERIALS AND METHODS
Cloning
The expression vector for mAOX4 was constructed using primers that allowed cloning into
the NdeI and SalI restriction sites of the bacterial expression vector pTrcHis after PCR
amplification of the full length mAox4 cDNA obtained from the Harderian gland of
C57BL/6N mice. The resulting plasmid was designated pMAOX4 and expresses the mAOX4
protein with an N-terminal His6-tag. For the expression of mAOX3, we used the plasmid
pMMA1 (Mahro et al., 2011). Codon-optimized genes for mAOX1 and mAOX2 were
synthesized (GeneArt, Thermo Fisher Scientific, Sunnyvale, CA). They contain the DNA
sequences of each gene with codons preferred by E. coli, but do not result in a change of the
amino acid sequence of the synthesized proteins. The mAox1 and mAox2 synthetic genes
were cloned into the NdeI and SalI restriction sites of the pTrcHis expression vector and the
resulting constructs were designated pMAOX1co and pMAOX2co.
Expression and Purification
For the heterologous expression of mAOX enzymes in E. coli, pMAOX4, pMAOX1co and
pMAOX2co were transformed into TP1000 (∆mobAB) cells (Palmer et al., 1996). Cultures
were grown at 30°C and 130 rpm, using 2 Liter flasks containing 1 Liter of LB-peptone
supplemented with 150 µg/ml ampicillin, 1 mM Na2MoO4 and 20 mM isopropyl β-D-
thiogalactoside (IPTG). Cells were harvested after 24 hours of growth by centrifugation at
5000g for 5 minutes and suspended in 50 mM sodium phosphate buffer pH 8.0, containing
300 mM NaCl. Two cycles of cell lysis were performed at 12°C and 1.35 kbar (TS Benchtop
Series Constant Systems, Northampton, UK). After the first cycle, cell lysates were treated
with 1 mg/ml DNase I for 5 min. At the end of the process, cell lysates were cleared by
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centrifugation at 18000 g for 1 h at 4°C. The supernatants were loaded onto Ni-NTA
(QIAGEN, Valencia, CA) columns using 0.3 ml of resin per liter of culture. The matrix was
washed with 20 column volumes of the following two buffers consecutively: 1) 50 mM
sodium phosphate pH.8.0, 300 mM NaCl and 10 mM imidazole; 2) 50 mM sodium phosphate
pH.8.0, 300 mM NaCl and 20 mM imidazole. Proteins were eluted with 50 mM sodium
phosphate pH.8.0, 300 mM NaCl and 250 mM imidazole. For the further purification steps,
the elution buffer was exchanged to 50 mM Tris-HCl, 200 mM NaCl and 1 mM EDTA (pH
8.0) with the use of PD-10 columns (GE Healthcare, Chalfont St. Giles, Buckinghamshire,
UK). The purified enzymes were concentrated by ultrafiltration (Amicon 50-kDa MW cut-
off; Milipore Corporation, Billerica, MA). This was followed by size exclusion
chromatography on Superdex 200 columns (GE Healthcare) equilibrated in 50 mM Tris-HCl,
200 mM NaCl and 1 mM EDTA (pH 8.0). Each fraction was analyzed by SDS-
polyacrylamide gel electrophoresis (PAGE) and only the fractions containing dimeric AOX
proteins were combined. Purified proteins were stored at -80 °C until further use.
Chemical in vitro sulfuration
Purified enzymes were subjected to an in vitro chemical sulfuration step using a slight
modification of a previously described protocol (Wahl and Rajagopalan, 1982). Proteins
dissolved in buffer (50 mM Tris-HCl, pH 8.0, containing 200 mM NaCl and 1 mM EDTA)
were incubated in an anaerobic chamber (Coy Laboratory Products, Grass Lake, MI) with
sodium sulfide (2 mM) as the sulfur source and sodium dithionite (1 mM) to facilitate
reduction. Methyl viologen (25 µM) was used as an anaerobic indicator of the reaction. After
1.5 h incubation at 4°C, reactions were stopped and the enzyme was equilibrated in 50 mM
Tris-HCl, 200 mM NaCl and 1 mM EDTA (pH 8.0) using a PD-10 column.
