The ORF slr0091 of Synechocystis sp. PCC6803 encodes a high-light induced aldehyde dehydrogenase converting apocarotenals and alkanals

Article abstract of DOI:10.1111/febs.12361

Oxidative cleavage of carotenoids and peroxidation of lipids lead to apocarotenals and aliphatic aldehydes called alkanals, which react with vitally important compounds, promoting cytotoxicity. Although many enzymes have been reported to deactivate alkanals by converting them into fatty acids, little is known about the mechanisms used to detoxify apocarotenals or the enzymes acting on them. Cyanobacteria and other photosynthetic organisms must cope with both classes of aldehydes. Here we report that the Synechocystis enzyme SynAlh1, encoded by the ORF slr0091, is an aldehyde dehydrogenase that mediates oxidation of both apocarotenals and alkanals into the corresponding acids. Using a crude lysate of SynAlh1-expressing Escherichia coli cells, we show that SynAlh1 converts a wide range of apocarotenals and alkanals, with a preference for apocarotenals with defined chain lengths. As suggested by in vitro incubations and using engineered retinal-forming E. coli cells, we found that retinal is not a substrate for SynAlh1, making involvement in Synechocystis retinoid metabolism unlikely. The transcript level of SynAlh1 is induced by high light and cold treatment, indicating a role in the stress response, and the corresponding gene is a constituent of a stress-related operon. The assumptions regarding the function of SynAlh are further supported by the surprisingly high homology to human and plant aldehyde dehydrogenase that have been assigned to aldehyde detoxification. SynAlh1 is the first aldehyde dehydrogenase that has been shown to form both apocarotenoic and fatty acids. This dual function suggests that its eukaryotic homologs may also be involved in apocarotenal metabolism, a function that has not been considered so far. Aldehyde dehydrogenases play an important role in detoxification of reactive aldehydes. Here, we report on a cyanbacterial enzyme capable in converting two classes of lipid-derived aldehydes, apocaotenals and alkanals. The corresponding gene is a constituent of a stress-related operon, and homology to eukaryotic enzymes points to a yet not considered possibility of their being involved in scavenging of apocarotenals.

Full text of DOI:10.1111/febs.12361

The ORF slr0091 of Synechocystis sp. PCC6803 encodes
a high-light induced aldehyde dehydrogenase converting
apocarotenals and alkanals
1
1
1,2
Danika Trautmann , Peter Beyer and Salim Al-Babili
1
2
Faculty of Biology, Albert-Ludwigs University of Freiburg, Germany
Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal, Kingdom
of Saudi Arabia
Keywords
Oxidative cleavage of carotenoids and peroxidation of lipids lead to apoca-
rotenals and aliphatic aldehydes called alkanals, which react with vitally
important compounds, promoting cytotoxicity. Although many enzymes
have been reported to deactivate alkanals by converting them into fatty
acids, little is known about the mechanisms used to detoxify apocarotenals
or the enzymes acting on them. Cyanobacteria and other photosynthetic
organisms must cope with both classes of aldehydes. Here we report that the
Synechocystis enzyme SynAlh1, encoded by the ORF slr0091, is an aldehyde
dehydrogenase that mediates oxidation of both apocarotenals and alkanals
into the corresponding acids. Using a crude lysate of SynAlh1-expressing
Escherichia coli cells, we show that SynAlh1 converts a wide range of apoca-
rotenals and alkanals, with a preference for apocarotenals with defined chain
lengths. As suggested by in vitro incubations and using engineered retinal-
forming E. coli cells, we found that retinal is not a substrate for SynAlh1,
making involvement in Synechocystis retinoid metabolism unlikely. The
transcript level of SynAlh1 is induced by high light and cold treatment,
indicating a role in the stress response, and the corresponding gene is a con-
stituent of a stress-related operon. The assumptions regarding the function
of SynAlh are further supported by the surprisingly high homology to
human and plant aldehyde dehydrogenase that have been assigned to alde-
hyde detoxification. SynAlh1 is the first aldehyde dehydrogenase that has
been shown to form both apocarotenoic and fatty acids. This dual function
suggests that its eukaryotic homologs may also be involved in apocarotenal
metabolism, a function that has not been considered so far.
aldehyde dehydrogenase; alkanals;
apocarotenoids; carotenoid cleavage;
cyanobacteria
Correspondence
S. Al-Babili, BESE Division, King Abdullah
University of Science and Technology,
Thuwal, Kingdom of Saudi Arabia
Fax: +966 2 802 0103
Tel: +966 2 808 2762
E-mail: salim.babili@kaust.edu.sa
(Received 6 April 2013, revised 26 May
2013, accepted 28 May 2013)
doi:10.1111/febs.12361
Introduction
Aldehyde dehydrogenases (ALDHs, aldehyde:NAD
(
lism [3]. Furthermore, ALDHs contribute to the
response of cells to oxidative stress [4–6]. Under such
conditions, reactive oxygen species (ROS) are formed,
leading to lipid peroxidation and giving rise to lipid-
derived aldehydes. These products are reactive electro-
philes that promote cytotoxicity by forming adducts
with various vitally important nucleophiles, such as
+
P) oxidoreductase, EC 1.2.1.5) constitute a widely
distributed superfamily of enzymes that oxidize alde-
hydes into the corresponding acids by transferring
+
+
electrons to the co-enzymes NAD and NADP [1,2].
ALDHs contribute to various metabolic pathways, e.g.
amino acid, retinoid or c-aminobutyric acid metabo-
Abbreviations
ALDH, aldehyde dehydrogenase; ROS, reactive oxygen species.
