Báo cáo khoa học: Analysis of the NADH-dependent retinaldehyde reductase activity of amphioxus retinol dehydrogenase enzymes enhances our understanding of the evolution of the retinol dehydrogenase family pot

Analysis of the NADH-dependent retinaldehyde reductase
activity of amphioxus retinol dehydrogenase enzymes
enhances our understanding of the evolution of the retinol
dehydrogenase family
Diana Dalfo
´
, Neus Marque
´
s and Ricard Albalat
Departament de Gene
`
tica, Facultat de Biologia, Universitat de Barcelona, Spain
Retinoic acid (RA) regulates critical physiologic pro-
cesses in vertebrates, such as anterior–posterior pattern
formation, cell proliferation, tissue differentiation,
morphogenesis, and embryonic development [1]. The
main source of retinoids stems from the enzymatic
cleavage of dietary b-carotenes, which produces retin-
aldehyde. This, in turn, is reduced to retinol, which is
subsequently esterified to retinyl esters and stored in
the liver [2]. Upon demand, these esters can be hydro-
lyzed to retinol, which is released into the circulation
to be used in target tissues, to undergo oxidation into
retinaldehyde, and to be further transformed into RA.
Retinol dehydrogenase and retinaldehyde reductase
activities are therefore major players in retinoid meta-
bolism, making essential contributions to the physio-
logic control of RA availability. In vertebrates, retinol
dehydrogenase activity has been classically associated
with two distinct protein families, the short-chain
Keywords

cephalochordates; chordate evolution;
retinaldehyde reductases; retinoic acid
metabolism; retinol dehydrogenases
Correspondence
R. Albalat, Departament de Gene
`
tica,
Facultat de Biologia, Universitat de
Barcelona, Av. Diagonal, 645, 08028
Barcelona, Spain
Fax: +34 934034420
Tel: +34 934029009
E-mail:
Website: />indexen.htm
(Received 9 March 2007, revised 4 May
2007, accepted 30 May 2007)
doi:10.1111/j.1742-4658.2007.05904.x
In vertebrates, multiple microsomal retinol dehydrogenases are involved in
reversible retinol ⁄ retinal interconversion, thereby controlling retinoid meta-
bolism and retinoic acid availability. The physiologic functions of these
enzymes are not, however, fully understood, as each vertebrate form has
several, usually overlapping, biochemical roles. Within this context, amphi-
oxus, a group of chordates that are simpler, at both the functional and
genomic levels, than vertebrates, provides a suitable evolutionary model for
comparative studies of retinol dehydrogenase enzymes. In a previous study,
we identified two amphioxus enzymes, Branchiostoma floridae retinol dehy-
drogenase 1 and retinol dehydrogenase 2, both candidates to be the
cephalochordate orthologs of the vertebrate retinol dehydrogenase
enzymes. We have now proceeded to characterize these amphioxus
enzymes. Kinetic studies have revealed that retinol dehydrogenase 1 and

retinol dehydrogenase 2 are microsomal proteins that catalyze the reduc-
tion of all-trans-retinaldehyde using NADH as cofactor, a remarkable com-
bination of substrate and cofactor preferences. Moreover, evolutionary
analysis, including the amphioxus sequences, indicates that Rdh genes were
extensively duplicated after cephalochordate divergence, leading to the gene
cluster organization found in several mammalian species. Overall, our data
provide an evolutionary reference with which to better understand the
origin, activity and evolution of retinol dehydrogenase enzymes.
Abbreviations
AKR, aldo-keto reductase; AR, aldose reductase; CRAD, cis-retinol/androgen dehydrogenase; ER, endoplasmic reticulum; GFP, green
fluorescent protein; HAR, human aldose reductase; HSD, hydroxysteroid dehydrogenase; HSI-AR, human small intestine aldose reductase;
MDR, medium-chain dehydrogenase ⁄ reductase; NLS, nuclear localization sequence; PAN2, pancreas protein 2; RA, retinoic acid; RRD,
mouse retinal reductase; SDR, short-chain dehydrogenase ⁄ reductase.
FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS 3739
retinol dehydrogenases [short-chain dehydrogenase ⁄
reductase (SDR)-RDHs] and the medium-chain
alcohol dehydrogenases [medium-chain dehydrogenase ⁄
reductase (MDR)-ADHs] [3,4]. Despite the many bio-
chemical studies on these two protein families, the major
enzyme(s) responsible for the in vivo oxidation of retinol
remains uncertain. In previous studies, we analyzed
the functionality and evolution of the MDR-ADH
family [5–8,9]. Here, we focus on the contribution of
SDR-RDH enzymes to retinol ⁄ retinal metabolism.
Classically, RDH enzymes have been regarded as a
complex vertebrate group of microsomal proteins
that catalyze the conversion of retinol to retinaldehyde
in vitro using NADH as cofactor [4]. However, the
RDH family contains many enzymes with diverse
substrate specificities towards cis and trans isomeric

forms, and, mostly, toward steroids. Hence, attempts to
determine the physiologic contribution of each RDH
enzyme to RA metabolism have been impaired by the
variety of enzymes as well as by the overlaps in substrate
recognition.
Substantial multiplicity and redundancy is also pre-
sent in the reductive direction of the pathway. Four
vertebrate protein families have been associated with
retinaldehyde reduction. Members of the SDR-RDH
group such as RDH2, RDH5 and 17b hydroxysteroid
dehydrogenase type 9 (17bHSD9) [10–12], non-RDH
SDR enzymes, including mouse retinal reductase
(RRD), retinal short-chain dehydrogenase/reductase 1
(retSDR1), photoreceptor outer segment all-trans
retinol dehydrogenase (prRDH), retinal reductase 1
(RalR1) and pancreas protein 2 (PAN2) [13–17],
MDR-ADH forms such as ADH1, ADH4 [18] and
amphibian ADH8 [19], and members of the aldo-keto
reductase superfamily, including human aldose reduc-
tase (AR), human small intestine aldose reductase
(HSI-AR) and chicken aldo-keto reductase (AKR)
[20,21], all catalyze retinaldehyde reduction in vitro.
To shed light on the evolutionary origin and physio-
logic basis of the RDH and retinaldehyde reductase
multiplicity of vertebrates, analysis of the cephalochor-
date amphioxus is invaluable. Cephalochordates are
useful organisms for comparative analyses, as their low
gene complexity and archetypical body plan organiza-
tion suggest that they retain many ancient characteris-
tics. Amphioxus did not undergo the extensive gene