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Polyacrylamide Gel Electrophoresis
SDS-polyacrylamide gel electrophoresis (PAGE) was performed with 10% gels under
reducing conditions (β-mercaptoethanol) using a standard protocol (Laemmli, 1970). Gels
were stained with Coomassie Brilliant Blue R (Sigma, Munich, Germany). The molecular
weight of the separated proteins was determined by comparison with a protein standard
(molecular weight marker, MWM; Fermentas/Thermo Fisher Scientific).
Native-PAGE was performed with 7% gels under non-reducing and non-denaturing
conditions. Samples were separated at a constant voltage of 40 V. Gels were stained with
Coomassie Brilliant Blue R. The in-gel enzymatic activity was visualized by staining with
500 µM substrate (phthalazine or pyridoxal-5-monophosphate) and 1 mM Nitro blue
tetrazolium chloride (NBT) in 50 mM Tris buffer.
Ultraviolet-Visible Spectroscopy Absorption Spectroscopy
UV-visible absorption spectra of enzymes were recorded in 500 µl quartz cuvettes using a
Shimadzu UV-2600 spectrophotometer (Somerset, NJ). The specific molar extinction
coefficient of 21000 M-1cm-1 at 450 nm was used to determine the concentration of proteins.
NADH reduction was achieved by the addition of 1 mM NADH. Spectra were recorded
under anaerobic conditions to avoid the re-oxidation of enzymes by oxygen.
Steady-state Kinetics
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Steady state kinetics of the purified enzymes were measured in 800 µl of 50 mM Tris-HCl
buffer (pH 8.0) containing 1 mM EDTA at 37 °C. The substrates pyridoxal 5-phosphate
(converted to pyridoxic acid), N1-methylnicotinamide (converted to 2-pyridone and 4-
pyridone (Stanulović and Chaykin, 1971), salicylaldehyde (converted to salicylic acid), 4-
(dimethylamino)cinnamaldehyde (p-DMAC) (converted to 4-(dimethylamino)cinnamic acid),
vanillin (converted to vanillic acid) and retinaldehyde (converted to retinoic acid), were used
in a concentration range around the Km of each substrate (the substrate and product of each
reaction are shown in the Supplemental Figure S1). 2,6 dichlorophenolindophenol (DCPIP) at
a concentration of 100 µM was used as the electron acceptor, which is converted to the
colorless DCPIPH2 leuco-form in a two-electron reduction step. The total enzyme
concentration varied between 0.05 and 0.2 µM, depending on the AOX enzyme used.
Enzyme activity was monitored at 600 nm with a computer-assisted spectrophotometer
(Shimadzu UV-2600) coupled to a temperature controlled cell holder (Shimadzu TCC-240A).
Enzymatic activities were converted to turnover number (kcat) using the molar extinction
coefficient of 16100 M-1cm-1 for oxidized DCPIP. Three individual measurements were
performed for each substrate concentration. Kinetic parameters were obtained by nonlinear
fitting of the Michaelis-Menten equation. All fitting analyses were performed with the
OriginPro 8.1G software (OriginLab, Northampton, MA).
Superoxide Production
The amount of superoxide produced during the enzymatic reactions was measured by
monitoring the reduction of cytochrome c (Sigma-Aldrich) at 550 nm and 37°C. Reaction
mixtures (800 µl) contained 100 µM cytochrome c and 50 µM substrate in 50 mM Tris-HCl
buffer (pH 8.0), 1 mM EDTA. The enzyme concentrations varied between 0.05 - 0.1 µM
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depending on the substrates (p-DMAC or vanillin) used. Enzymes were added to the mixture
to initiate the reaction and the absorbance change per min-1 was recorded with a
spectrophotometer (Shimadzu UV-2600). Superoxide formation was calculated using an
extinction coefficient of 21000 M-1cm-1 for cytochrome c. AOX activity with p-DMAC and
vanillin was determined by following the direct reduction of substrates at 398 and 347 nm,
respectively, in the presence of oxygen. The extinction coefficients of 30500 M-1cm-1 (p-
DMAC) and 25100 M-1cm-1 (vanillin) were used to convert the absorbance changes to units
of enzyme activity.