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An apocarotenal and alkanal aldehyde dehydrogenase
D. Trautmann et al.
nucleic acids and proteins [7]. ALDHs alleviate the
harmful consequences of oxidative stress by scavenging
such aldehydes [4,8].
stress [25]. In Synechocystis sp. PCC 6803, high-light
treatment results in induction of the apocarotenoid
15,15′-oxygenase SynACO [26], previously named
Diox1 [27]. This cyanobacterial enzyme catalyzes in vi-
tro cleavage of the C15–C15′ double bond in various
apocarotenoids with chain lengths ranging from C25 to
C35, to yield the C20 aldehyde retinal and a dialdehyde
with a chain length between C and C [27]. Sequence
Apocarotenals are an additional class of aldehydes
that serve as substrates for ALDHs [9,10]. Apocarote-
nals arise by oxidative cleavage of double bonds within
the conjugated double bond system of carotenoids,
which are essential isoprenoid pigments that mediate
photoprotection and contribute to light harvesting in
photosynthetic organisms. Moreover, carotenoids are
generally important as antioxidants and precursors of
signaling molecules. Apocarotenals are formed either
enzymatically by carotenoid cleavage oxygenases [11–
5
15
comparisons and in vitro enzymatic investigation of
the Anabaena homolog NosACO [28] indicate that for-
mation of retinal from longer apocarotenals is a com-
mon activity in cyanobacteria.
The enzymatic activity of CYP120A1, the sole cyto-
chrome P450 enzyme of Synechocystis, is a strong indi-
cation of functional retinoid metabolism in this
cyanobacterium. In silico prediction indicated that all-
trans retinoic acid binds to the enzyme, as also shown
by structural elucidation of CYP120A1 containing
bound all-trans retinoic acid [29,30]. Investigation of
the CYP120A1 activity led to its characterization as
an all-trans retinoic acid hydroxylase. CYP120A1 thus
resembles mammalian homologs involved in retinoic
acid metabolism and plant cytochrome P450 enzymes
that catalyze the breakdown of abscisic acid [26].
Here, we set out to identify the supposed Synecho-
cystis aldehyde dehydrogenase that is expected to oxi-
dize apocarotenals into their corresponding acids and
to provide a metabolic link between the retinal-form-
ing enzyme SynACO and the all-trans retinoic acid
hydroxylase CYP120A1. Although we have not identi-
fied a retinal dehydrogenase, we describe the first
reported aldehyde dehydrogenase that acts on both
classes of lipid-derived aldehydes, i.e. apocarotenals
and fatty aldehydes (also called alkanals), aliphatic
aldehydes that are thought to be derived from alkanes.
1
3] or by co-oxidation initiated through lipoxygenase
14], or non-enzymatically through a variety of mecha-
[
nisms and reactions initiated by ROS [15]. The best
known apocarotenal is the rhodopsin chromophore
retinal, a C20 aldehyde that arises from central cleav-
age of b-carotene at the C15–C15′ double bond, cata-
lyzed by members of the carotenoid cleavage
oxygenase family in animals and fungi [16,17]. In ver-
tebrates, retinal is converted by retinal dehydrogenases
into retinoic acid [3], which acts as a morphogen.
ALDHs have also been shown to be involved in for-
mation of other apocarotenoic acids, such as bixin, a
seed-specific pigment of Bixa orellana [18], and neuros-
poraxanthin, a C35 apocarotenoic acid of the fungi
Neurospora crassa and Fusarium fujikuroi [9,10].
Cyanobacteria synthesize and accumulate various
carotenoids, such as b-carotene and its dihydroxylated
derivative zeaxanthin, ketocarotenoids, such as echine-
none and canthaxanthin, and carotenoid glycosides,
such as myxoxanthophylls [19]. Because they rely on
oxygenic photosynthesis, cyanobacteria must cope with
enhanced levels of ROS, when challenged by unfavor-
able conditions such as salinity, drought and high light
intensities. Perception of ROS triggers the synthesis of
detoxifying enzymes, including superoxide dismutase,
catalase, ascorbate peroxidase, glutathione reductase
and glutathione S-transferase, as well as the produc-
tion of antioxidants such as ascorbate, a-tocopherol,
glutathione and carotenoids [20,21]. Among the latter,
xanthophylls in particular appear to exert a protective
role against oxidative stress [22]. It is likely that oxida-
tive stress results in the cleavage of carotenoids, giving
rise to apocarotenals [23]. This scenario may explain
the induction of carotenoid and apocarotenoid cleav-
age oxygenases that are capable of further metaboliz-
ing these degradation products. For instance, the
Anabaena sp. PCC 7120 carotenoid cleavage dioxygen-
ase NosCCD (previously named NSC1) [24], which
converts apocarotenals into volatile compounds such
as bÀionone and geranial, is induced by high-light
Results
SynAlh1, the YLO-1 homolog of Synechocystis
The Neurospora crassa enzyme YLO-1 has been shown
to catalyze the oxidation of apocarotenals to yield the
corresponding apocarotenoic acids [9]. A BLAST
search of YLO-1 against the Synechocystis genome
identified the enzyme encoded by ORF slr0091, named
here SynAlh1, as the best match. Wider sequence
comparison (Table S1) showed a higher similarity of
SynAlh1 to mammal members of the ALDH3 subfam-
ily than to the fungal YLO-1, i.e. 63% and 60% to
human ALDH3B1 and fatty aldehyde dehydrogenase
(FALDH) iso2, respectively, while the similarity to
YLO-1 is ~ 50%. The homology to the mammal reti-
nal dehydrogenases, e.g. human ALDH1A1-3, was less
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D. Trautmann et al.
An apocarotenal and alkanal aldehyde dehydrogenase
pronounced, with values between 36 and 39%. The clos-
est plant homologs of SynAlh1 also belong to the
ALDH3 subfamily, e.g. Arabidopsis thaliana ALDH3I1
with 52% similarity. The phylogenetic tree presented in
Fig. 1 shows that the cyanobacterial ALDHs constitute
a group together with enzymes from non-photosynthetic,
carotenoid-free bacteria such as Clostridium botulinum
and Bacillus cereus, and plant ALDH3s. The clade of
bacterial and plant ALDHs is more closely related to
human homologs than to the fungal YLO-1. Human
retinal dehydrogenases form a separate branch that is
clearly separated from the other ALDHs considered in
this analysis. Taken together, ALDHs, especially those
of the ALDH3 subfamily, appear to be universal, exist-
ing in bacteria, plants, fungi and mammals.
chromatography. However, purification was hampered
by the insolubility of the enzyme, and, in the best case,
only traces of purified thioredoxin-tagged SynAlh1
were obtained. Therefore, we used crude lysates of
E. coli cells expressing thioredoxin-tagged SynAlh1 to
test the enzymatic activity, using apocarotenals of vari-
ous chain lengths, i.e. b-apo-8′- (C ), b-apo-10′- (C ),
3
0
27
b-apo-12′- (C ), b-apo-14′- (C ) and b-apo-15′-carote-
2
5
22
nal (retinal, C ), as substrates. As shown in Fig. 2,
0
2
the enzyme catalyzed almost complete conversion of
substrates with a minimum chain length of C25 after
30 min incubation, as indicated by the change in color
due to oxidation of the conjugated carbonyl function.
