From the Department of Biochemistry and Molecular
Biology, Universitat Autònoma de Barcelona, E-08193 Bellaterra,
Barcelona, Spain and the § Department of Medical
Biochemistry and Biophysics, Karolinska Institutet,
S-17177 Stockholm, Sweden
Received for publication, November 20, 2000, and in revised form, January 9, 2001
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ABSTRACT |
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Enzymes of the short chain and medium chain
dehydrogenase/reductase families have been demonstrated to participate
in the oxidoreduction of ethanol and retinoids. Mammals and amphibians contain, in the upper digestive tract mucosa, alcohol dehydrogenases of
the medium chain dehydrogenase/reductase family, active with ethanol
and retinol. In the present work, we searched for a similar enzyme in
an avian species (Gallus domesticus). We found that chicken
does not contain the homologous enzyme from the medium chain
dehydrogenase/reductase family but an oxidoreductase from the aldo-keto
reductase family, with retinal reductase and alcohol dehydrogenase
activities. The amino acid sequence shows 66-69% residue identity
with the aldose reductase and aldose reductase-like enzymes. Chicken
aldo-keto reductase is a monomer of Mr 36,000 expressed in eye, tongue, and esophagus. The enzyme can oxidize aliphatic alcohols, such as ethanol, and it is very efficient in
all-trans- and 9-cis-retinal reduction
(kcat/Km = 5,300 and 32,000 mM Short chain dehydrogenases/reductases
(SDR),1 medium chain
dehydrogenases/reductases (MDR), and aldo-keto reductases (AKR) are
three oxidoreductase superfamilies that catalyze the oxidation and
reduction of a broad range of alcohol and aldehyde compounds of
physiological or pharmacological significance (1-3).
In MDR, the alcohol dehydrogenase (ADH) family constitutes a complex
system grouped into several classes (4, 5). ADH1 is the classical
enzyme responsible for liver ethanol metabolism, which also exhibits
activity with retinoids (6, 7). ADH2 is a liver enzyme that may
marginally contribute to ethanol and retinoid metabolisms (8, 9). ADH3
is a glutathione-dependent formaldehyde dehydrogenase
(FALDH) inactive with retinoids (6). ADH4 exhibits a unique epithelial
distribution and is abundant in the upper gastrointestinal tract mucosa
and eye (10-12). It is very efficient with retinoids rather than with
ethanol, and it has been proposed to have a role in retinoic acid
synthesis (6, 13-15). ADH7 has been described only in chicken embryo, exhibiting activity with retinoids (16). ADH8 is an
NADP(H)-dependent form with tissue distribution and
substrate specificity similar to those of mammalian ADH4, which has
been described in amphibians (17).
The SDR superfamily contains an alcohol dehydrogenase that accounts for
ethanol metabolism in Drosophila (18) and several mammalian
forms that exhibit activity with retinoids (15, 19, 20). Within the AKR
superfamily, several groups of enzymes with related structure and
function have been established (3). Among them, the aldose reductase
(AR), aldehyde reductase, and hydroxysteroid dehydrogenase (HSD)
families have been profoundly characterized in mammals. The AR family
has been suggested to be involved in the development of secondary
diabetic complications (21) because of its ability to reduce glucose to
sorbitol (22), a hyperosmotic compound. Other roles in aldehyde
detoxification, osmotic homeostasis, steroid conversion, and
catecholamine metabolism have been also proposed for AR (23-26).
Several forms including human ARL1 (aldose
reductase-like 1) (27), small
intestine reductase (28), mouse vas deferens reductase (29), mouse
fibroblast growth factor-regulated protein (30), and Chinese hamster
ovary reductase (31) have been described as a distinct group (3)
sharing 70% amino acid identity with AR sequences.
Vitamin A or retinol (ROL) is the precursor of retinal (RAL) and of
retinoic acid (RA). RAL is necessary for vision, and RA is essential
for growth, development, and cellular functions (32, 33). Biosynthesis
of RA from vitamin A implies reversible oxidation of ROL to RAL and
irreversible oxidation of RAL to RA. The latter compound, in the
all-trans- and 9-cis-isomeric forms, exerts its regulatory function bound to nuclear receptors (34). In photoreceptor cells, 11-cis-RAL binds to opsin to form rhodopsin. When one
photon is absorbed by rhodopsin, 11-cis-RAL is isomerized to
all-trans-RAL, which dissociates from opsin. In
all-trans-RA and 9-cis-RA synthesis from ROL, and
in rhodopsin regeneration, several oxidoreduction steps occur, which
are catalyzed by MDR and/or SDR enzymes, important for the regulation
of these processes (15, 20). No activity with retinoids has been ever
reported in enzymes from any AKR group.
In this study we have recognized an activity in the upper digestive
tract of chicken, similar to that of ADH from the MDR family previously
identified in mammals (11, 35) and amphibians (17). However, this avian
enzyme was not an ADH of the MDR family, but, surprisingly, an AKR.
Thus, we have here characterized the first member of the AKR
superfamily with NADP(H)-dependent retinal reductase and
ethanol dehydrogenase activities.