duplications that occurred during early vertebrate evo-
lution [22], but rather exhibits an RA-signaling system
and a retinoid content comparable to that of verte-
brates [23,24]. In a previous study, we identified two
enzymes, RDH1 and RDH2, that belong to the SDR-
RDH group in the species Branchiostoma floridae [25].
Here we present experimental data showing that these
two enzymes are endoplasmic reticulum (ER)-associ-
ated proteins that may participate in retinoid metabo-
lism, by catalyzing retinaldehyde reduction. Moreover,
phylogenetic analysis indicates that most vertebrate
RDHs derive from lineage-specific tandem duplications
of an ancestral form that may resemble the current
amphioxus enzymes. The novel vertebrate RDH
enzymes would have evolved new biochemical activities
in retinoid and steroid metabolism after cephalochor-
date divergence, thereby contributing to the increased
physiologic complexity of the vertebrate subphylum.
Results
Enzymatic properties of recombinant RDH1 and
RDH2
Amphioxus RDH1 and RDH2 proteins tagged at the
N-terminus with the hemagglutinin (HA) epitope were
produced in COS-7 cells and purified in the microsom-
al fraction. The enzymatic activity of this fraction was
assayed against retinoids. Given that most vertebrate
RDHs can catalyze cis-retinol and ⁄ or trans-retinol oxi-
dation, these were the substrates initially evaluated.
Indeed, mouse RDH1 (kindly provided by J. L. Napoli,
University of California, Berkeley, CA, USA) was used

to monitor the retinol oxidation assay. Unexpectedly,
the oxidative activity observed for the amphioxus
enzymes was below the detection capacity of the assay,
< 0.002 nmol (Fig. 1A–C), although a wide range of
conditions were used: from pH 6 to 9, 5–12.5 lm
all-trans-retinol, 0.5–2 mm NAD
+
and NADP
+
, and
10–100 lg of microsomes obtained from independent
assays. Negligible activity was also observed when
9-cis-retinol was assayed (data not shown). Next, we
analyzed whether RDH1 and RDH2 exhibited reduc-
tase activity (Fig. 1D,E). Retinol production in vitro
increased 2.5-fold and 25-fold for RDH1 and RDH2,
respectively, in the presence of NADH, as compared
to controls. However, these differences were not detec-
ted with NADPH, even though COS cells showed
intrinsic NADPH-dependent retinal reductase activity
[12]. The specific activity of each amphioxus enzyme
was 0.25 nmol of retinolÆmin
)1
Æ(mg of microsomes)
)1
for RDH1 incubated with 15 lm all-trans-retinal, and
1.4 nmol of retinolÆmin
)1
Æ(mg of microsomes)
)1

for
RDH2 with 12.5 lm all-trans-retinal (Table 1). Fur-
thermore, to examine whether RDH forms had isomer
specificity, we also assayed the RDH1 and RDH2
activities toward 9-cis-retinal. However, only residual
9-cis-retinal reductase activity, less than 0.03 nmolÆ
min
)1
Æmg
)1
, was detected for these two enzymes
(Fig. 1F,G). The reaction products were extracted and
Amphioxus retinol dehydrogenase enzymes D. Dalfo
´
et al.
3740 FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS
analyzed by RP-HPLC, and the kinetic constants of
the two RDHs for all-trans-retinal were determined
(Fig. 1H,I; Table 1). The apparent K
m
values of
RDH1 and RDH2 (8.7 lm and 8.9 lm, respectively)
were similar, whereas the maximum specific activities
(0.3 nmolÆmin
)1
Æmg
)1
and 2.3 nmolÆmin
)1
Æmg

)1
, respect-
ively) and the maximum specific activities ⁄ K
m
ratios
(0.03 and 0.26, respectively) were > 7.5-fold higher for
RDH2 than for RDH1. Finally, the apparent cofactor
K
m
values of amphioxus enzymes (Fig. 1J,K; Table 1)
were 224 lm and 98 lm NADH for RDH1 and
RDH2, respectively, whereas no significant activity
was detected with NADPH. This cofactor preference is
consistent with the presence and absence of specific
amino acids at certain positions in the amphioxus
enzymes (Fig. 2A): both enzymes contain the D and T
residues at the equivalent positions of cow RDH5 for
NADH specificity, and lack any positively charged
amino acid at the corresponding position of the rat
RDH2 K64, which may be essential for NADPH
preference [26].
Activity of recombinant RDH1 and RDH2
in intact cells
Amphioxus RDH1 and RDH2 and mouse RDH1 were
expressed in COS-7 cells to evaluate their activities
with retinoids in an intact cell system. In agreement
Fig. 1. Enzymatic activity of amphioxus
RDH1 and RDH2 enzymes. The biochemical
activity of the microsomal fraction of COS-7
cells transfected with amphioxus HA-RDH1-

expressing (A, D, F), HA-RDH2-expressing
(B, E, G) and mouse Rdh1-expressing (C)
constructs was analyzed. For oxidative reac-
tions (A–C), the microsomal fraction (15 lg)
was incubated with all-trans-retinol (10 l
M)
and NAD
+
(1 mM) at pH 8.0 for 15 min at
37 °C. For retinal reduction, the microsomal
fraction (15 lg) was incubated with 10 l
M
all-trans-retinal (D, E) or 9-cis-retinal (F, G)
and NADH (1 m
M) at pH 6.0 for 15 min at
37 °C. Elution was monitored at 380 nm for
retinal detection (A–C) and 325 nm for ret-
inol (D–G) detection. The values for all-trans-
retinaldehyde reduction of amphioxus RDH1
(H) and RDH2 (I) were determined at 1 m
M
NADH using eight concentrations of sub-
strate, from 0.5 to 20 l
M and from 0.5 to
15 l
M for RDH1 and RDH2, respectively.
The apparent K
m
values for cofactor NADH
were determined at 15 l