Quantification of molybdenum and iron
The molybdenum and iron contents of the purified AOX enzymes were determined by
inductively coupled plasma optical emission spectrometry (ICP-OES) with an Optima 2100
DV (PerkinElmer Life and Analytical Sciences, Waltham, MA). 500 µl of protein samples at
a final concentration of 5-10 µM were wet-ashed in an equal volume of 65% nitric acid by
incubation at 100°C overnight. The samples were diluted with 4 ml of distilled water. A
multielement standard solution with known accuracy (standard solution XVI; Merck
Millipore, Darmstadt, Germany) was used for the calibration. Measurements were performed
in triplicate for all samples.
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RESULTS
Purification of the mAOX enzymes from E. coli.
To compare the spectroscopic and kinetic properties of mAOX1, mAOX3, mAOX4
and mAOX2, we expressed the four mouse enzymes in a heterologous expression system in
E. coli. Efficient expression of the recombinant mAOX3 protein in E. coli cells has been
described previously (Schumann et al., 2009; Mahro et al., 2011) and has resulted in a
calculated yield of 0.8 mg/Liter (Table 1). Under the same expression conditions, the yield of
the mAOX1 protein from the corresponding native cDNA was relatively poor (Schumann et
al., 2009). To overcome this problem, we generated an expression construct based on an E.
coli codon-optimized mAox1 gene that resulted in a 150-fold increased average yield of
mAOX1 (Table 1). In contrast to the previously described purification protocols, both
mAOX1 and mAOX3 were purified by a simplified two-step purification method using Ni-
NTA and size exclusion chromatography. This resulted in increased yields of proteins
characterized by an equal level of purity.
In contrast, the expression systems for mAOX2 and mAOX4 were not established
before. The construct used for the expression of mAOX4 in E. coli TP1000 contained the
original cDNA amplified from the Harderian gland of C57BL/6N mice. For the expression of
mAOX2, we generated an E. coli codon optimized construct in analogy to the one described
for mAOX1. As detailed in the case of mAOX1 and mAOX3, the mAOX2 and mAOX4
proteins were purified from 12 liters of E. coli TP1000 cells. Similar to mAOX1 and
mAOX3, the mAOX4 and mAOX2 enzymes were purified by the 2-step purification
procedure involving Ni-NTA and size-exclusion chromatography. Upon size-exclusion
chromatography, mAOX1, mAOX2 mAOX3 and mAOX4 eluted as a single major peak,
which corresponded to the catalytically active dimeric forms of each enzyme with a
molecular mass of 300 kDa (Figure 2). Monomers were also detectable for all AOX enzymes
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as minor peaks with an elution volume around 12.5 ml. In the case of AOX4, a substantial
amount of protein showed the tendency to aggregate, however, the aggregates were well
separated from the main dimeric peak upon size exclusion chromatography (Figure 2). The
total yield of mAOX4 with an average of 1.1 ± 0.4 mg protein per liter of E. coli culture was
slightly higher than that of mAOX2, resulting in 0.8 ± 0.2 mg protein per liter of E. coli
culture (Table 1).
Relative to other molybdoenzymes, AOXs are characterized by the presence of a terminal
sulfido ligand in the equatorial position of Moco. This sulfido ligand is essential for the
catalytic activity of both types of enzymes and it is inserted by a specific Moco sulfurase
(Anantharaman and Aravind, 2002; Mendel and Leimkuhler, 2015; Terao et al., 2016). As the
E. coli and eukaryotic Moco sulfurases are substantially distinct from each other, expression
of mammalian AOXs in E. coli generally results in a low saturation level of the terminal
Mo=S group (Mahro et al., 2011; Foti et al., 2016). However, after purification, the sulfido
ligand can be ligated to the Moco of these enzymes by using a chemical sulfuration procedure
(Wahl and Rajagopalan, 1982). Consistent with the above considerations, expression of the
mAOX3 and mAOX4 in E. coli resulted in the isolation of two proteins with a low catalytic
activity, which could be increased more than 10-fold following the chemical sulfuration step
(Table 1). In the case of the two codon-optimized constructs used for mAOX1 and mAOX2
expression in E. coli, they resulted in the production of two catalytically inactive proteins
(Table 1). However, mAOX1 and mAOX2 regained significant activity upon chemical
sulfuration.