Avian Tissues--
Chicken (Gallus domesticus)
tissues were obtained immediately after death from a local
slaughterhouse. The tissues were cut, cleaned, rinsed in ice-cold
distilled water, frozen in liquid nitrogen, and stored at Enzyme Purification--
Tissue samples (tongue and esophagus,
390 g) were pooled and homogenized in 10 mM sodium
phosphate, 2 mM dithiothreitol, pH 7.0. The homogenate was
centrifuged (27,000 × g, 30 min, 4 °C), filtered
through glass wool, ultracentrifuged (85,000 × g, 30 min, 4 °C), and filtered again. The supernatant was dialyzed against the initial buffer and applied to a CM-Sepharose (Amersham Pharmacia Biotech) column (2.5 × 40 cm) equilibrated with the same buffer. The column was washed with 400 ml of buffer followed by a linear gradient (500 ml) of increasing NaCl concentration (0-250
mM). The fractions exhibiting octanol dehydrogenase
activity were pooled and concentrated with an Amicon concentrator
(Diaflo PM10 membranes). The preparation was dialyzed against the
initial buffer and applied to a 2',5'-ADP-agarose (Amersham Pharmacia
Biotech) column (1 × 20 cm). The column was washed with the same
buffer, and the active fractions were pooled, concentrated, dialyzed
against 10 mM Tris-HCl, 2 mM dithiothreitol, pH
8.2, and applied to an HPLC system (Waters 600) equipped with a Q
column. Elution of the enzyme was performed with a linear gradient of
sodium acetate (0-300 mM). The active fractions were
dialyzed against 10 mM Tris-HCl, 2 mM
dithiothreitol, pH 8.2, and applied to a fast protein liquid chromatography (Amersham Pharmacia Biotech) equipped with a
Sephacryl-S200HR column. Protein concentrations were determined by the
Bio-Rad protein assay method, using bovine serum albumin as a standard. Homogeneity was assessed by electrophoresis on SDS-polyacrylamide gel
(36) and the silver stain technique.
Starch Gel Electrophoresis--
Electrophoresis on starch gel
was performed as described (11), except that the gel buffer contained
0.74 mM NADP instead of NAD. Gel slices were activity
stained with 1 M ethanol or 0.1 M 2-buten-1-ol
and 0.1 mM NADP.
Mass Spectrometry--
Matrix-assisted laser desorption
ionization time-of-flight mass spectra were acquired on a Bruker Biflex
spectrometer equipped with a pulsed nitrogen laser (337 nm) in linear
positive ion mode and using a 19 kV acceleration voltage. Samples were
prepared by mixing equal volumes of a saturated solution of the matrix in 0.1% trifluoroacetic acid in water/acetonitrile, 2:1, and a protein
solution at a concentration range of 1-10 µM. From this mixture, 1 µl was spotted on the sample slide and allowed to
evaporate to dryness. Mass spectra were calibrated externally using
appropriate protein standards.
Protein Sequence Analysis--
Protein for sequence analysis was
reduced with dithiothreitol and carboxymethylated with iodoacetate as
described for ADH (17). Aliquots were then submitted to proteolytic
digestions with trypsin, Lys-C protease, N-Asp protease, or Glu-C
protease in 0.1 M ammonium bicarbonate or to treatment with
CNBr in 70% formic acid. Peptide mixtures were purified by reverse
phase HPLC on C18 columns with a gradient of acetonitrile in 0.1%
aqueous trifluoroacetic acid. Fractions obtained were submitted to
analysis in an ABI Procise HT sequencer, after prescreening in a
Voyager DEPRO matrix-assisted laser desorption ionization
time-of-flight mass spectrometer to secure finding relevant fragments
for sequence analysis. Several peptides were also submitted to
collision-induced dissociation sequence analysis with a Micromass
quadrupole time-of-flight tandem mass spectrometer equipped with a
nano-ES ionization
inlet.2
cDNA Cloning and Sequence Analysis--
Poly(A)+
RNA was isolated from chicken tongue and esophagus with the Quickprep®
Micro mRNA Purification Kit (Amersham Pharmacia Biotech). The first
strand of the cDNA was synthesized from 2 µg of RNA
poly(A)+ with You-Prime first strand beads (Amersham
Pharmacia Biotech) using a T17 primer or the
R0R1-(dT)17 primer adaptor (37). The resulting
pools of cDNA were used as templates in a polymerase chain reaction
with CHICK1 (5'-ATGCCIGTIYTIGGIYTIGGIACITGG-3', amino acid positions
16-24) and CHICK2 (5'-TGIGGIACIGGDATIGCICKCCART-3', amino acid
positions 302-310), designed from peptide sequences.
To obtain the 5' and 3' cDNA ends, the rapid amplification of
cDNA ends method (37) was followed. For the amplification of
poly(A) end, CHICK3 (5'-AGGTTCTGATCCGGTTC-3', amino acid
positions 244-249), CHICK4 (5'-GACTTCGAGCTGTCTAA-3', amino acid
positions 285-290) specific primers, and R0
(5'-AAGGATCCGTCGACATC-3') and R1 (5'-GACATCGATAATACGAC-3'),
corresponding to the primer-adaptor sequence, were used in two nested
PCR.