M and 12.5 lM all-
trans-retinaldehyde for RDH1 (J) and RDH2
(K), respectively, using six concentrations of
cofactor, from 0.005 to 1.5 m
M. Assays
were performed with 15 lg of microsomes
for 15 min at 37 °C. Each point represents
the average of three replicates.
D. Dalfo
´
et al. Amphioxus retinol dehydrogenase enzymes
FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS 3741
with the biochemical analysis of the microsomal-puri-
fied enzymes, amphioxus RDH1 and RDH2 catalyzed
the reduction of all-trans-retinaldehyde into all-trans-
retinol in intact cells (Fig. 3A), whereas RDH1- and
RDH2-transfected cells showed no differences from
mock-transfected cells in the generation of all-trans-
retinaldehyde from all-trans-retinol (Fig. 3B). Mock-
transfected COS-7 cells reduced all-trans-retinaldehyde,
indicating that the cells harbor reductases. Transfec-
tion with amphioxus RDH1 cDNA produced a net
47 pmol and 99 pmol of retinol per mg of total protein
after 1 h of incubation with 10 lm and 20 lm of
retinal, respectively. RDH2 enzymes were more effi-
cient than RDH1 enzymes, and generated 64 and
134 pmol of retinol in the same assay conditions.
Overall, transfection with RDH1 and RDH2 cDNA
increased the level of retinaldehyde reduction by
 35% and  50%, respectively, as compared to the

mock-transfected cells. Mouse RDH1, in contrast,
oxidized retinol to retinaldehyde (Fig. 3B) but did not
support retinaldehyde reduction. Indeed, mouse RDH1
decreased the amount of retinol generated in the assays
with retinaldehyde incubation (data not shown), sug-
gesting that the substrate used by this enzyme was the
retinol generated from retinaldehyde by endogenous
COS-7 cell reductase activity.
Intracellular localization
COS-7 cells were transiently transfected with con-
structs expressing the amphioxus RDH1 and RDH2
enzymes fused to several peptides: HA epitope, green
fluorescent protein (GFP) and b-galactosidase. In
agreement with the RDH1 and RDH2 purification in
the microsomal fraction (Fig. 2D), immunostaining of
cells expressing HA-RDH1 and HA-RDH2 proteins
revealed a typical pattern of ER-associated proteins,
with no nuclear staining or plasma membrane localiza-
tion observed (Fig. 2B,C). GFP fusion was used to
visualize the intracellular localization in living cells,
thereby avoiding any artefacts caused by the cell
fixation process. RDH2 fused to GFP either at the
C-terminus (RDH2
1)335
-GFP) or at the N-terminus
(GFP-RDH2
1)335
) of the enzyme (Fig. 2E,I) exhibited
a pattern that overlapped with the ER-Tracker Blue
White DPX, which was used as a specific ER marker

in living cells (Fig. 2F,J). The subcellular localization
of the RDH2 enzyme (RDH2
1)335
) fused to the b-ga-
lactosidase protein was also consistent with a typical
pattern of ER-associated proteins (Fig. 2M).
Table 1. All-trans-retinal activity and kinetic constants of B. floridae
RDH enzymes compared with those of known vertebrate retinal
reductases. Values are from this work, B. floridae RDH1 (BfRDH1)
and BfRDH2, or from the literature [6-10,13,16,17,25]. ND, not
determined. HAR, human aldose reductase.
All-trans-retinal NADH
Specific
activity
(nmolÆ
min
)1
Æmg
)1
)
Maximum
specific
activity
(nmolÆ
min
)1
Æmg
)1
)
K

m
(lM)
K
m
(lM)
BfRDH1 0.25 0.3 ± 0.06 8.7 ± 4.1 224 ± 81
BfRDH2 1.4 2.3 ± 0.5 8.9 ± 3.8 98 ± 25
RalR1 ND 18 ± 0.05 0.5 ± 0.05 1300 ± 200
PAN2 ND 27 ± 1 0.08 ± 0.02 1060 ± 70
RRD ND 40 ± 1 2.3 ND
retSDR1 0.04 ND ND ND
HAR ND 15 ± 1
a
10 ± 2 ND
HIS-AR ND 193 ± 4
a
19 ± 4 ND
Chicken
AKR
ND 170 ± 15
a
32 ± 4 ND
RDH2 0.25 ND ND ND
17bHSD9 0.17 ND ND ND
RDH5 16 ND ND ND
a
k
cat
values in min
)1

.
Fig. 2. ER subcellular localization of amphioxus RDH1 and RDH2 proteins. (A) Sequence alignment of amphioxus RDH1 and RDH2 enzymes.
Amino acid substitutions are shown and identities are represented by dashes. The active site (YTVAK) and the cofactor-binding motifs are
marked in bold. The D43, T67 and A69 residues, involved in cofactor specificity, are indicated by asterisks. Flanking the N-terminal hydropho-
bic segment, the LERGR motif is underlined. Arrows indicate the truncated RDH2 forms fused to GFP or to b-galactosidase proteins. (B, C)
Immunostaining with an antibody to HA of COS-7 cells transfected with constructs encoding HA-RDH1 and HA-RDH2, respectively, and
examined using confocal microscopy. (D) Western blot of the pellets after 13 000 g (lanes 2 and 4) and 100 000 g centrifugations (lanes 1
and 3, microsomal fractions) of homogenates of COS cells transfected with HA-RDH1 (lanes 1 and 2) and HA-RDH2 (lanes 3 and 4). (E–L)
In vivo localization of RDH2 in the ER of the cells. COS-7 cells were transfected with constructs encoding RDH2
1)335
-GFP (E), RDH2
1)28
-
GFP (G), RDH2
1)58
-GFP (H), GFP-RDH2
1)335
(I), GFP-RDH2
295)335
(K) and GFP (L). ER-tracker Blue White DPX marker was used to specific-
ally visualize the ER in living cells (F, J). (M–R) Localization of RDH2-b-galactosidase chimeric proteins. COS-7 cells were transfected with
constructs encoding the full-length (RDH2
1)335
) (M) and four C-terminal truncated RDH2 forms (RDH2
1)229
, RDH2
1)165
, RDH2
1)137
and

RDH2
1)58
) (N–Q, respectively) fused to the NLS-b-galactosidase protein, and with the pb-galactosidase-N2 empty vector (R). The pb-galac-
tosidase-N2 vector contains a nuclear localization sequence (NLS) 5¢ to the LacZ gene. The SV40 NLS localizes the b-galactosidase codified
by the empty vector to the nucleus. Cells were immunostained with an antibody to b-galactosidase and examined using a Zeiss Axiophot
fluorescence microscope.
Amphioxus retinol dehydrogenase enzymes D. Dalfo
´
et al.
3742 FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS
To analyze the contribution of protein domains to
the ER anchorage, the localization of the full-length
enzyme was compared with those of five truncated
forms. The pattern of the full-length construct was
essentially identical to that of the C-terminal truncated
RDH2 forms (RDH2
1)229
, RDH2
1)165
, RDH2
1)137
,
and RDH2
1)58
RDH2
1)28
) fused either to b-galactosi-
dase (Fig. 2N–Q) or to GFP (Fig. 2G,H), whereas the
b-galactosidase and GFP controls showed a signal,
mainly in the nucleus (Fig. 2L,R). The observation