HPLC analysis confirmed this activity by revealing for-
mation of apocarotenoic acids, which eluted earlier
than the aldehydes in the separation system used and
showed the expected hypsochromic UV/Vis spectral
shifts. LC-MS analysis was used to confirm the reac-
tion. The m/z values observed supported the formation
of apocarotenoic acids (shown in Fig. 2A for b-apo-
10′-apocarotenoic acid: C H O ). However, the
SynAlh1 oxidizes apocarotenals in vitro
The homology of SynAlh1 to Neurospora YLO-1 indi-
cates that it may be an apocarotenal dehydrogenase.
To check this possibility and to perform in vitro
assays, we expressed the enzyme coupled to a 6xHis or
thioredoxin tag, and attempted to purify it by affinity
2
7
36
2
enzyme did not convert all of the apocarotenals tested.
Experiments with retinal (C ) and b-apo-14′-carotenal
2
0
Fig. 1. Phylogeny of SynAlh1 and homologs. Cyanobacterial enzymes are outlined in green. Node labels represent bootstrap values; the
lengths of branches represent exchanges per sequence. Micromonas Alh was used as an out-group. Amino acid sequences of ALDHs were
taken from the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov): huALDH1A2, human NP_000684.2;
huALDH1A1, human NP_000680; huALDH1A3, human NP_003879; NeuYlo-1, XP_957628 from Neurospora crassa OR74A; huFALDH iso2,
human NP_000373.1; huFALDH iso1, human NP_001026976.1; huALDH3A1, human NP_000682.3; huALDH3B2, human NP_000686.2;
huALDH3B1, human NP_000373.1; AtALDH3I1, NP_567962.1 from Arabidopsis thaliana; AtALDH3H1, NP_849770 from
Arabidopsis thaliana; CbALDH, ZP_02613185.1 from Clostridium botulinum NCTC2916; BcYwdH, ZP_04322346.1 from Bacillus cereus
m1293; McyAlh1, YP_001655212.1 from Microcystis aeruginosa NIES-843; ProAlh1, YP_001550253.1 from Prochlorococcus marinus str.
MIT9211; ScoAlh1, YP_171740.1 from Synechococcus elongatus PCC6301; SynAlh1, NP_442494.1 from Synechocystis sp. PCC6803;
CyAlh1, YP_002375503.1 from Cyanothece sp. PCC7424; TriAlh1, YP_722319.1 from Trichodesmium erythraeum IMS101; NosAlh1,
NP_487712 from Anabaena sp. PCC7120; AcaAlh1, YP_001517422.1 from Acaryochloris marina MBIC11017; Micromonas Alh,
XP_002506086 from Micromonas sp. RCC299.
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An apocarotenal and alkanal aldehyde dehydrogenase
D. Trautmann et al.
A
B
C
Fig. 2. In vitro activity of heterologously
expressed SynAlh1 in incubations with
apocarotenals. (A) UFLC analysis of in vitro
assays using a crude lysate of SynAlh1-
expressing E. coli and empty vector
controls: a, b-apo-8′-carotenal; c, b-apo-10′-
carotenal; e, b-apo-12′-carotenal; g,
8
′,8-diapocarotene-8′,8-dial. The incubation
led to the formation of: b, b-apo-8’-
carotenoic acid; d, b-apo-10’-carotenoic acid;
f, b-apo-12’-carotenoic acid; h, 8’,8-
diapocarotene-8’,8-dioic acid. Structures of
the products are depicted. Absorption
spectra of substrates and products are
shown in the insets. (B) Color change of b-
apo-8′-carotenal (control; orange) due to
conversion into b-apo-8′-carotenoic acid
(
yellow) by SynAlh1. (C) LC-MS analysis of
b-apo-10′-carotenoic acid (C27
36 2
H O ;
peak d).
(C ), the chain lengths of which are below the mini-
mum chain length of C , did not yield apocarotenoic
cleavage oxygenases, CarT [31], CarX [17] or CarS
[32], which mediate formation of b-apo-4′-carotenal
(C ), b-apo-15′-carotenal (C , retinal) or b-apo-
2
2
2
5
acids.
3
5
20
Carotenoid-derived dialdehydes may arise through
double cleavage. To test the ability of SynAlh1 to con-
vert such products, we incubated the enzyme with
12′-carotenal (C ), respectively. For this purpose,
25
SynAlh1 was introduced into pBAD-CarT, pBAD-CarX
or pThio=CarS downstream of the respective oxygenase
cDNA, to yield pBAD-CarT-SynAlh1, pBAD-CarX-
SynAlh1 and pThio=CarS-SynAlh1, respectively. The
obtained plasmids allow expression of the respective
oxygenase and SynAlh1 as separate proteins in one
operon. Transformation of pThio=CarS-SynAlh1 and
induction in b-carotene-accumulating E. coli cells led to
formation of b-apo-12′-carotenoic acid (peak c, Fig. 3)
from b-apo-12′-carotenal (peak b, Fig. 3) upon expres-
sion of CarS alone, while control cells transformed with
empty plasmid accumulated b-carotene (peak a) but no
apocarotenoids. In agreement with the in vitro data,
co-expression of SynAlh1 with CarT yielded neurospora-
xanthin (b-apo-4′-carotenoic acid), but co-expression with
the retinal-forming enzyme CarX did not yield retinoic
acid (Fig. S2).