The UV-Vis spectra of mAOX1, mAOX3, mAOX4 and mAOX2 displayed the
characteristic features of molybdo-flavoenzymes, showing an absorption maximum at 450
nm and a shoulder at 550 nm (Figure 2 A-D insets). The ratios of A280/A465 of 5.0 and of
A465/A550 of 3.0 are characteristics for metalloflavoproteins including xanthine oxidase
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(XO) and AOX. A ration of A465/A550 of 3.0 corresponds to a FAD to iron ratio of 1:4,
while the ratio of A280/A465 of 5.0 is indicative for a high purity of the enzyme. Generally,
the 280/450 nm absorption ratios of mAOX1, mAOX4, and mAOX2 were 5.7, 5.4, and 5.0,
respectively (Table 1), which is consistent with a high level of enzyme purity. The similar
450/550 ratios determined for the enzymes are in line with the FAD and [2Fe2S] saturation
levels (Table 1).
SDS-polyacrylamide gels performed with the purified enzymes under reducing
conditions with β-mercaptoethanol and showed the presence of one major 150 kDa band in
the case of mAOX1 and mAOX3. Under the same conditions, mAOX4 and mAOX2 were not
fully reduced, as slower migrating bands, representing the corresponding multimeric forms,
were readily detectable (Figure 2A-D). The bands around 130 kDa, 80 kDa, and 50 kDa
identified for all enzymes (Kundu et al., 2007; Mahro et al., 2011), corresponded to
degradation products, as confirmed by electrospray mass spectroscopy analysis (data not
shown).
The levels of molybdenum and iron saturation for purified AOX1, AOX3, AOX4 and
AOX2 were determined by inductively coupled plasma optical emission spectroscopy (Figure
3). In the case of mAOX1, molybdenum saturation levels around 40% were calculated, while
approximately 50% values were detected for mAOX3, mAOX4 and mAOX2. The iron
saturation levels were around 60% for all the enzymes and reflected the saturation with
2x[2Fe2S] clusters. In conclusion, heterologous expression of the four mAOX enzymes in E.
coli resulted in incomplete but comparable overall metal and cofactor saturation. The
incomplete saturation with Moco and [2Fe2S] clusters has been reported before for other
molybdoflavoenzymes expressed in E. coli (Mahro et al., 2011; Hartmann et al., 2012; Foti et
al., 2016). For the purification of AOX enzymes from native sources like mouse livers,
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however, cofactor saturation levels were generally not reported for comparison (Terao et al.,
2001).
Direct comparison of the activities of the mAOX enzymes with selected substrates.
To compare the kinetic parameters of mAOX1, mAOX3, mAOX4 and mAOX2, the
steady-state kinetics of the four enzymes were determined with pyridoxal, vanillin, 4-
(dimethylamino)cinnamaldehyde (p-DMAC), N1-methylnicotinamide, salicylaldehyde, and
retinaldehyde as substrates. These substrates were selected, since they are of physiological
and toxicological interest and were reported before to be used as substrates for rat, human,
rabbit or mouse AOX enzymes isolated from liver extracts (Stanulović and Chaykin, 1971;
Stell et al., 1989; Obach, 2004; Kitamura et al., 2008; Terao et al., 2009) The kinetic
parameters were determined by a spectrophotometric assay based on the 2-electron reduction
of the electron acceptor DCPIP to DCPIPH2. The KM and kcat values calculated for each AOX
enzyme and substrate are shown in Table 2. To obtain better comparability of the data across
the four AOXs, the kinetic constants were corrected and normalized for a 100% molybdenum
saturation level of the purified enzymes.
As for mAOX1, the most efficient substrate is p-DMAC with a catalytic efficiency of
34.1 min-1 µM (Table 2). Vanillin shows the highest kcat value (449.9 ± 26.4 min-1), although
the affinity of this substrate for the enzyme is relatively low, as indicated by the high KM
value (104.05 ± 17.67 µM). Similar kcat values are evident following challenge of mAOX1
with salicylaldehyde and retinaldehyde. In spite of the consistent values for kcat, the catalytic
efficiency calculated for mAOX1 with retinaldehyde is 5-times higher than with
salicylaldehyde. Pyridoxal and N1-methylnicotinamide are not efficient mAOX1 substrates,
as indicated by the inability to measure detectable enzymatic activity.