D. Dalfo
´
et al. Amphioxus retinol dehydrogenase enzymes
FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS 3743
that nuclear or cytosolic staining was not increased for
any construct suggested that the N-terminal segment
would be sufficient to target and anchor the protein to
the ER membranes. Furthermore, we analyzed the
contribution of the C-terminal end to ER localization.
We fused the last 41 amino acids of the amphioxus
RDH2 enzyme (a region equivalent to the reported
C-terminal segment of mouse RDH1 [27]) to GFP:
GFP-RDH2
295)335
. The protein did not localize to the
ER of transfected COS cells, but showed a diffuse
signal similar to that of the GFP control (compare
Fig. 2K,L).
Evolution of the RDH group
tblastn comparisons showed that the sequences most
similar to the amphioxus enzymes were those of the
vertebrate RDHs (E-value ¼ 2e-75 and 1e-71 with Pan
troglodytes, similar to sterol ⁄ retinol dehydrogenase, for
RDH1 and RDH2, respectively). In the phylogenetic
analysis, amphioxus RDH branched outside a clade
comprising the ‘classic’ SDR-RDH1 ⁄ 2 ⁄ 3 ⁄ 4 ⁄ 5 ⁄ 6 ⁄ 7 ⁄ 9
(RDH1–7 ⁄ 9) members, which includes six human
enzymes [similar-RDH2, RDH4, orphan short-chain
dehydrogenase/reductase (SDR-O), RDH, RDH5 and
dehydrogenase/reductase member 9 (DHRS9)], eight

rat forms (similar-RDH1, RDH2, similar-RDH2,
RDH3, SDR-O, 17b-HSD19, RDH5 and DHRS9) and
11 mouse proteins [RDH1, RDH9, RDH6, truncated-
RDH, similar-RDH, cis-retinol/androgen dehydrogen-
ase (CRAD)-L, RDH7, SDR-O, 17b-HSD19, RDH5
and DHRS9] (Fig. 4A). Except for DHRS9, the genes
encoding these enzymes were not spread over several
chromosomes, but rather clustered in the human gen-
ome at 12q13–14 and in the syntenic regions of rat
and mouse chromosomes 7 and 10, respectively [28]
(Fig. 4B). DHRS9 genes are located in human chromo-
some 2, rat chromosome 3 and mouse chromosome 2,
which would be paralogous to chromosomes 12, 7 and
10, respectively [29]. The overall analysis, examining
the topology of the phylogenetic tree and the position
of each gene inside the cluster, was informative regard-
ing the orthology relationships of the distinct enzymes,
and allowed us to define five RDH classes (Fig. 4A). It
is of note that other vertebrate SDRs (some reported
as RDH enzymes), such as RDH8, RDH10, RDH11,
RDH12, RDH13, RDH14, similar to epidermal retinal
Fig. 4. (A) Phylogenetic relationship of the RDH1–7 ⁄ 9 and other retinoid ⁄ steroid active SDR forms from human (Hs), rat (Rn) and mouse
(Mm) genomes with the amphioxus RDH enzymes. A neighbor-joining tree was generated with the
CLUSTALX program, and confidence in
each node was assessed by 1000 bootstrap replicates. The RDH1–7 ⁄ 9 cluster comprises all the vertebrate sequences that group with the
amphioxus enzymes. Additional enzymes involved in retinoid ⁄ steroid metabolism appear to be distantly related (less than 55% of sequence
identity in the region used for the tree reconstruction, data not shown), and were therefore considered to be members of distinct SDR
groups. The bootstrap values defining each group are shown (black numbers). Internally, the RDH1–7 ⁄ 9 enzymes grouped into five classes,
I–V. The bootstrap values defining each class are shown (red numbers). (B) Structural organization of the human, rat and mouse RDH clus-
ters using the Map Viewer website from NCBI. The name of the each Rdh gene (black boxes) is indicated. Alternative names for each gene

are listed in supplementary Table S2. Orthology relationships among genes of several species are indicated (continuous lines). Notice that
Rdh5 genes are in the same chromosome but outside the RDH clusters (dotted line), and that Dhrs9 genes are located in distinct chromo-
somes. Genes flanking the RDH sequences are also depicted (green boxes). TAC3, tachykinin 3; KIAA0352 (ZBTB29), zinc finger and BTB
domain containing 39; ADMR, adrenomedullin receptor; PRIM1, primase polypeptide 1; NACA, nascent-polypeptide-associated complex
alpha-polypeptide; CD63, CD63 antigen; BLOC1S1, biogenesis of lysosome-related organelles complex-1, subunit 1; ABCB11, ATP-binding
cassette, subfamily B, member 11; LRP2, low-density lipoprotein receptor-related protein 2.
Fig. 3. Synthesis of retinol and retinaldehyde in transfected COS-7
cells expressing amphioxus RDH enzymes. (A) Different concentra-
tions (5, 10 and 20 l
M) of all-trans-retinaldehyde were added to
COS-7 cells expressing RDH1 (black bars) and RDH2 (white bars).
Retinol was extracted from the cells and analyzed by HPLC. The
bars represent the net retinol production per mg of total protein
after 1 h of incubation with the three substrate concentrations. (B)
Retinol oxidation in transfected cells was also evaluated for RDH1,
RDH2 and mouse RDH1 (gray bars), which was used as a positive
control of the reaction. The bars represent the net retinal produc-
tion per mg of total protein after 1 h of incubation with 5, 10 and
20 l
M all-trans-retinol.
Amphioxus retinol dehydrogenase enzymes D. Dalfo
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et al.
3744 FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS
A
B
D. Dalfo
´
et al. Amphioxus retinol dehydrogenase enzymes
FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS 3745