8
,8′-diapocarotene-8,8′-dial (crocetin aldehyde, C ,
20
peak g in Fig. 2A) and 8,12′-diapocarotene-8,12′-dial
C ). As shown in Fig. 2A, we observed formation of
(
1
5
the di-apocarotenoic acid crocetin (peak h). Similarly,
SynAlh1 converted both aldehyde groups in b-apo-8′,
1
2-carotenedial, into the respective di-apocarotenoid
acid (data not shown). As indicated by the chain
length of both dialdehydes, the requirement for a
minimal length of C25 as observed for apocarotenals
appears to be irrelevant for conversion of dialdehydes.
We also tested apocarotenoids such as 3-OH-,
4
-oxo-b-apo-8′-carotenal and apo-8′-lycopenal are pro-
duced from the carotenoids zeaxanthin, echinenone
and lycopene, respectively. We observed formation of
apocarotenoic acids from all substrates with a minimal
chain length of C25 (Fig. S1) (see Table S2 for a list of
substrates tested).
SynAlh1 oxidizes fatty aldehydes in vitro
To demonstrate SynAlh1 activity in vivo, we
expressed the enzyme in carotenoid-accumulating
E. coli strains together with one of the carotenoid
The sequence similarity of SynAlh1 to eukaryotic
ALDHs that convert fatty aldehydes indicates that the
3
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D. Trautmann et al.
An apocarotenal and alkanal aldehyde dehydrogenase
half as high (30–40 nmol/mL). Hexanal and b-apo-
4′-carotenal showed the lowest product formation
(5–10 nmol/mL).
SynAlh1 transcript levels increase under stress
conditions
Metabolism of apocarotenals and alkanals by SynAlh1
indicates a function in detoxification of aldehydes that
arise under conditions leading to oxidative stress, e.g.
high light, and hence expression of SynAlh1 may be
induced by high-light treatment. To confirm this, we
exposed Synechocystis liquid cultures to high light
and cold stress, and determined transcript levels of
SynAlh1 by real-time RT-PCR. Fig. 5A shows the
results of the high-light experiment. The mean SynAlh1
transcript level increased to ~ 2-fold within 30 min,
continued increasing to ~ 17-fold after 1 h, and
decreased to 4 to 6-fold after 4 h of exposure.
Fig. 3. UFLC analysis of in vivo conversion of b-apo-12′-carotenal
(C25) in carotenoid-accumulating E. coli cells. Transformation of a
b-carotene strain with the pThio empty vector results in
accumulation of b-carotene (A), transformation with the oxygenase
CarS expression vector pThio=CarS results in accumulation of
b-apo-12′-carotenal (B), and transformation with the double
construct pThio=CarS-SynAlh1 results in formation of b-apo-12′-
carotenoic acid (C). The spectra of the products are given in the
insets.
For cold stress treatment, we lowered the growth
temperature from 30 to 20 °C, and harvested the
cultures at various time points. As shown in Fig. 5B,
SynAlh1 transcript levels increased due to cold stress;
however, the induction was slower and less pro-
nounced (~ 6 to 7-fold) than upon high-light stress.
Shifting the growth temperature back to 30 °C led to
transcript levels similar to those observed before the
treatment.
enzyme may also act on these substrates. To test this
hypothesis, we incubated crude lysates of SynAlh1
over-expressing cells with dodecanal and determined
the activity of the enzyme photometrically by measur-
ing NADH production. GC-MS analysis was used to
confirm the identity of the product formed, revealing
The strong high-light induction of SynAlh1 tran-
script levels indicates a function in the stress response
of the cell. This presumed role is further supported by
+
the expected molecular ion [M ] of m/z 214 for do-
decanoic acid (C H O ). Various other alkanals were
1
2
24
2
also tested (see Table S2), from C (acetaldehyde) to
2
C18 (octadecanal), and the two C aromatic aldehydes
7
4-OH-benzaldehyd and 3,4-di-OH-benzaldehyde. As
shown in Table S2, SynAlh1 oxidized only linear fatty
aldehydes with a minimal chain length of C (hexanal).
6
To address the question of the substrate preference
of SynAlh1, i.e. apocarotenoids versus fatty aldehydes,
we determined the relative effectiveness of conversion
in time-course experiments. Apocarotenal oxidation
was quantified by HPLC, while alkanal oxidation was
measured by monitoring NADH formation photomet-
rically. The results obtained are shown in Fig. 4. At
equimolar substrate concentrations (80 lM), the initial
velocities of product formation were comparable with
both substrate classes, with the exception of b-apo-4′-
carotenal, which showed very ineffective conversion.
The various reactions reached their individual plateaux
at various levels. b-apo-8′-carotenal and b-apo-10′-car-
otenal showed the highest product yields (~ 60 nmol/
mL), followed by the alkanals nonanal, dodecanal,
hexadecanal and octadecanal, with yields approximately
Fig. 4. Product formation curves for various substrates by SynAlh1
in crude lysate incubations. All substrates were applied at
a
concentration of 80 lM; see Experimental procedures for assay
composition. Product formation was determined at the indicated
time points either by using the kinetic mode of the photometer
(
alkanals) or by HPLC quantification (apocarotenals). Values are
means Æ SE of at least three replicates. The curves were fitted
using the equation for one-phase exponential curves in the
GraphPad Prism program (GraphPad Software, La Jolla, CA, USA).