To obtain the 5' end, the first strand of cDNA was synthesized
using a sequence-specific oligonucleotide primer (CHICK5,
5'-AAGTTTGAGATTCCAATGG-3', amino acid positions 162-168) with AMV
reverse transcriptase (Roche Molecular Biochemicals), at 42 °C. A
poly(A) tail was added to the cDNA pool with deoxynucleotide
terminal transferase (Amersham Pharmacia Biotech). The second strand of
cDNA was synthesized with the R0R1-(dT)17
primer adaptor and Expand High Fidelity DNA polymerase kit (Roche
Molecular Biochemicals). The amplification of 5' end was performed
using CHICK6 (5'-ACTTAGTGACAACGAAGAGG-3', amino acid positions 79-84),
CHICK7 (5'-CCCAACCCCAACACCGGCAT-3', amino acid positions 19-25),
R0 and R1 primers, in two nested PCR.
PCR amplifications were performed with the Expand High Fidelity kit
(Roche Molecular Biochemicals). The PCR products were directly
sequenced or phosphorylated with T4 polynucleotide kinase (Roche
Molecular Biochemicals), cloned into the SmaI site of
pBluescript II SK(+), and sequenced (AlfExpress, Amersham Pharmacia Biotech).
Northern Blot Hybridization--
Total RNA was isolated from
frozen tissues by the guanidinium thiocyanate method (38).
RNA-poly(A)+ was isolated from 20-µg aliquots of total
RNA with the Quickprep® Micro mRNA Purification Kit (Amersham
Pharmacia Biotech). RNA samples were run in 1× MOPS, 2.2 M
formaldehyde, 1% agarose gel and transferred onto Nylon membranes
(Schleicher & Schuell) by means of a TurboblotterTM
(Schleicher & Schuell). RNA was immobilized by a 20-s pulse of UV light
in a Stratalinker (Stratagene). A 870-bp chicken AKR DNA-specific probe
(50 ng) was labeled with 50 µCi of [ Enzyme Kinetics--
Activity was determined by monitoring the
change in NADPH concentration at 25 °C in a Varian Cary 219 spectrophotometer by measurements at 340 nm, in 0.1 M
sodium phosphate, pH 7.5. Glutathione-dependent formaldehyde dehydrogenase activity was measured as reported (39). One
unit of activity corresponds to 1 µmol of reduced coenzyme formed or
utilized per min, at 25 °C, based on an absorption coefficient of
6220 M
Kinetic parameters were obtained from activity measurements, with
substrate concentrations that ranged from at least 0.1× Km to 8× Km. Each individual
rate measurement was run in duplicate. Three independent determinations
were performed for each kinetic constant. Kinetic constants were
calculated using ENZFITTER (Elsevier Biosoft) and expressed as the
means ± S.D.
Protein Modeling and Retinoid Docking--
A three-dimensional
model of chicken AKR was constructed by adopting its amino acid
sequence into the known fold of human AR (Protein Data Bank entry code
1ADS) (41) using the ICM program (version 2.7, Molsoft LLC, 1997), as
described (14). The quality of the model was assessed with the PROCHECK
program (42).
To study the interaction of retinoids with AKR, the Protein Data Bank
coordinates of human AR and the chicken AKR model were used. To prepare
human AR structure for docking simulations, hydrogen atoms were added,
methyl groups were rotated, and the most severe atomic contacts were
minimized. RAL in an extended conformation (carbon Multiple Alignments and Phylogenetic Trees--
Protein and DNA
sequences were searched in the GenBank, EMBL, PIR, and SwissProt data
bases with the FASTA (45) and BLAST (46) algorithms. The Internet AKR
Web page was regularly consulted to update information. Sequence
alignments were performed using the GCG program PILEUP. Bootstrap (1000 trials) neighbor-joining phylogenetic trees were calculated by means of
the CLUSTALW program (47) and displayed with TREEVIEW program (48).
Enzymatic Forms with ADH Activity in Chicken Tissues--
Several
ADH-type bands were detected on starch gel electrophoresis (Fig.
1) because of their
NADP(H)-dependent activity staining with 2-buten-1-ol. A
putative FALDH (or ADH3) appeared as an anodic band with variable
intensity in all tissues analyzed. The nature of this band was assessed
by glutathione-dependent activity staining with
formaldehyde in duplicate gel slices (not shown). Moreover, a cathodic
band detected in liver homogenates could correspond to ADH1 (49). In
addition, a previously unidentified anodic band with lower mobility
than FALDH appeared in homogenates from eye, tongue and esophagus when
2-buten-1-ol or ethanol was used as a substrate.