dehydrogenase 2 (RaDH9), epidermal retinal dehydro-
genase 2 (eRaDH2), retSDR1, DHRS4, 17b-HSD11,
17b-HSD12, 11b-HSD11, 11b-HSD12 and short-chain
dehydrogenase/reductase 10 isoform B (SCDR10B),
branched outside the vertebrate–cephalochordate clade
(Fig. 4A) and were located in diverse nonparalogous
mammalian chromosomes. These enzymes would be
therefore distantly related to the RDH1–7 ⁄ 9 forms and
should be considered members of separate enzyme
families. Indeed, the position of the amphioxus enzy-
mes in the phylogenetic tree implied that these families
are ancient, pre-dating the emergence of the chordate
phylum.
Discussion
The biochemical characterization revealed that the
amphioxus RDH enzymes catalyzed all-trans-retinal
reduction (Table 1, Figs 1 and 3). Unfortunately, com-
parison with other RDHs was limited to rat RDH2,
mouse 17bHSD9 and RDH5, as the reductase capacity
of most vertebrate RDHs has not been assayed. Thus,
we also compared amphioxus data with those from
other non-RDH vertebrate retinal reductases, such as
retSDR1, RalR1, PAN2, RRD, human AR, HSI, and
chicken AR (Table 1), although the comparison was
also hindered by the variety of assay conditions used.
We have shown that amphioxus enzymes show iso-
mer preference, trans versus cis retinal forms, as occurs
with the vertebrate RalR1, PAN2, RRD, retSDR1,
prRDH, HSI and chicken AKR enzymes. Moreover,
although retinol ⁄ retinal interconversion is a reversible

reaction, neither amphioxus RDH1 or RDH2 showed
significant activity towards retinol in the in vitro assays
with microsomal proteins or in the intact cell systems.
This strict preference towards the reductive direction
has been reported for other retinoid-active enzymes
(e.g. the vertebrate retinal reductases RRD [13], HAR
[20,21] and prRDH [15]), whereas other enzymes
(RalR1 [30], PAN2 [17], HSI and chicken AKR
[20,21]) also catalyze retinol oxidation, albeit with
considerably lower efficiency. The specific activities
towards all-trans-retinal of amphioxus enzymes were
0.25 nmolÆmin
)1
Æmg
)1
for RDH1 and 1.4 nmolÆmin
)1
Æ
mg
)1
for RDH2; these were 6.3-fold and 23-fold
higher, respectively, than that reported for retSDR1
(0.04 nmolÆmin
)1
Æmg
)1
), a photoreceptor enzyme that
reduces all-trans-retinal in the visual cycle [14]. The
specific activity of RDH2 was 5.6-fold and 10.8-fold
higher than that of rat RDH2 (0.25 nmolÆmin

)1
Æmg
)1
)
[10] and mouse 17bHSD9 (0.13 nmolÆmin
)1
Æmg
)1
) [12],
respectively, whereas the activity of amphioxus RDH1
was comparable to those of these enzymes. In contrast,
the amphioxus enzymes showed lower retinaldehyde
reductase efficiency than some vertebrate enzymes. The
specific activity of mouse RDH5 with all-trans-retinal
(16 nmolÆmin
)1
Æmg
)1
[11]) was higher than that of
either RDH1 or RDH2; RalR1 [30], PAN2 [17] and
RRD [13] showed lower K
m
and higher maximum spe-
cific activity values for all-trans-retinal (Table 1) and
therefore higher maximum specific activity ⁄ K
m
ratios,
which are a measure of the catalytic effectiveness of
the enzymes; the AKR members (human AR, HSI and
chicken AKR) [20,21] showed similar K

m
values but
higher maximum specific activities (Table 1), which
also implied higher maximum specific activity ⁄ K
m
ratios, i.e. greater effectiveness, for the vertebrate
AKR than for the cephalochordate forms. Overall,
these data support our finding that amphioxus RDH
shows retinal reductase activity within the range repor-
ted for diverse vertebrate enzymes.
The most significant difference between the amphi-
oxus and the other retinal reductases was, nevertheless,
their preference for the NADH cofactor. To our
knowledge, these are the first SDR retinaldehyde
reductases reported to use NADH instead of NADPH.
Conventionally, cofactor preference had been directly
related to the oxidative or reductive direction of the
reaction. Therefore, it was assumed that oxidative
RDHs would be NADH-dependent, whereas NADPH
enzymes would catalyze the reductive reaction. This
hypothesis was based on the ratios between the oxid-
ized and reduced forms of the coenzymes [31]. It
appears, however, that cofactor ratios vary greatly
among organs and cell types, and that the redox status
can be greatly influenced by external factors [32].
Noticeably, amphioxus enzymes have the capacity to
reduce retinaldehyde to retinol in intact cells (Fig. 3),
suggesting that the endogenous NADH level in COS-7
cells is enough to allow this reduction. Our data sup-
port the contention that coenzyme preference does not

necessarily constrain the direction of the reaction. In
fact, several RDHs (e.g. human, mouse and bovine
RDH10 [33] and human RDH-E2 [32]) prefer NADP
to NAD as a cofactor.
Structurally, most of the cytosolic SDR enzymes are
composed of 250–280 amino acid residues [34,35],
whereas the membrane-associated SDR enzymes are
extended at both the N-terminal and C-terminal ends
by up to about 350 amino acids [36,37]. Amphioxus
RDH1 and RDH2 were 332 and 335 amino acids long,
respectively, and their subcellular localization in trans-
fected COS-7 cells concurred with that of ER-associ-
ated proteins. The observation that nuclear or
cytosolic staining was not increased for any C-terminal
truncated constructs indicated that the N-terminal
Amphioxus retinol dehydrogenase enzymes D. Dalfo
´
et al.
3746 FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS
segment was sufficient to target and anchor the protein
to the ER membranes. As the shortest segment was
only 28 amino acids long (from the initial methionine
to the LERGR motif), it can be assumed that the sign-
aling sequence for ER localization falls in this region
of the protein. In addition to the targeting function,
signal sequences are also crucial in protein topogenesis,
as they participate in the final cytosolic ⁄ lumenal orien-
tation. The most prominent determinant of signal ori-
entation is the distribution of charged amino acids at
either end of the hydrophobic sequence. According to