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An apocarotenal and alkanal aldehyde dehydrogenase
D. Trautmann et al.
A
B
Fig. 5. Expression levels of SynAlh1 determined by real-time RT-PCR analysis. Changes in the transcript levels at the indicated time points
are shown as n-fold relative to the untreated Synechocystis culture at time point zero. Values are means and SEM of at least three
repetitions. (A) High-light treatment. Cultures were shifted from 20 to 280 lmol photons mÀ2ÁsÀ1. (B) Cold treatment. Cultures were shifted
from 30 °C (standard temperature) to 20 °C for 24 h, and then shifted back to 30 °C.
the presence of genes encoding stress-related proteins
A
that surround the SynAlh1 (slr0091) locus (Fig. 6A),
including two heat-shock chaperones (slr0086/DnaK
and slr0093/DnaJ), a carotene ketolase (slr0088/CrtO)
and the tocopherol biosynthetic enzymes c-tocopherol
methyl transferase (slr0089) and 4-hydroxyphenylpyru-
vic acid dioxygenase (slr0090). The co-localization of
B
these genes may be indicative of their being constitu-
ents of a stress-responsive operon. To test this possibil-
ity, we designed RT-PCR experiments in which the
intergenic regions plus adjacent parts of the surround-
ing genes can be amplified only if the two respective
genes are co-transcribed. As a negative control, we
tested the intergenic region between slr0086 on the left
border of the putative operon and sll0078 on the
reverse strand, and also performed all PCR reactions
on RNA but without reverse transcription. The results
of the PCR reactions, shown in Fig. 6B, demonstrate
that all genes from slr0086 to slr0096 are co-tran-
scribed as a single RNA molecule. However, it is likely
that also other smaller transcripts are produced from
this region, as indicated by the existence of predicted
Fig. 6. (A) Genomic context of SynAlh1 (slr0091). The genes that
transcription start sites within the operon (Fig. 6A).
are putatively part of an operon include slr0086 (heat-shock protein
DnaK), slr0088 (CrtO, b-carotene ketolase), slr0089 (c-tocopherol
Discussion
methyltransferase), slr0090 (4-hydroxyphenylpyruvate dioxygen-
ase), slr0091 (SynAlh1), slr0092 (glycosyl transferase), slr0093 (heat-
shock protein DnaJ), slr0095 (O-methyltransferase), slr0096 (sulfate
transporter), slr0498 (unknown function) and slr0854 (photolyase).
Transcription start sites as described by Mitschke et al. [47] are
indicated by arrows. (B) RT-PCR analysis to determine operon size.
Primers were designed to yield products covering intergenic regions
and PCR reactions were performed using cDNA, genomic DNA
Oxidative cleavage of carotenoids is a widespread
process that is expected to take place not only in all
organisms that synthesize these pigments but also in
animals that obtain carotenoids from dietary sources.
This reaction is usually mediated by carotenoid cleav-
age oxygenases that initiate the synthesis of biologically
important compounds such as retinoic acid, trisporic
acids, abscisic acid and strigolactones [32,34,35]. How-
ever, ROS also promote carotenoid cleavage. This is
followed by complex rearrangements leading to prod-
ucts that are identical to those produced enzymatically.
Apocarotenoids are electrophiles, and are therefore
reactive with the many nucleophiles common in cells.
However, very little is known about the mechanisms
(
positive control) and RNA (negative control) templates. Amplicon
bands yielded in PCRs with cDNA indicate that the respective genes
are transcribed together.
used for their detoxification. One possibility is their
oxidation to acids, as investigated here for SynAlh1.
We also investigated the activity of SynAlh1 on the
assumption that the reaction has a wider biological
significance. The oxidation of apocarotenals may
3
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D. Trautmann et al.
An apocarotenal and alkanal aldehyde dehydrogenase
represent the missing link between a retinal-forming
enzyme that exists in Synechocystis (SynACO) [27] and
a cytochrome P450 that catalyzes the hydroxylation of
all-trans retinoic acid (CYP120A1) [26]. SynAlh1 may
be the enzyme that provides the retinoic acid substrate
for CYP120A1. For this purpose, we performed in vi-
tro incubations with numerous apocarotenals, and
showed that SynAlh1 is an apocarotenal dehydroge-
nase. However, our data indicate that the apocarotenal
substrate must have a minimum chain length of C .
family 3 act on lipid peroxidation-derived aldehydes.
To our knowledge, enzymes of this family have not
been tested with the apocarotenals that are present in
plants and may occur in animal systems. Therefore,
investigation of the conversion of apocarotenals by
these enzymes may unravel new physiological and met-
abolic functions that have been overlooked in the past.
Real-time RT-PCR expression analysis of SynAlh1
indicates a function in the stress response. The gene
expression was up-regulated ~ 17-fold upon high-light
treatment and by ~ 6-fold upon cold stress. These find-
ings suggest, that similar to homologous ALDHs from
A. thaliana (ALDH3H1 and ALDH3I1) [37,38],
SynAlh1 activity is part of the Synechocystis response
to oxidative stress conditions. In support of this,
SynAlh1 is located in a cluster of genes that are known
to be involved in the stress response. Moreover, our
RT-PCR experiments demonstrate that SynAlh1 is
transcribed as part of an operon consisting of eight
ORFs (slr0086–slr0096), including genes encoding pro-
teins involved in tocopherol and carotenoid biosynthe-
sis and protection of cells from oxidative stress [33,39–
41]. Taken together, the catalytic activity of SynAlh1
as well as the expression pattern and locus of the cor-
responding gene indicate a function as a detoxifying
enzyme involved in the oxidative stress response.
2
5
This excludes retinal (C ) as a substrate and thus a
2
0
role of SynAlh1 in Synechocystis retinoid metabolism.
To confirm this result, we also designed in vivo systems
based on carotenoid-accumulating E. coli strains that
also express a carotenoid cleavage enzyme, yielding
retinal, a C25 and a C35 apocarotenal. In agreement
with the chain length restriction described above,
introduction of SynAlh1 led to C25 and C35 apocarote-
noid acids but not to retinoic acid.
The pronounced homology of SynAlh1 to alkanal-
converting aldehyde dehydrogenases such as human
ALDH3B1 [4,36] or A. thaliana ALDH3I1 [37]
prompted us to investigate these substrates. SynAlh1
converted aliphatic fatty aldehydes with a minimum
size of C6 (hexanal). SynAlh1 is the first aldehyde
dehydrogenase described so far that converts both sub-
strate classes, i.e. apocarotenals and fatty aldehydes.
The in vitro activity with apocarotenals and alkanals
raised the question regarding the main substrates of
SynAlh1. Direct comparison was hindered by the lack
of availability of a single analytical system that is
capable of detecting acids from both substrate classes.