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In the case of mAOX3, the kcat values obtained with the various substrates considered
are generally lower than the ones calculated for mAOX1. Similar to mAOX1, mAOX3 shows
the highest catalytic efficiency when challenged with p-DMAC (196.73 min-1µM). In
addition, the calculated KM values indicate that mAOX3 is characterized by a significantly
higher affinity for vanillin and salicylaldehyde than mAOX1. The most remarkable difference
between the two AOX enzymes is represented by the ability of AOX3 to recognize N1-
methylnicotinamide as a substrate. Finally, mAOX2 also showed the highest catalytic
efficiency with p-DMAC as substrate with a value of 125.28 min-1µM. Overall, the kcat values
of mAOX2 were about half with vanillin, p-DMAC and retinaldehyde as compared to
mAOX3, while for N1-methylnicotinamide a 2-fold higher kcat value was obtained in
comparison to mAOX3.
Regardless of the substrate considered and with the exception of salicylaldehyde,
mAOX4 is the enzyme characterized by the lowest kcat values. Similar to what was observed
for mAOX1, N1-methylnicotinamide is not recognized as a substrate by mAOX4. In addition,
AOX4 shows the highest the catalytic efficiency upon challenge with p-DMAC (36.3 min-
1µM). Surprisingly and contrary to the idea that AOXs are involved in the metabolism of
vitamin B6 (KEGG pathways, http://www.genome.jp/kegg-bin), pyridoxal is not recognized
as a substrate by any of the four mouse AOX enzymes. To confirm this important
observation, we further evaluated the ability of mAOX1, mAOX3, mAOX4 and mAOX2 to
oxidize pyridoxal, using an active enzyme staining assay based on native polyacrylamide gel
electrophoresis under non-reducing conditions (Marelja et al., 2014). In fact, this assay allows
long incubation times with the selected AOX substrate and it is characterized by an
unmatched level of sensitivity under these experimental conditions. After separation, the four
mAOXs migrated as single bands confirming the purity of our preparations (Figure 4). More
importantly, no signal was observed in correspondence to the mAOX1, mAOX3, mAOX4
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and mAOX2 protein bands upon overnight incubation of the gels with pyridoxal as substrate.
In contrast, easily detectable bands were observed when the gels are challenged with
phthalazine as a substrate even after a much shorter incubation time (Figure 4). Taken
together, these results fully confirm the observation that pyridoxal is not a substrate of any of
the mAOX enzymes and reveal that the enzymes unlikely play a significant role in the
catabolism of vitamin B6 (Stanulović and Chaykin, 1971; Garattini et al., 2008).
mAOX3 is the sole enzyme with NADH oxidase activity.
We investigated differences at the flavin site of the mAOX enzymes. AOX enzymes
are characterized by the exclusive use of molecular oxygen as electron acceptor. In contrast,
the other molybdo-flavoenzyme, xanthine dehydrogenase (XDH), uses NAD+ as the electron
acceptor in its dehydrogenase form, while it solely transfers the reducing equivalents to
molecular oxygen upon conversion into its oxidase form (XO) (Okamoto et al., 2013).
Despite the inability to react with NAD+, XO can be reduced by NADH (Olson et al., 1974).
The ability of XO to interact with NADH, but not with NAD+, is a consequence of the
different redox potentials of the FAD cofactor in the XDH and XO forms. For these reasons,
we determined the relative ability of the four mAOX enzymes to oxidize NADH in the
absence of oxygen (Figure 5). Approximately 10 µM of each enzyme was incubated with 500
µM NADH under anaerobic conditions and the corresponding absorption spectra were
recorded after 1 and 20 minutes of incubation. NADH was able to reduce only mAOX3 and
complete reduction was observed after 20 min of incubation. The result supports the data
from the crystal structure showing that the environment around FAD is different in mAOX3
relative to mAOX1, mAOX4 or mAOX2 (Coelho et al., 2012).
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mAOX2 produces the highest rate of superoxide anions.