the ‘positive-inside’ rule, the most positively charged
flanking transmembrane segment is usually found on
the cytosolic side of the membrane [38,39]. Amphioxus
RDH did not show positively charged amino acids at
the N-terminus of the signaling sequence, but rather
contained the L24ERGR motif at the C-terminus
of the hydrophobic sequence, thereby resembling
the R19ERQV, R19ERKV, R19VRQV and
R19DRQ(S ⁄ C) sequences of a number of vertebrate
RDHs [40]. This motif would predict, therefore, a
cytosolic orientation of the amphioxus enzymes.
For several RDH enzymes, the C-terminal trans-
membrane segment forms a hydrophobic helix-turn-
helix that is sufficient to retain them in the ER, e.g.
CRAD1 [41]. We fused the last 41 amino acids of the
amphioxus RDH2 enzyme (a region equivalent to the
reported C-terminal segment of mouse RDH1 [27]) to
GFP. This protein did not localize to the ER of trans-
fected COS cells (Fig. 2K), and therefore the C-ter-
minal end of amphioxus RDH2 was not sufficient for
ER targeting. Amphioxus RDH2 was structurally
more similar to the enzymes that rely exclusively on
the N-terminal hydrophobic segment as membrane
anchor (e.g. mouse RDH1 [27], human 11bHSD1 [42],
human 11bHSD2 [43] and human RalR1 [16]) than to
the other RDHs such as bovine RDH5 [44], mouse
RDH4 [45] and mouse CRAD1 [41,46], which would
be anchored to both the N-terminal and C-terminal
hydrophobic segments.
Finally, evolutionary analysis including the amphi-

oxus enzymes highlighted the relevance of using evolu-
tionary criteria rather than biochemical classifications
for gene nomenclature and family description. The
phylogenetic tree and the genomic organization now
permit a proper definition of the vertebrate RDH1–7 ⁄ 9
group and reveal an internal classification of mamma-
lian RDH1–7 ⁄ 9 enzymes into five classes, pointing to
recurrent gene tandem duplications as the most likely
mechanism for the cluster organization of the Rdh
genes. In a recent study [47], Belyaeva & Kedishvili
proposed a model for the evolution of the vertebrate
RDH1–7 ⁄ 9 group (referred to as the RDOH-like SDR
group in their article). On the basis of a comparative
genomic and phylogenetic analysis that included sev-
eral vertebrate species, these authors suggested that
early in vertebrate evolution, an initial tandem duplica-
tion of the Rdh ancestor gave rise to the ‘ Dhrs9 ⁄ Rdhl–
11-cis-RDH-homolog’ cluster. The 11-cis-RDH-homolog
gene was afterwards duplicated by a mechanism that
implied translocation of the new copy to another
region of the genome to generate the 11-cis-RDH ⁄ Rdh5
gene. Later on, the 11-cis-RDH ⁄ Rdh5 gene underwent
several tandem duplication events in its new chromoso-
mal location, which led to the appearance of the cur-
rent RDH cluster in tetrapods. However, an alternative
evolutionary model is possible (Fig. 5). We hypothesize
that an initial tandem duplication of an Rdh ancestor
gave rise to a two-gene cluster, which was further
duplicated, probably as a result of the genome duplica-
tion events that took place during early vertebrate evo-

lution [22]. During fish evolution, one gene was lost,
leading to the ‘Rdh5 (AAH97151) + Dhrs9 (Rdhl-
like)+Rdhl’ combination currently found in zebrafish
[47]. In amphibians and mammals, extra tandem dupli-
cations produced the RDH clusters found in Xenopus,
human, mouse, rat, dog and cow [47] (Fig. 4B). Even-
tually, the mammalian Rdhl ⁄ CX410306 ortholog was
lost. The closer phylogenetic relationship of RDH5 ⁄ 11-
cis-RDH to Rdhl ⁄ CX410306 enzymes than to RDH4-
SDR-O-RDH forms [47] is consistent with this model.
Furthermore, the observation that the Ddrs9 genes and
the Rdh5-RDH cluster are located in paralogous chro-
mosomes also supports our hypothesis.
In conclusion, the analysis of amphioxus enzymes
contributes to improving our understanding of the
functional complexity of vertebrate gene families
regarding retinoid metabolism. However, to date, no
convincing enzymes for retinol oxidation have been
found among cephalochordate RDH members. The
full genome sequence of amphioxus, currently being
released, will allow comprehensive searches for novel
candidates, which may also have relevant physiologic
roles in the retinoid pathway of vertebrates.
Experimental procedures
Expression of HA-RDH, GFP-RDH, and
b-galactosidase-RDH proteins
To produce RDH1 and RDH2 proteins tagged at the
N-terminus with the HA epitope, the full-length coding
sequences of the Rdh1 and Rdh2 genes were PCR-amplified
(oligonucleotides 1–2, and 3–4, respectively; the oligonucleo-

tide sequences used in this study are provided in supple-
mentary Table S1) from plasmids containing the Rdh1 and
D. Dalfo
´
et al. Amphioxus retinol dehydrogenase enzymes
FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS 3747
Rdh2 cDNAs and cloned in the pACT2 vector (Clontech,
Mountain View, CA, USA). The HA-tagged Rdh1 and
Rdh2 coding fragments were released from the pACT2 vec-
tor and cloned into the pCDNA3 vector (Invitrogen, Carls-
bad, CA, USA). To fuse amphioxus RDH2 either at the
N-terminus or C-terminus of the GFP, the full-length
coding region, two C-terminal truncated RDH2 forms and
one N-terminal truncated RDH2 form were PCR-amplified
(RDH2
1)335
, oligonucleotides 5 and 6, amino acids 1–335,
full-length; RDH2
1)58
, oligonucleotides 5 and 7, amino
acids 1–58, truncated just after the cofactor-binding
sequence GXXXGXG; RDH2
1)28
, oligonucleotides 5 and
8, amino acids 1–28, truncated after the LERGR motif;
RDH2
295)335
, oligonucleotides 9 and 6, amino acids
295–335) and cloned into the pEGFP-N2 and pEGFP-C2
vectors (Clontech). To produce both the full-length and the