Therefore, two analytical methods were used. In addi-
tion, the complicated biphasic (micellar) systems used
hindered more refined kinetic analysis at various sub-
strate concentrations. The results obtained in time-
course experiments performed using one concentration
To shed more light on the biological function of
SynAlh1, we generated knockout lines that showed
alteration in pigment pattern, growth and photosyn-
thetic performance (unpublished data). However,
probably due to the microevolution observed in
Synechocystis strains held in laboratories [42], we
observed this phenotype only in a certain genomic back-
ground, and, moreover, the extent of these changes
decreased over time. Given the importance of the scav-
enging function presumably exerted by SynAlh1, it
appears likely that Synechocystis does not rely on a sin-
gle enzymatic activity for scavenging of toxic aldehydes.
(80 lM) for all of the substrates indicate that apoca-
rotenals are the preferred substrates under these
in vitro conditions. Nevertheless, the considerable con-
version of alkanals indicates that SynAlh1 may cata-
lyze the oxidation of both types of lipid-derived
aldehydes in cells. This assumption is supported by the
presence of homologs from other cyanobacteria as well
as from non-photosynthetic, carotenoid-free bacteria
that show similarities to SynAlh1 ranging from ~ 72%
Experimental procedures
Cloning of SynAlh1
The SynAlh1 coding sequence (slr0091) was amplified from
Synechocystis sp. PCC 6803 genomic DNA using primers
5′-ATGAATACTGCTAAAACTGTTGTTGC-3′ and 5′-CT
AGGAGAACAACTTTTTGATTAAATTC-3′. The PCR
product was inserted into pSC-A (Stratagene, Amsterdam,
Netherlands) to produce pSC-Alh1. The gene equipped
with tow flanking BamHI restriction sites was obtained by
PCR, using primers 5′-TGTGGATCCATGAATACTGCT
AAAACTG-3′ and 5′-TGTGGATCCCGAGAACAACTT
TTTGATTA-3′. The PCR product was cloned into pQE60
(accession number YP_0023755503.1 from the cyano-
bacterium Cyanothece sp.) to 69% (accession number
ZP_02613185.1 from Clostridium botulinum). Also, the
broad substrate specificity of SynAlh1 reflects the high
homology to both apocarotenal-converting YLO-1 and
eukaryotic ALDHs converting alkanals. Nevertheless,
the closest homologs of SynAlh1 belonging to ALDH
FEBS Journal 280 (2013) 3685–3696 ª 2013 FEBS
3691

An apocarotenal and alkanal aldehyde dehydrogenase
D. Trautmann et al.
(
Qiagen, Hilden, Germany) at the BamHI restriction sites.
octadecanals were produced by oxidation of the respective
fatty alcohols as follows: 0.5 g hexadecanol or octadecanol
The resulting plasmid pQE60-Alh1, encoding a His-tagged
SynAlh1 protein, was sequenced to confirm integrity and
orientation. To create a His-tagged SynAlh1 fused with
thioredoxin the Alh1 fragment was amplified from pQE60-
Alh1 using primers 5′-GCACTGCAGATGAATACTGCT
powder, 45 mL pentane and 1.2 g MnO
2
were stirred over-
night, and subsequently the MnO particles were removed
2
by centrifugation (5000 g, 4 ꢁC for 2 min). To separate
aldehydes from underivatized fatty alcohols, TLC was per-
formed using TLC Silica 60 plates (Merck, Darmstadt,
Germany) developed using light petroleum/diethyl ether
(4 : 1 v/v). To visualize the aldehyde band, a small part of
the plate’s brim was sprayed with Schiff’s reagent while
protecting the rest of the plate using a glass cover. The
aldehyde band was scraped off the plate, collected in
petroleum ether, and silica particles were removed by cen-
trifugation (5000 g, 4 ꢁC for 2 min). Using a Schiff’s
reagent assay and a standard curve with known concentra-
tions of dodecanal, the concentrations of hexadecanal and
octadecanal solutions were measured photometrically at
556 nm.
AAAAC-3′ (adding
a
5′ PstI restriction site) and
′- CTAATTAAGCTTAGTGATGGTGATG-3′ (adding a
′ HindIII restriction site after the 6xHis tag and stop
5
3
codon). The PstI/HindIII-digested PCR product and
pThio-Dan1, a derivative of pBAD/Thio (Invitrogen, Pais-
ley, UK) carrying the pUC18 polylinker, were ligated to
yield pThio-Dan1-Alh1-His. Plasmids for in vivo experi-
ments were generated by excision of the Alh1 fragment
from pQE60-Alh1 using BamHI and creation of blunt ends
using T4 DNA polymerase (New England Biolabs, Schwal-
bach, Germany), followed by cloning into PmeI-digested
vectors pBAD-CarX, pThio=CarS and pBAD-CarT, encod-
ing various carotenoid cleaving oxygenases.
For a 200 lL assay volume, substrate micelles were pro-
duced by mixing ethanolic apocarotenoids (80 lM final con-
centration) with 50 lL of ethanolic 4% octyl-b-glucoside
solution, drying in a vacuum centrifuge, and dissolving the
resulting gel in 100 lL of 2 x incubation buffer (200 mM
pyrophosphate, 200 mM NaCl, pH 7.5, 2 lL of 100 mM
ꢀ
PCR was performed using Phusion high-fidelity DNA
polymerase (Finnzymes, Espoo, Finland) according to the
manufacturer’s instructions. PCR fragments were purified
TM
TM
from agarose gel bands using an illustra GFX PCR DNA
and gel band purification kit (GE Healthcare, Freiburg,
Germany). For ligations, T4 DNA ligase (New England Biol-
abs) was used according to the manufacturer’s instructions.