Purification of a Chicken Enzymatic Form with ADH
Activity--
The novel enzyme form (later recognized as an AKR) was
purified from tongue and esophagus (Table
I). In the CM-Sepharose chromatography,
an octanol dehydrogenase activity peak was detected in the gradient
fractions. In agreement with the results of starch gel electrophoresis,
another peak of activity corresponding to FALDH, which overlapped with
the major protein peak, was eluted by the initial washing and,
therefore, well separated from the novel enzymatic form. The
2',5'-ADP-agarose resin bound weakly the chicken enzyme, which eluted
in the washing step, delayed with respect to the major protein peak.
The HPLC Q column step resulted in a 2-fold increase in the specific
activity, but contaminating proteins remained in the fractions
corresponding to the octanol dehydrogenase peak. Finally, the fast
protein liquid chromatography purification step with a Sephacryl
S200-HR column resulted in a homogeneous enzyme, as demonstrated by
SDS-polyacrylamide gel electrophoresis (Fig.
2). The purified preparation showed a
specific activity of 0.92 unit/mg with octanol (Table I).
With the Sephacryl S200-HR column chromatography, a molecular
mass of 36.2 kDa was estimated for the native enzyme using a set
of proteins as standards. Moreover, molecular mass values of 36 and
36.6 kDa were determined by SDS-polyacrylamide gel electrophoresis (Fig. 2) and mass spectrometry, respectively. Thus, it can be concluded
that the native form is a monomer.
cDNA and Protein Sequences of the Novel Enzymatic
Form--
Before any data from peptide sequences were available, the
first attempt to clone the cDNA was based on degenerated primers deduced from consensus sequences of vertebrate ADH, which were used in
the PCR with cDNA from tongue and esophagus. This strategy only
produced DNA fragments corresponding to chicken ADH1, which could be
marginally expressed in tongue and esophagus, because no active ADH1
was found by starch gel electrophoresis in these tissues (Fig. 1).
These preliminary results suggested that no enzyme homologous to
mammalian ADH4 or amphibian ADH8 is expressed in the upper digestive
tract from chicken.
We therefore decided to investigate the nature of the novel chicken
enzyme by protein analysis. However, attempts at direct sequencer
degradation gave no result, suggesting that the N terminus was blocked.
Therefore, different sets of the carboxymethylated protein were cleaved
with proteolytic enzymes. After fractionation with reverse phase HPLC
and subsequent sequencer analysis, scans against data banks showed that
the peptides obtained were homologs to fragments of AKR enzymes. Hence,
the present enzyme was concluded to be a novel type of AKR, with
ADH-like activity. Because of the initial failure to get a cDNA
sequence with conventional ADH primers, impurities in the protein
preparation, and initial unclarities between protein and cDNA data,
the protein analysis was continued until essentially the entire amino
acid sequence had been determined by sequencer analysis of 57 peptides
from five separate digests (with CNBr, trypsin, N-Asp, Lys-C, or Glu-C
proteases), covering 311 of 316 residues, i.e. all residues
except positions 65-69. Finally, the N terminus was proven to be
acetylated by collision-induced dissociation analysis in a quadrupole
time-of-flight mass spectrometer of the N-terminal fragment from the
Glu-C protease digest, which started with acetyl-Ala, by removal of the
initiator Met from the primary translation product.2
Using oligonucleotide primers designed according to the peptide
sequences, a DNA fragment of 870 bp was obtained by the PCR. The rapid
amplification of cDNA ends method generated three additional DNA
fragments corresponding to the 5' end (104 and 105 bp) and the 3' end
(625 bp). The DNA fragments encompassed a 1504-bp full-length cDNA.
It included a 44-bp 5'-flanking region, an open reading frame of 951 bp, encoding a 317-amino acid primary translation product,
corresponding to the 316-amino acid acetylated mature form and the
initiator Met, and a 509-bp 3'-untranslated region with a
poly(A) tail (Fig. 3). The end
results of the protein and cDNA data were in complete
agreement.
The amino acid sequence of chicken AKR shows high identity with those
of the AR (66-69%) and AR-like (68-69%) families (Table II). The N-terminal region of chicken AKR
contains one additional amino acid residue with respect to those of AR
and AR-like members. However, the amino acid numbering of AR has been
used for the novel enzyme. A phylogenetic tree was built by the
bootstrap neighbor-joining method using aldo-keto reductase sequences
(Fig. 4). An unrooted representation of
the tree emphasized four robust clusters constituted by AR, AR-like,
HSD, and aldehyde reductase sequences. The chicken AKR positioned below
the split of the AR and AR-like clusters.
The novel enzyme contains the catalytic tetrad (Asp43,
Tyr48, Lys77, and His110)
characteristic of other AKR forms (50), and the residues strictly (Asn160, Gln183, and Ser263) and
highly conserved (Ser159, Tyr209,
Leu212, Lys262, Arg268,
Glu271, and Asn272) involved in NADP(H) binding
(3). In contrast to the conserved nature of the coenzyme-binding site,
the residues considered important for substrate specificity differ
notably between chicken AKR and AR or AR-like sequences (Table II) (see below).
Northern Blot Analysis--
Chicken AKR mRNA (1.8 and 4.0 kilobases) was found in the eye and the upper digestive tract (tongue
and esophagus) but not in small intestine (Fig.