The mAOX enzymes investigated here are all oxidases that use molecular oxygen as
electron acceptor and do not react with NAD+. In the reduction spectra, the formation of a
flavin semiquinone was also not observed (Schumann et al., 2009; Mahro et al., 2011) (data
not shown). AOXs have been reported to produce large amounts of H2O2 as the final reaction
product. However, potential and enzyme-specific differences in the rate of superoxide
.-
production have never been investigated before (Kundu et al., 2007). The production of O2
can be conveniently monitored by including cytochrome-c in the reaction mixture. In fact,
.-
.-
cytochrome-c reduction by O2 is considerably faster than spontaneous O2 dismutation. The
.-
amount of O2 produced in the reaction mixture was estimated by comparison to a blank
reaction run under the same conditions in the presence of superoxide dismutase. The
experiment was performed in the presence of 50 µM vanillin and p-DMAC as substrates, to
compare possible influences of the substrate on the rate of superoxide production. To obtain
.-
the percentage of O2 production, the reduction of cytochrome c (U/µmol enzyme) was
related to the overall rate of substrate consumption (U/µmol enzyme) (Table 3) determined
by the product formation using vanillin and p-DMAC as substrates. The results in Table 3
show that mAOX2 is characterized by the highest rate of superoxide production per substrate
.-
turnover. Indeed, the enzyme produces approximately 40% of O2 during turnover with
.-
vanillin and p-DMAC. In terms of O2 production, mAOX2 is followed by mAOX1 with a
superoxide production rate of around 30% per mol of substrate converted. The lowest
percentage of these radical species is observed with mAOX3 and mAOX4 which produce
.-
around 20% of superoxide radicals during substrate turnover. Since the same ratios of O2
/substrate converted were obtained with different substrates used in the assay, the ratio seems
to be independent of the substrate used.
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DISCUSSION
AOX is gaining increased importance as a drug metabolizing enzyme. However,
AOX mediated drug metabolism is not very well understood so far. Conventional methods to
predict human AOX1 metabolism from preclinical animal model data are often not suitable
due to interspecies differences in AOX activity. Mice, rats, rabbits and dogs generally used in
the prediction of drug metabolism fail to accurately predict AOX metabolism because they
express a different complement of AOX enzymes. For example, rodents such as mice and rat
express AOX1 and AOX3 in the liver whereas dogs express none (Garattini et al., 2009).
Humans, on the other hand, express a single AOX1 enzyme (Garattini et al., 2009). In order
to understand how to predict AOX clearances in humans, we need to understand the
structural determinants of substrate specificity among the different enzymes. Human AOX1
is 85% identical to mouse AOX1, while the other enzymes differ by 61-65% among each
other (Garattini and Terao, 2011). Combinations of homology modeling and molecular
dynamics simulations demonstrated major differences among mAOX1, mAOX3, mAOX4,
and mAOX2 at the protein surface and in the substrate-binding region (Cerqueira et al.,
2015). In particular, specific amino acids characteristic for each enzyme in the substrate-
binding site were predicted to be responsible for differences in the catalytic activity, substrate
and inhibitor specificities (Vila et al., 2004).
Here we report on the purification of the four mouse AOX enzymes in the
heterologous expression system represented by the E. coli TP1000 strain to directly
investigate differences among the different enzymes. The major aim of the study was to
compare the catalytic activities of mAOX1, mAOX3, mAOX4 and mAOX2 directly and
without cross-contaminations of the other enzymes, as it might have been the case with the
native enzymes purified from mouse tissues. A study directly comparing the purified
enzymes has not been reported before and is helpful to explain differences in previous studies
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using animal systems like rats, mice or rabbits, all expressing the four enzymes (Stanulović
and Chaykin, 1971; Kundu et al., 2012). We optimized and applied the same method for the
purification of the four mouse AOXs. The established method is based on a two-step
purification protocol involving Ni-NTA and size exclusion chromatography. The procedure
resulted in pure enzymes and an increased yield as compared to the previous purification
procedures described for recombinant mAOX1 and mAOX3 (Schumann et al., 2009; Mahro
et al., 2011). The high yields of mAOX1 and mAOX2 are a consequence not only due to the
changes in the purification method, but also to the use of a codon-optimized expression
vector and a chemical sulfuration step. The combination of these two factors gave rise to a
150-fold increase in the yield of mAOX1.
We profiled the four mouse AOX enzymes with three potential substrates of
physiological significance, i.e., retinaldehyde, N1-methylnicotinamide and pyridoxal, as well
as three other molecules of toxicological relevance, i.e. vanillin, p-DMAC and
salicylaldehyde. Overall, mAOX1 shows the highest kcat values for most of the substrates
tested. The only exception is represented by retinaldehyde, which is a substrate recognized by
the four enzymes with similar affinity. This last result substantially confirms and
complements the results obtained on native mAOX1 and mAOX3, which demonstrated only
minor differences in the Km and kcat of the two enzymes for retinaldehyde (Vila et al., 2004).