+
NAD ). Then 5 lL of crude lysate and water were added
to give a final volume of 200 lL. Assays were incubated at
2
8 °C for 30 min, reactions were stopped by adding 400 lL
acetone, and lipophilic compounds were extracted using
light petroleum/diethyl ether (1 : 2 v/v). For conversion
rates, the assays were scaled up to a final volume of
Recombinant expression of SynAlh1 and
preparation of crude lysate
4
00 lL (apocarotenals) or 1000 lL (alkanals). Apocarote-
For protein expression, a Tuner/Gro7 E. coli strain was gen-
erated by transformation of pGro7 (TaKaRa Bio Europe,
Mobitec, Goettingen, Germany) into Tuner (DE3) cells
noic extracts were subjected to HPLC analysis using
a-tocopherol acetate as internal and external standards to
correct for losses during extraction, and using integrated
peak areas, a b-carotene standard curve and specific molar
extinction coefficients to calculate conversions. Assays with
alkanals were analyzed spectrophotometrically using
the kinetic mode of a Shimadzu UV 2501 PC photometer
(Novagen, Darmstadt, Germany). Tuner/Gro7 cells were
transformed with pThio-Dan1-Alh1-His. Then 50 mL of 2 x
YT medium (Sigma-Aldrich, Taufkirchen, Germany) were
inoculated with 3 mL of overnight pre-culture, and grown at
2
8 °C until the attenuance at 600 nm was 0.5–0.8. Then pro-
(
Shimadzu Corp., Kyoto, Japan) by detecting the NADH
tein expression was induced using 0.2% w/v arabinose and
incubation at 28 °C for 4 h. Cells were harvested by centri-
fugation at 5000 g for 10 min. The pellet was resuspended in
increase during the reaction at 340 nm.
1
mL lysis buffer (50 mM NaH
2 4
PO , 300 mM NaCl, 1 mg/mL
In vivo assays
lysozyme, 1 mM dithiothreitol, 0.1% v/v Triton X-100, pH
8
1
1
.0) and incubated on ice for 30 min. After sonication with
0–15 pulses, the lysate was centrifuged for 30 min at
2 000 g to remove insoluble matter. The supernatant was
Carotenoid-accumulating E. coli strains were generated by
transforming XL-1 Blue E. coli cells with p-b, pGamma or
pTorulin, resulting in production of b-carotene, c-carotene
or torulene, respectively. p-Beta [17], or pTorulin [10] strains
were then transformed with pBAD-CarX-Alh1, pThio=
CarS-Alh1 or pBAD-CarT-Alh1 and suitable control plas-
mids. The 50 mL LB medium containing appropriate
antibiotics, and, in the case of the pTorulin strains, also
containing isopropyl b-D-thiogalactoside to induce caroten-
oid synthesis, were inoculated with 3 mL of overnight
pre-culture and grown at 28 °C until the attenuance at
600 nm was 0.5–0.8. Then protein expression was induced
transferred to a new tube and used for in vitro assays.
In vitro assays
Apocarotenoic substrates were kindly provided by BASF
(Ludwigshafen, Germany) and purified by TLC using TLC
Silica 60 plates (Merck, Darmstadt, Germany). Alkanals
except hexadecanal and octadecanal were purchased from
Sigma-Aldrich (Taufkirchen, Germany). Hexadecanal and
3
692
FEBS Journal 280 (2013) 3685–3696 ª 2013 FEBS

D. Trautmann et al.
An apocarotenal and alkanal aldehyde dehydrogenase
using 0.2% w/v arabinose and incubation at 25 °C over-
night. Cells were harvested by centrifugation at 5000 g for
diameter, 0.25 lm polyethylene glycol film; Phenomenex,
Aschaffenburg, Germany) using an initial temperature of
100 °C for 2 min, increasing to 300 °C with a ramp of
15 °C/min, and then kept at 300 °C for 2 min. The
constant He carrier gas flow was 1 mL/min, the split flow
was 50 mL/min, and the inlet temperature was 260 °C.
Standard electrospray ionization was used, with an ion
source potential of 70 eV and a source temperature of
1
0 min, and carotenoids were extracted by resuspending the
pellet in 150 lL of 50 mM HEPES pH 8.0, 20 lL
formaldehyde and 4 mL methanol, and sonication. Then
6
mL chloroform and water were added to achieve phase
separation. The chloroform phase was collected, reduced to
dryness, and resolved in 80 lL chloroform for HPLC analysis.
2
00 °C. The compounds were identified by comparison of
the mass spectra with the National Institute of Standards
and Technology database (http://www.nist.gov).
Analytical methods
HPLC analysis of extracts from in vitro assays was per-
formed using a Shimadzu UFLC XR system equipped with
an SPD-M20A diode array detector. The separation was
Stress treatments and expression analyses
performed using
a
YMC C30 reversed-phase column
Synechocystis sp. PCC 6803 cultures were grown in liquid
(
150 mm long, 3.0 mm internal diameter, 5 lm particles;
BG11 medium [43] in Erlenmeyer flasks with constant illu-
À2 À1
YMC Europe, Dinslaken, Germany) using solvent A (1 : 1
v/v methanol/t-butylmethyl ether), and solvent B (120/40/4
v/v/v methanol/water containing 0.5% w/v ammonium ace-
tate/t-butylmethyl ether). The column was developed at a
flow rate of 0.8 mL/min, starting with 100% B, a 5 min gra-
dient to 40% B, a 10 min gradient to 20% B and a 2.5 min
gradient to 2% B, followed by holding at 2% B for 1.5 min.
Finally, the column was re-equilibrated to 100% B for
mination (20 lmol photons m Ás ) at 30 °C. For high-
light treatment, the light intensity was increased to
À2 À1
280 lmol photons m Ás . For cold stress, the temperature
was reduced to 20 °C. Harvesting, RNA isolation, reverse
transcription and real-time RT-PCR were performed as
described previously [26]. The following primers and probes
were used: Syn 16S forward, 5′-CAGTAGCTGGTCTGA
GAGGATGAG-3′; Syn 16S reverse, 5′-ACTGCTGCCTC
CCGTAGGA-3′; Syn 16S locked nucleic acid probe, VIC/
YY-5′-CCACACTGGGACTGAG-3′; Syn Alh1 forward,
5′-GGCAATTCAGATGCTTTTCG-3′; Syn Alh1 reverse,
5′-ACTAACTAACCGTGACCATTG-3′; Syn Alh1 locked
nucleic acid probe, FAM-5′-ATCCAGCCCATAGCCCT
3′-BHQ1. An ABI Prism 7000 (Applied Bioystems, Foster
City, CA, USA) was used for real-time RT-PCR.