5). The 1.8-kilobase transcript is likely to represent the mature mRNA. No signal was detected in brain, skeletal muscle, heart, or liver (not shown). These results are consistent with those obtained by starch gel electrophoresis and activity staining (Fig. 1), which also detected active enzyme in eye,
tongue, and esophagus, indicating a tissue-specific expression of the
chicken AKR gene.
Substrate Specificity of Chicken AKR--
Kinetic constants of
chicken AKR were determined with several substrates (Tables
III and
IV). Chicken AKR exhibited a marked NADP(H) preference, suggesting carbonyl reduction as the most likely
physiological reaction (Table III). Compounds such as
D,L-glyceraldehyde and glyoxal, characteristic
substrates for the aldo-keto reductases, were relatively poor
substrates for chicken AKR. Moreover, no activity was found with aldose
sugars (glucose, galactose, ribose, and xylose) at concentrations up to
150 mM or with steroids (corticosterone, epiandrosterone,
dehydroisoandrosterone, 5
An interesting property of chicken AKR is its ADH activity, because no
data on aliphatic alcohols as substrates for AKR enzymes are found in
the literature. All aliphatic alcohols assayed were substrates that
saturated the enzyme. The Km values decreased steadily as the number of carbons in the aliphatic chain increased, whereas only a small variation was detected in
kcat values (Table III).
Among the results obtained with different substrates, the most striking
are those concerning retinoids (Table IV), because the AKR superfamily
has not been reported before to be active with these physiological
compounds. Chicken AKR has the ability to reduce all-trans-,
9-cis-, and 13-cis-RAL isomers and to oxidize all-trans- and 9-cis-ROL. Km
values are similar for all the RAL isomers, but
kcat values differ notably. The best substrate was 9-cis-RAL with a kcat value 5- and 20-fold higher than those for all-trans-RAL and for
13-cis-RAL, respectively, and with an extremely high
kcat/Km ratio (32000 mM Molecular Model of Chicken AKR--
A molecular model of the
chicken AKR tertiary structure was built (Fig.
6A). Following amino acid
identity (Table II) and phylogenetic criteria (Fig. 4), the structures
of the AR and AR-like enzymes were considered the most valid templates.
The coordinates of human AR (41), mouse fibroblast growth
factor-regulated protein (51), and Chinese hamster ovary reductase (52)
three-dimensional structures were used, but no significant differences
were found between the three calculated models for chicken AKR (the
root mean square deviation was 0.9-1 Å), and thus the one obtained from the human AR template was used as a working object.
The model shows that the residues responsible for the NADP(H) over
NAD(H) preference, i.e. Lys262 and
Arg268, are conserved in chicken AKR. However, an Asp
residue is found at position 264, which is never occupied by residues
with acidic side chains in other members of the family. In the
crystallographically determined structure, the main chain nitrogen at
position 264 faces the 2'-phosphate of NADP(H) and establishes a
hydrogen bond with the coenzyme (41). Our model shows that the distance
between the charged group of Asp264 in chicken AKR and the
NADP(H) 2'-phosphate is 4.1 Å and, therefore, a negligible effect on
cofactor binding in terms of electrostatic energy should be expected.
The substrate-binding pocket of the chicken AKR model is highly
hydrophobic. It includes mostly aromatic (Trp20,
Phe47, Tyr48, Trp79,
Tyr111, Tyr209, and Trp219) and
aliphatic (Ala45, Leu121, Leu122,
and Ile298) residues. At the edge of the active site,
position 47, typically occupied by apolar aliphatic residues, and
position 111, with a conserved Trp in AR, are considered responsible
for the sugar and steroid specificity (3, 53). Significantly, chicken
AKR, inactive with pentose or hexose sugars and steroids, contains aromatic and bulky residues at these positions, Phe47 and
Tyr111 (Table II). The absence of Cys residues at positions
298 and 303 could also contribute to the kinetic properties of
chicken AKR with glyceraldehyde and aldosugars. Thus, human AR
C298S and C303S mutants show a decrease in catalytic efficiency
with glucose and xylose, and the C298S mutant shows an increased
Km with glyceraldehyde (54).
The presence of Leu121 and Leu122, in loop A
and Ile298, Pro299, Val300,
Pro301, Gln302, and Ser303 in loop
C (Table II), in addition to the Phe47 and
Tyr111 substitutions, confer a distinct geometry to the
substrate-binding cleft with respect to human AR (Fig. 6, B
and C) that could be correlated with the substrate
specificity of chicken AKR. The cleft of the chicken enzyme is
structured as a funnel shaped cavity that is suitable for binding
hydrophobic substrates containing the carbonyl reactive group at
the end of an aliphatic chain, such as retinoids, but unsuitable for
bulky polycyclic compounds, such as steroids (Fig. 6B). In
contrast, the structure of human AR active site (Fig. 6C) is
appropriate for steroid binding as well as for retinoids (see below).