four C-terminal truncated RDH2 enzymes fused at the
N-terminus of b-galactosidase, the coding regions of Rdh2
were generated by PCR amplification: RDH2
1)335
(oligonu-
cleotides 10–11; amino acids 1–335, full-length), RDH2
1)229
(oligonucleotides 10–12; amino acids 1–229, lacking the
C-terminal end), RDH2
1)165
(oligonucleotides 10–13; amino
acids 1–165, truncated just before the active site, YXXXK),
RDH2
1)137
(oligonucleotides 10–14; amino acids 1–137,
truncated after the GLVNNAG region), and RDH2
1)58
(oligonucleotides 10–15; amino acids 1–58, truncated just
after the cofactor-binding sequence GXXXGXG). The
design of these constructs was based on the predicted trans-
membrane segments of the RDH2 enzyme given by the
tmpred [48], das [49] and hmmtop [50] programs (data not
shown). The five PCR fragments were cloned in the pbGal-
N2 vector, in frame at the 5¢ end of the coding region of
LacZ. This vector expresses b-galactosidase protein fused
to a nuclear localization sequence (NLS) driven by the
strong human cytomegalovirus immediate early promoter,
and was created by cloning the NLS-LacZ gene from the
PSP-1.72b-galactosidase plasmid [51] into a pEGFP-N2
vector from which the Gfp coding sequence had been

removed. The SV40 NLS localizes b-galactosidase to the
nucleus. All the constructs were verified by sequencing.
For subcellular localization experiments, COS-7 cells
(African green monkey kidney cells; ECACC, Porton
Down, Wiltshire, UK) were grown in DMEM with Gluta-
MAX II (Invitrogen) and 4500 mgÆL
)1
d-glucose, supple-
mented with 10% fetal bovine serum, 100 UÆmL
)1
penicillin
G and 100 lgÆmL
)1
streptomycin in a 5% CO
2
humidified
atmosphere at 37 °C. Cells were seeded on glass coverslips
into 12-well plates (5 · 10
4
cells per well) and transfected
24 h later with 0.5 lg of purified plasmid DNA per well,
using 2.3 lL of FuGene6 (Roche, Basel, Switzerland). Cells
were transfected with constructs encoding HA-RDH1,
HA-RDH2, and the full-length and the four C-terminal
Fig. 5. Hypothetical model of RDH1–7 ⁄ 9
group evolution leading to the current ver-
tebrate multiplicity. Fish and amphibian
arrangements are those described for Danio
rerio and Xenopus tropicalis in [47].
Amphioxus retinol dehydrogenase enzymes D. Dalfo

´
et al.
3748 FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS
truncated RDH2-b-galactosidase proteins. Twenty-four hours
later, cells were fixed with 4% formaldehyde for 15 min at
room temperature, and permeabilized with methanol for
5 min at room temperature. Nonspecific binding was
blocked with 1% BSA for 1 h at room temperature. Cells
were then incubated for 1 h at room temperature with a
1 : 200 dilution of rabbit anti-HA serum (Sigma-Aldrich,
St Louis, MO, USA) or with a 1 : 1000 dilution of rabbit
anti-b-galactosidase serum (ICN, Costa Mesa, CA, USA).
Cells were then incubated with a 1 : 200 dilution of Rhod-
amine Red-X-conjugated donkey anti-(rabbit IgG)
(Molecular Probes, Eugene, OR, USA) for 1 h at room
temperature in the dark. After each incubation with anti-
body, cells were washed twice in NaCl ⁄ P
i
· 1 for 5 min at
room temperature. The analyses of HA-RDH1 and
HA-RDH2 constructs were performed with a SP2 Leica
confocal microscope (Leica Microsystems GmbH, Wetzlar,
Germany), and those of RDH2-b-galactosidase constructs
were done with a Zeiss Axiophot fluorescence microscope
(Carl Zeiss, Oberkochen, Germany).
GFP fusion proteins were used to visualize the intracellu-
lar localization in living cells. Twenty-four hours after
transfection with Rdh2
1)335
-pEGFP-N2, Rdh2

1)58
-pEGFP-
N2, Rdh2
1)28
-pEGFP-N2, pEGFP-C2-RDH2
1)335
and
pEGFP-C2-RDH2
295)335
constructs (RDH2
1)335
-GFP,
RDH2
1)58
-GFP, RDH2
1)28
-GFP, GFP-RDH2
1)335
and
GFP-RDH2
295)335
, respectively), GFP localization was ana-
lyzed with a Leica DMIL fluorescence microscope. Alter-
natively, transfected cells on coverslips were rinsed with
NaCl ⁄ P
i
· 1 and incubated with a 4 lm prewarmed solu-
tion of ER-Tracker Blue White DPX (Molecular Probes)
for 30 min at 37 °C. Coverslips were rinsed and mounted
using a drop of Vectashield H-1000 (Vector Laboratories,

Burlingame, CA, USA). Analyses were done with a Zeiss
Axiophot fluorescence microscope.
Enzymatic activity and kinetic constants
To analyze the enzymatic activity of HA-RDH1 and
HA-RDH2 proteins, COS-7 cells were seeded into Petri
dishes (1 · 10
6
cells per plate), and transfected 24 h later
with 2.4 lg of plasmid DNA and 14.4 lL of FuGene6
(Roche). Control experiments were performed with cells
transfected with equal amounts of the empty vector. After
72 h of transfection with HA-constructs, COS-7 cells were
collected by centrifugation, 5 min at 400 g, Eppendorf
centrifuge 5702, rotor 5702R (Eppendorf, Hamburg,
Germany), resuspended in 20 mm Hepes, 150 mm KCl,
1mm EDTA, 10% sucrose and 2 mm dithiothreitol
(pH 7.5), and homogenized by sonication for 1 min (Sonifi-
er 250; Branson, Danburg, CT, USA). Debris and unbro-
ken cells were removed by two centrifugation steps at
13 000 g for 15 min at 4 °C, Eppendorf minispin. The
microsomes were subsequently collected by ultracentrifuga-
tion at 100 000 g for 2 h at 4 °C (Optima TL ultracentri-
fuge, rotor TLS55; Beckman Coulter Inc., Fullerton, CA,
USA), and resuspended in 0.1 m potassium phosphate,
0.1 mm EDTA, 0.1 mm dithiothreitol and 20% glycerol.
Protein concentrations were determined by the Bradford
method (Bio-Rad, Hercules, CA, USA), using BSA as
standard. Microsomes were stored at ) 80 °C until analysis.
For western blot analysis, proteins were resolved by
12.5% SDS ⁄ PAGE and transferred onto a nitrocellulose