So far, one or more unspecified mouse AOX enzymes have been deemed to be involved in
nicotinamide metabolism thanks to their ability to generate pyridones (Stanulović and
Chaykin, 1971). Interestingly, our data indicate that mAOX1 and mAOX4 do not recognize
N1-methylnicotinamide as a substrate. In contrast, N1-methylnicotinamide is a 5-fold more
efficient substrate of mAOX2 relative to mAOX3. This indicates that liver N1-
methylnicotinamide metabolism observed in mouse liver cytosolic extracts is due to mAOX3,
the major form of hepatic AOX, and not to mAOX1, which is expressed at much lower levels
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in this tissue (Stanulović and Chaykin, 1971; Kitamura et al., 2008). The metabolism of N1-
methylnicotinamide by human AOX1 has not been investigated with the purified enzyme
before. Studies on humanized mice extracts showed some discrepancies in respect to product
formation in comparison to the products in mice, thus, we only can speculate here whether
N1-methylnicotinamide is indeed a substrate of hAOX1 (Kitamura et al, 2008). A recent
computational study predicted structural differences in the substrate binding funnel of the
four mouse AOXs (Cerqueira et al., 2015). In particular, mAOX1 has been proposed to be
endowed with the widest substrate binding region, while mAOX4 has been predicted to
contain the narrowest one. The experimental results obtained with N1-methylnicotinamide are
not entirely consistent with the predictions. In fact, neither mAOX1 nor mAOX4 utilize this
substrate, while N1-methylnicotinamide is a good mAOX2 and mAOX3 substrate (Mahro et
al., 2013).
Pyridoxal is one of the forms of vitamin B6 and a potential physiological substrate of
human AOX1 (KEGG pathways). Previous data indicated that the native form of mAOX3,
purified from liver, was capable of oxidizing pyridoxal into pyridoxic acid, while the native
form of mAOX4, purified from the Harderian gland, was unable to oxidize this aldehyde into
the corresponding carboxylic acid (Terao et al., 2009). The results of the current study
demonstrate, however, the inability of the four recombinant mAOX enzymes to recognize
pyridoxal as a substrate. At present, we do not have any explanation for the discrepancy
observed between the results obtained with native and recombinant mAOX3. However, from
the results on the mouse enzymes it only can be predicted that pyridoxal is also not a
substrate of the human enzyme. This needs to be further investigated in future studies.
We used our purified mAOX enzymes to test the reactivity of the above substrates at
.-
the FAD site. In particular, we determined NADH oxidase activity and the amount of O2
radical production from molecular oxygen. NADH is found in all living cells and it is the
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major source of the electrons transported by the mitochondria and responsible for the
synthesis of ATP. Hepatic AOX preparations have been shown to oxidize NADH thereby
.-
generating the reducing equivalents necessary for O2 production from molecular oxygen
(Kundu et al., 2007; Maia et al., 2007; Choughule et al., 2015). However, most of these
studies were based on the use of hepatic cytosolic extracts obtained from rats or rabbits
containing mixtures of AOX1 and AOX3. Even a study by Maia et al. 2015, which
investigated nitrate reductase activity using rat liver and human liver extracts, investigated
the NADH oxidase activity only with the rat liver extracts and not with the human source
(Maia et al., 2015). Our results indicate that mAOX3 is the only enzyme which can be
reduced by NADH. mAOX3 is the major AOX enzyme expressed in mouse liver, hence, it is
possible that the associated NADH oxidase is capable of producing significant amounts of
.-
O2 in this tissue. As mAOX1 is the human AOX1 orthologue, it is likely that hAOX1 is also
incapable of reacting with NADH. The different reactivity with NADH of AOX1 and AOX3
can be explained by the crystal structure of the enzymes, where a different loop at the FAD
site of hAOX1 and mAOX3 became visible. The crystal structure of hAOX1 revealed that a
loop close to the FAD pocket (T1230RGPDQ1235) is flipped by almost 180° relative to mouse
AOX3. Thus, the NADH binding site might be blocked by this loop in AOX1 enzymes and
likely also in the AOX4 and AOX2 enzymes, for which no structural data are available so far.