Docking Simulations--
The interaction of RAL with chicken AKR
was studied by docking simulations in our three-dimensional model. The
molecule of all-trans-RAL fitted the substrate-binding
pocket, and many features suggest that this interaction may correspond
to a productive complex. The position of the RAL molecule in the
substrate-binding pocket was found to be such that the re
face of the aldehyde group is parallel to the nicotinamide ring (23,
53). The RAL aliphatic chain does not clash against any of the
voluminous side chains that line the cavity, and the
The absence of activity of chicken AKR with steroids was studied by
docking simulations with testosterone (not shown). The substrate-binding pocket could harbor the testosterone molecule; however, unfavourable contacts were found with Phe47 (Fig.
6B), the side chain of which was sandwiched between the protein main chain and the steroid. Interestingly, these contacts disappeared in the docking simulation when Phe was substituted by Val,
the residue found in human AR at position 47.
An alcohol dehydrogenase, named ADH4, active with ethanol and
retinoids and present in the eye and digestive tract organs has been
described in mammals (6, 11, 56, 57). In the stomach of amphibians, an
alcohol dehydrogenase with similar substrate specificity, but a
separate origin has been reported (ADH8) (17). Interestingly, ADH8
exhibits a NADP(H) preference over NAD(H), a feature unique to this ADH
in the MDR superfamily from animals. We, therefore, searched for a
similar enzyme, either NAD(H) or NADP(H)-dependent, in an
avian species, G. domesticus. An
NADP(H)-dependent enzyme, active with ethanol and
retinoids, was found in digestive organs and in the eye of chicken.
But, unexpectedly, it does not correspond to an ADH of the MDR
superfamily but to a completely different enzyme, a reductase of the
AKR superfamily. We have characterized this chicken AKR enzyme and
showed that it exhibits distinct structural and functional properties.
Chicken AKR showed a Km value of 6 mM
with D,L-glyceraldehyde, 100-300-fold higher
than that reported for human AR with this substrate (20-70
µM) (22, 54, 58) but similar to the values exhibited by
human AR-like and Chinese hamster ovary enzymes (2 and 28 mM, respectively) (27, 31). An important feature of chicken
AKR is the absence of activity with glucose, whereas AR enzymes are
typically active with this compound (22, 25). In contrast, with the
exception of human AR-like enzyme (27), AR-like forms are inefficient
(59) or not active at all (25, 31) with glucose.
Chicken AKR is not active with steroids containing hydroxyl or ketone
groups in their 3 Chicken AKR is active with aliphatic aldehydes and the corresponding
alcohols, including ethanol. To our knowledge, activity with ethanol
has never been reported for any member of the AKR family. The higher
hydrophobicity of the substrate-binding site of chicken AKR, as
compared with other enzymes of the family, is probably at the basis of
its distinct specificity. The localization of avian AKR in upper
digestive tract tissues suggests a possible contribution as a metabolic
barrier against aliphatic alcohols and aldehydes present in the food
stuffs. This role was previously proposed for mammalian ADH4, also
expressed in these tissues, and with similar kinetic constants toward
aliphatic substrates (6, 11).
Another unexpected feature is that chicken AKR can transform retinoids
with high catalytic efficiency. The
kcat/Km values of chicken AKR
with all-trans-RAL and 9-cis-RAL are similar to
those for human AR with its best physiological substrates (22, 23), and
they support a physiological role of chicken AKR in RAL reduction.
Chicken AKR is the fastest 9-cis-RAL reductase known. Its
kcat (860 min In the light of these results, it seems reasonable to propose a new and
critical biological function for the AKR superfamily, the metabolism of
retinoids. Chicken AKR, like mammalian ADH4, may perform two different
roles in regard to retinoid metabolism: 1) regeneration of rhodopsin
and involvement in the homeostasis of RAL through the reduction of
all-trans-RAL to all-trans-ROL in the eye tissues
and 2) conversion of retinoids and the regulation of RA synthesis,
essential in tissue differentiation and maintenance in ocular and
digestive tract tissues.
The upper digestive tract and the eye of vertebrates need an
oxidoreductase capable of metabolizing ROL and RAL. In mammals, an
NAD(H)-dependent ADH (ADH4) of the MDR family seems to
participate in this function. In amphibians, an
NADP(H)-dependent ADH (ADH8), also of the MDR family,
accounts for this activity. In the present avian line, a different
structure, an NADP(H)-dependent aldo-keto reductase, is
used for this function. It is relevant to consider that this
specificity toward retinoids may be also present in previously well
characterized AKR members from other vertebrate groups, including
human. In agreement to this, our docking simulations of the
crystallographically determined human AR structure with retinoids
suggest a productive binding of these compounds.
Activity-wise, SDR, MDR, and AKR superfamilies have evolved
convergently, although they structurally exhibit divergence with distant relationships (3, 60). In SDR and MDR, the convergence has
given rise to paradigmatic cases in the metabolisms of ethanol and
retinoids. Thus, the widespread MDR ADH is the one responsible for
ethanol metabolism in vertebrates, yeast, and bacteria. However, in
Drosophila, which lacks an MDR ADH, an SDR ADH exerts that function (61). Moreover, several forms of SDR and MDR are ROL dehydrogenases or RAL reductases, apparently involved in physiological retinoid metabolism (15, 20). We have shown here that AKR has also
evolved convergently with SDR and MDR in terms of ethanol and ROL
metabolism. The (1·min
1, respectively). This
finding represents the inclusion of the aldo-keto reductase family,
with the (
/
)8 barrel structure, into the scenario of
retinoid metabolism and, therefore, of the regulation of vertebrate
development and tissue differentiation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C.
For enzyme analysis, tissues were thawed, fragmented, and homogenized
in 10 mM Tris-HCl, 2 mM dithiothreitol, pH 7.6 (1:1 w/v).
-32P]dCTP (3000 Ci/mmol, Amersham Pharmacia Biotech) by the random priming method
(Prime-a-Gene labeling system, Promega). Membrane prehybridization and
hybridization were performed in 0.2 M sodium phosphate, 1 mM EDTA, 1% bovine serum albumin, 1% SDS, pH 7.2, at
65 °C, overnight. Membranes were washed in 40 mM sodium
phosphate, 1 mM EDTA, 1% SDS, pH 7.2, at 65 °C, for
1 h and exposed to Hyperfilm-MP (Amersham Pharmacia Biotech)
overnight at
80 °C with two intensifying screens.
1·cm
1 at 340 nm.
Steroids were dissolved in 0.3 M methanol. Methanol was not
a substrate nor an inhibitor of chicken AKR under the present
conditions (maximum methanol concentration reached in the assay was 45 mM). ROL oxidation and RAL reduction were performed according to a published procedure (40), using reported absorption coefficients (14).
angles of single
bonds = 180°) was placed into the substrate-binding pocket in
the same orientation as the ternary ligand testosterone (43). Nonrigid
docking simulations were based upon a Monte Carlo procedure (44),
allowing free movement of the substrate, of its rotable bonds, and of
the
angles of the residues inside a 5 Å radius from the docked
substrate. Additional distance restraints were imposed: 1.0-2.4 Å between the aldehyde oxygen of RAL and the catalytic hydroxyl group of
Tyr48, and 2.0-2.4 Å between C-15 of RAL and C-4
of NADPH. Similarly, docking simulations with testosterone were
performed. Energy values were calculated with the ICM program as
reported (14).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
[in a new window]
Fig. 1.
Starch gel electrophoresis of chicken tissue
homogenates. Activity staining using 2-buten-1-ol as a substrate
and NADP as cofactor. T, tongue; H, heart;
Ey, eye; B; brain; K, kidney;
I, small intestine; Es, esophagus; L,
liver; P, purified chicken AKR.
Purification of chicken AKR
View larger version (53K):
[in a new window]
Fig. 2.
SDS-polyacrylamide gel electrophoresis of
purified chicken AKR. Lane 1, enzyme preparation eluted
from the HPLC column. Lane 2, purified enzyme after the fast
protein liquid chromatography step.
View larger version (57K):
[in a new window]
Fig. 3.
Nucleotide and amino acid sequences of
chicken AKR. Nucleotide numbers are shown
above the lettered sequence, and amino acid residue numbers
are on the right side. The mature protein starts with
acetyl-Ala as shown, after removal of the initiator Met, and the
resulting protein sequence contains 316 amino acid residues. A putative
polyadenylation signal is underlined at position 1435.
Alignment of amino acid residues constituting the substrate-binding
pocket and A, B, and C loops (63) of different AKR enzymes
View larger version (25K):
[in a new window]
Fig. 4.
Phylogenetic tree of AKR superfamily.
AR, AR-like, aldehyde reductase, HSD, and related sequences are
included. Sequences were obtained from data banks. Branch lengths are
proportional to the number of residue substitutions. Bootstrap values
higher than 950 (over 1000 trials) are indicated as a percentage. *,
bootstrap value of 644.
View larger version (50K):
[in a new window]
Fig. 5.
Northern blot analysis of chicken AKR.
Membrane was prepared with poly(A)+ RNA (2 µg/lane).
T, tongue; Es, esophagus; I, small
intestine; Ey, eye.
-androstane-17
-ol-3-one, and
5-cholesten-3
-ol-7-one) at concentrations up to 150 µM. 3-Nitrobenzaldehyde was a very good substrate,
exhibiting the lowest Km and the highest catalytic
efficiency among the compounds tested. Aliphatic aldehydes such as
hexanal and 2-trans-hexenal were actively used by chicken
AKR, with no significant differences between the two isomers.
Kinetic constants of chicken AKR with aldehydes, alcohols, and
cofactors
Kinetic constants of chicken AKR with retinoids
1·min
1). Again,
9-cis-ROL was the best retinoid substrate for the oxidation reaction. No differences were found in the Km values of all-trans-ROL and 9-cis-ROL with respect to
their corresponding aldehydes. In contrast, the
kcat values were much lower for the alcohols
(Table IV).
View larger version (51K):
[in a new window]
Fig. 6.
Homology-based model and docking simulations
of chicken AKR. A, ribbon representation of the protein
structure with bound NADPH (green) and
all-trans-RAL (red). The residues at loops A, B,
and C conserved in AR sequences and substituted in chicken AKR are
represented as orange spheres (positions 47, 111, 113, 122, 124, 129, 130, 216, 223, 224, 291, 298-300, 302, 303, and 312). B and
C, accessible surface of the residues in the
substrate-binding pocket (yellow) of the chicken AKR model
(B) and crystal structure of human AR (41) (C).
D and E, All-trans-RAL
(red) bound to the model of active site of chicken AKR
(D) and human AR (E). The atomic distances
(Å) between groups involved in the catalytic reaction (between the O
of the aldehyde group and the hydroxyl of Tyr48 and the
N of His110 and between the C of the aldehyde and the C4 of the
nicotinamide ring) are indicated.
-ionone ring of
the retinoid showed complementarities with residues from loops A and C. The atomic distances between the oxygen of the aldehyde group and the
hydroxyl group of Tyr48, and the N
of His110
(catalytic residues) in the model were 1.8 and 3 Å, respectively. The
atomic distance between the C4 of the nicotinamide ring and the C15 of
RAL was 2.2 Å (Fig. 6D). The angles drawn by lines through
nicotinamide C4-nicotinamide H4R-RAL C15 and the nicotinamide H4R-RAL
C15-RAL oxigen were 150° and 100°, respectively. Moreover, 9-cis-RAL also bound to the substrate-binding pocket of
chicken AKR with distances to catalytic residues similar to those of
the all-trans isomer (not shown). These distances and angles
in the model are compatible with those determined for a transition
state (50, 55). All-trans-RAL also could be docked to the
substrate-binding pocket of the crystal structure of human AR. Although
this molecule adopted a different orientation to the one observed in
chicken AKR, the distances of the carbonyl group of
all-trans-RAL to the catalytic residues and NADPH predict a
productive binding (Fig. 6E).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, 17
, or 20
positions. In contrast, many AKR
enzymes, such as HSDs, human AR, Chinese hamster ovary reductase, and
mouse vas deferens reductase, catalyze steroid conversion (3, 22, 25,
31, 59), although the human AR-like enzyme is not active with
progesterone nor with 17
-hydroxyprogesterone (27). Therefore,
in relation to the kinetic properties with both sugars and steroids,
chicken AKR exhibits more similarities with the AR-like than with the
AR enzymes.
1) is several times
higher than those for the best MDR ADHs (7, 13, 14, 17), which are
faster enzymes than the SDR RAL reductases (15). The
kcat/Km (catalytic
efficiency) is also extremely high for 9-cis-RAL (32000 mM
1·min
1), only comparable
with the value of amphibian ADH8 with all-trans-RAL (33750 mM
1·min
1) (17). Moreover, the
catalytic efficiency of chicken AKR for all-trans-RAL is
similar to that of the most efficient ADHs (7, 13, 14, 17). In
conclusion, the present AKR kinetic constants, mostly
kcat and
kcat/Km, are similar or even
higher than those of the SDR and MDR enzymes active with retinoids,
further supporting a role of chicken AKR in RAL reduction.
/
)8 barrel of the AKR tertiary
structure, with a conserved core and variable loops in outer domains,
responsible for substrate binding, represents a highly adaptable
scaffold, able to evolve and develop distinct functions (62) as the
retinoid oxidoreduction, here demonstrated. The inclusion of the AKR
superfamily in the metabolism of retinoids defines a new scenario where
the triad SDR-MDR-AKR would participate in the cellular regulation of
the essential signaling function of retinoids.
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Note Added in Proof |
---|
Chicken AKR has been designed AKR1B12 based on cluster analysis including members of the AKR superfamily, as recommended by Jez et al. (Jez, J. M., Flynn, T. G., and Penning, T. M. (1997) Biochem. Pharmacol. 54, 639-647).
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FOOTNOTES |
---|
* This work was supported by Commission of the European Union Grant BIO4-CT97-2123, Spanish Dirección General de Enseñanza Superior e Investigación Científica Grants PM96-0069 and PB98-0855, and Swedish Medical Research Council Project 13X-3532.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ295030.
¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain. Tel.: 34-93-5813026; Fax: 34-93-5811264; E-mail: xavier.pares@uab.es.
Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M010478200
2 D. Hirschberg, E. Cederlund, A. P. Jonsson, B. Crosas, J. Farrés, X. Parés, T. Bergman, and H. Jörnvall, in preparation.
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ABBREVIATIONS |
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The abbreviations used are: SDR, short chain dehydrogenase/reductase; ADH, alcohol dehydrogenase; AKR, aldo-keto reductase; AR, aldose reductase; FALDH, glutathione-dependent formaldehyde dehydrogenase; HSD, hydroxysteroid dehydrogenase; MDR, medium chain dehydrogenase/reductase; RA, retinoic acid; RAL, retinal; ROL, retinol; HPLC, high performance liquid chromatography; PCR, polymerase chain reaction; bp, base pair(s).
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