2
min. Extracts of assays with dialdehydes were analyzed
using a Macherey-Nagel (Dueren, Germany) HPLC C18 col-
umn (250 mm long, 4.6 mm internal diameter) on a Waters
(Eschborn, Germany) HPLC system equipped with a
Waters 996 photodiode array detector, and solvent A (1 : 1
v/v MeOH/t-butylmethyl ether) and solvent B (120 : 40 : 4
v/v/v MeOH/water/t-butylmethyl ether). The column was
developed at a flow rate of 1 mL/min, starting with 100% B
for 7 min, followed by a 8 min gradient to 0% B, then
RT-PCR design for operon analysis
1
00% A was maintained for 20 min, followed by re-equili-
bration to 100% B for 10 min. For extracts from in vivo
assays, the column was developed using the same solvent
system at a flow rate of 0.6 mL/min as follows: the starting
conditions of 100% B were changed to 0% B in a gradient
over 20 min, then 0% B was maintained for 4 min, followed
by a re-equilibrating step to 100% B for 5 min.
RNA and cDNA were obtained as described previously [26].
To investigate whether two genes are transcribed together in
one molecule, PCRs were designed in order to amplify the
intergenic region plus adjacent parts of the surrounding
genes. PCR performed using cDNA as the template only led
to a PCR product if the two genes were transcribed together,
yielding one molecule of mRNA and subsequently one mole-
cule of cDNA. The intergenic region between slr0086 on the
left border of the putative operon and sll0078 on the reverse
strand was used as a negative control. The same PCRs per-
formed with genomic DNA as the template served as a posi-
tive control in which all amplicons were obtained, while
PCRs using RNA (negative control) yielded no PCR prod-
uct. The identity of the resulting RT-PCR fragments was
confirmed by sequencing and alignment. Table S3 lists the
oligonucleotides used for the RT-PCR analysis.
LC-MS analyses of HPLC-purified apocarotenoic acids
were performed as described previously [9], using a Thermo
LTQ mass spectrometer equipped with a Surveyor HPLC
system (Thermo Electron, Waltham, MA, USA).
For identification of fatty acid products by GC-MS anal-
ysis, in vitro alkanal assays were incubated for 2 h at 28 °C
and extracted using light petroleum/diethyl ether (1 : 2 v/v).
To protect carboxylic groups, methylation was performed
by boiling the dried extract in 40 lL trimethylsulfonium
hydroxide (Sigma-Aldrich, Taufkirchen, Germany) for
1
0 min. The sample was then applied to a Trace gas chro-
matograph (Thermo Finnigan, Thermo Scientific, Bonn,
Germany) coupled to a Trace DSQ II mass spectrometer
Homology analysis
(
Thermo Scientific). Fatty acids methyl esters were sepa-
Pairs of amino acid sequences were aligned using the Nee-
dle pairwise alignment tool (http://www.ebi.ac.uk/Tools/psa/)
rated on a ZB-WAX column (30 m long, 0.25 mm internal
FEBS Journal 280 (2013) 3685–3696 ª 2013 FEBS
3693

An apocarotenal and alkanal aldehyde dehydrogenase
D. Trautmann et al.
based on the Needleman–Wunsch alignment algorithm [44].
Multiple sequence alignments for phylogenetic trees were
calculated on the command line on Linux using the imple-
mented muscle algorithm [45], yielding an alignment in the
FASTA format. Phylogenetic trees were calculated using
the command line approach in QuickTree (http://www.
sanger.ac.uk/resources/software/quicktree/) on Linux. Quick-
Tree calculates phylogenetic trees on the basis of the neigh-
bor-joining algorithm [46]. Pseudo replicates were
calculated 1000 times and are given as bootstrap values at
the nodes of the tree in Fig. 1. The trees were graphically
visualized using FigTree software version 1.3.1 (http://tree.-
bio.ed.ac.uk/software/figtree/).
(ALDH3A1): biochemical characterization and
immunohistochemical localization in the cornea.
Biochem J 376, 615–623.
9 Estrada AF, Youssar L, Scherzinger D, Al-Babili S &
Avalos J (2008) The ylo-1 gene encodes an aldehyde
dehydrogenase responsible for the last reaction in the
Neurospora carotenoid pathway. Mol Microbiol 69,
1207–1220.
10 D ꢀı az-S ꢀa nchez V, Estrada AF, Trautmann D, Al-Babili
S & Avalos J (2011) The gene carD encodes the
aldehyde dehydrogenase responsible for
neurosporaxanthin biosynthesis in Fusarium fujikuroi.
FEBS J 278, 3164–3176.
1
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Acknowledgements
This work was supported by Deutsche Forschungsgeme-
inschaft grant number AL 892/1-4 and the Deutsche
Forschungsgemeinschaft Graduate School (GRK1305).
We are indebted to Hansgeorg Ernst (BASF, Ludwig-
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FEBS Journal 280 (2013) 3685–3696 ª 2013 FEBS
3695

An apocarotenal and alkanal aldehyde dehydrogenase
D. Trautmann et al.
Fig. S2. HPLC analysis of in vivo activity tests for
b-apo-4′-carotenal and retinal conversion by SynAlh1.
Table S1. Homology of selected ALDHs to the
Synechocystis enzyme SynAlh1.
Supporting information
Additional supporting information may be found in
the online version of this article at the publisher’s web
site:
Table S2. List of aldehyde substrates tested with
SynAlh1.
Fig. S1. UFLC analysis of in vitro incubations with
Table S3. List of oligonucleotides used for operon
analysis (see Fig. 6).
3
′-OH-b-apo-8′-carotenal, 3′-OH-b-apo-12′-carotenal,
-oxo-b-apo-8′-apocarotenal and 8′-apo-lycopenal.
4
3
696
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