However, studies on the reducibility of hAOX1 by NADH are missing in the literature so far.
All studies available were investigating rat liver or rabbit liver extracts, thus, from our data it
can be concluded that the NADH oxidase activity reported in these studies is mainly based on
the activity of AOX3.
So far mainly xanthine oxidase (XO) has been implicated to generate significant
.-
amounts of O2 during the course of their catalytic activity against recognized substrates
(Kundu et al., 2007). However, taking into account the relative levels of enzymatic activity of
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XO and AOX in human liver (Krenitsky et al., 1972), AOX1 has been calculated to generate
24-fold larger amounts of superoxide than XO. Thus, human AOX1 has been suggested to
represent a significant source of ROS in the cytosol and therefore might play a critical role in
in ROS-mediated tissue injury under specific conditions (Kundu et al., 2012). Given this
.-
background, we compared the production of O2 by purified mAOX1, mAOX3, mAOX4 and
.-
mAOX2. Our results indicate a different rate of O2 production by each mouse AOX enzyme
.-
in relation to the amount of substrate converted (in mol O2 produced per mol of substrate
converted). This ratio seems to be independent of the substrate used, since the same ratios of
.-
O2 /substrate converted were obtained with different substrates used in the assay. In
particular, mAOX2 is the most efficient producer of superoxide anions with a rate of 40%. As
the enzyme is located in the nasal mucosa, which is one of the main site of entrance for
infective agents, it is possible to speculate that mAOX2 produces reactive ROS to protect
mice from viral and bacterial infections. A similar role may be played by mAOX1 and/or
mAOX3 in the liver. Also mAOX1 was shown to produce a ratio of 30% superoxide radicals,
which is higher than the reported value of 15-20% for XO (Hunt and Massey, 1992). Thus,
also hAOX1 might be a major source of superoxide radical production in the human liver. In
.-
contrast, mAOX3 and mAOX4 produce a rate of 20% O2 radicals per mole of substrate
converted, a ratio which is significantly lower as compared to mAOX1 and mAOX2.
In conclusion, this study focuses on the development of a simple and efficient system
for the isolation and first comparative enzymatic characterization of the four mouse AOXs.
The system is likely to represent an important tool for the definition of the substrate
specificity of each enzyme. At the basic level, the identification of the common and specific
physiological substrates of each enzyme is likely to provide clues as to the functional role
played by mAOX1, mAOX3, mAOX4 and mAOX2 in the homeostasis of the mouse. In fact,
the existence of multiple and species-specific enzymatic forms of AOX is one of the most
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important and unresolved problems in the field. In particular, the presence of a single human
AOX enzyme, i.e. AOX1, and multiple as well as tissue-specific AOXs in other animal
species, with particular reference to mice and rats, has important ramifications not only in
terms of basic biology but also at the applied level. In fact, human AOX1 is of emerging
interest in the context of drug metabolism and development, as the enzyme is capable of
oxidizing and potentially inactivating an ever increasing number of heteroaromatic rings
which represent the principal components of numerous molecules of therapeutic and
toxicological interest (Pryde et al., 2010). Due to the existence of four different AOX
enzymes in mice and rats and a single AOX enzyme in humans, the use of rodents as pre-
clinical models for pharmacokinetic, pharmacodynamic and toxicological studies involving
drugs which are potential human AOX1 substrates are generally not suitable model systems
for these particular studies (Sanoh et al., 2015). In this context, we propose that the
availability of a system which allows the purification of large amounts of catalytically active
human (Foti et al., 2016) and mouse AOXs is likely to be the basis for screening tests aimed
at establishing whether new drug candidates and available drugs represent efficient substrates
for this class of enzymes. We therefore propose to directly use the purified enzymes for
studies on substrate conversion, since without cross-contaminations with other enzymes the
mode of substrate conversion and inhibition can be investigated directly in molecular detail.
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AUTHORSHIP CONTRIBUTIONS
Participated in research design: Kücükgöze, Terao, Garattini, Leimkühler
Conducted experiments: Kücükgöze
Performed data analysis: Kücükgöze, Leimkühler
Wrote or contributed to the writing of the manuscript: Kücükgöze, Terao, Garattini,
Leimkühler
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