membrane. Nonspecific binding was blocked with 10%
nonfat milk and 10% NaCl ⁄ P
i
· 10. The membrane was
incubated with a 1 : 1000 dilution of anti-HA mouse serum
(BAbCO, Richmond, CA, USA) for 1 h at room tempera-
ture, washed three times with MPT (0.5% nonfat milk,
10% NaCl ⁄ P
i
· 10 and 0.05% Tween-20) for 5 min at
room temperature, and then incubated with a 1 : 3000-fold
dilution of peroxidase-conjugated sheep anti-(mouse IgG).
The filters were washed again three times with MPT, and
antibody binding to HA-RDH1 and HA-RDH2 proteins
was visualized with an ECL western blotting analysis sys-
tem (Amersham Biosciences, Little Chalfont, UK).
Oxidative and reductive activities of RDH1 and RDH2
proteins were determined using commercial trans and cis
isomers of retinoids (Sigma-Aldrich), except for 9-cis-ret-
inol, which was synthesized by chemical reduction of 9-cis-
retinal with NaBH
4
(kindly provided by X. Pare
´
s, Universi-
tat Auto
`
noma de Barcelona, Bellaterra, Spain). Purity was
checked by HPLC. Stock solutions of retinoids were pre-
pared in ethanol. Ethanol-dissolved retinoids were solubi-

lized in the reaction buffer by a 10 min sonication in the
presence of equimolar delipidated BSA (Sigma-Aldrich).
Concentrations of ethanol in the reaction mixture did not
exceed 1%. Retinoid concentrations were determined on
the basis of the corresponding extinction coefficients at the
appropriate wavelengths in an aqueous buffer, as previously
described [52]. Catalytic activity was assayed in 90 mm
potassium phosphate and 40 mm KCl at pH 6.0 for reduc-
tive activity, and at pH 8.0 for oxidative activity in silicon-
ized Eppendorf tubes. Reactions were started by the
addition of cofactor and carried out for 15 min at 37 °Cin
0.5 mL. The amount of protein used in the reaction mixture
was 15 lg. The reaction was terminated by the addition of
an equal volume of cold methanol supplemented with
20 lm butylated hydroxytoluene. Retinoids were extracted
using solid-phase extraction on a Waters Oasis HLB
Extraction cartridge (Waters, Milford, MA, USA), follow-
ing the manufacturer’s instructions, and analyzed using a
Waters Alliance HPLC System. The elution was monitored
on a Waters 2695 Alliance ⁄ PDA Waters 2996 at 380 nm
for retinal isomers and 325 nm for retinol isomers. Reti-
noids were separated using an RP-HPLC column (Kromasil
100 C18 5 lm25· 0.46 cm; Teknokroma, Sant Cugat
del Valle
`
s, Spain) with acetonitrile ⁄ acetate ammonium 1%
(85 : 15) as mobile phase. The flow rate was 1.8 mLÆmin
)1
.
Under these conditions, elution times were as follows:

10.9 min for 9-cis-retinol, 11.4 min for all-trans-retinol,
D. Dalfo
´
et al. Amphioxus retinol dehydrogenase enzymes
FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS 3749
14.8 min for 9-cis-retinal, and 15.3 min for all-trans-retinal.
Retinoids were quantitated by comparing their peak areas
with a calibration curve constructed from peak areas of a
series of standards. The peak detection limit was about
2 pmol of retinoid.
The apparent K
m
values for the reduction of all-trans-ret-
inal were determined at 1 mm NADH using eight concentra-
tions of substrate (from 0.5 to 20 l m and from 0.5 to 15 lm
for RDH1 and RDH2, respectively). The apparent K
m
val-
ues for cofactor NADH were determined at 15 lm and
12.5 lm of all-trans-retinal for RDH1 and RDH2, respect-
ively, using six concentrations of cofactor, from 0.005 to
1.5 mm. Kinetic analyses were performed with 15 lg of pro-
tein. The background level of product formed in control
reactions using microsomes from COS-7 cells transfected
with an empty vector was taken into account and subtracted
from each experimental data point. Kinetic constants
obtained by nonlinear Marquardt’s regression analysis were
calculated from at least three independent experiments using
the commercial grafit curve-fitting software (Erithacus
Software Ltd, Horley, UK) and expressed as means ± SD.

To measure activity in intact COS-7 cells, 24 h after
transfection the medium was replaced with fresh medium
containing delipidated BSA (5 mgÆmL
)1
) and all-trans-
retinol or all-trans-retinal at three concentrations (5, 10
or 20 lm). After 6 h of incubation, cells were lysed, and
retinoids were extracted and analyzed by RP-HPLC as
described above.
Evolutionary analysis
Accession numbers for all the sequences used in this study
are provided in supplementary Table S2. Protein sequence
alignments and a neighbor-joining tree were generated with
clustalx [53] and drawn with the treeviewppc program
[54]. Confidence in each node was assessed by 1000 boot-
strap replicates. The N-terminal and C-terminal regions of
several SDR enzymes were highly variable in length and
sequence, and produced unreliable alignments. These
regions were therefore excluded from the phylogenetic
analyses, and only alignments from amino acids 30–293,
referred to the amphioxus sequences, were considered.
Human similar-RDH2, rat similar-RDH1 and similar-
RDH2, and mouse truncated RDH and similar-RDH
sequences were excluded from the phylogenetic analysis, as
they were pseudogenes and ⁄ or partial RDH sequences. The
genomic organization of the RDH group was deduced
using the Map Viewer website from NCBI: http://
www.ncbi.nlm.nih.gov/mapview/.
Acknowledgements
We thank X. Pare

´
s, J. Farre
´
s, S. Martras and O. Galle-
go for their valuable help in the biochemical analyses.
We also thank the Serveis Cientı
´
fico-Te
`
cnics (UB) for
their assistance with HPLC analysis. The authors
acknowledge the critical comments raised by the review-
ers, which have substantially improved the manuscript.
This study was supported by a grant from the Minis-
terio de Ciencia y Tecnologı
´
a, BMC2003-05211 (Spain)
and by an FPI fellowship from the MEC (Ministerio de
Educacio
´
n y Cultura) to D. Dalfo
´
and a grant from the
Universitat de Barcelona to N. Marque
´
s.
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Supplementary material
The following supplementary material is available
online:
Table S1. Oligonucleotides used for the PCR amplifi-
cation of the different constructs.
Table S2. Accession numbers of the sequences used in
this study.
This material is available as part of the online article
from
Please note: Blackwell Publishing is not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corres-
ponding author for the article.
Amphioxus retinol dehydrogenase enzymes D. Dalfo
´
et al.
3752 FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS