(Received for publication, October 5, 1994; and in revised form, December 7, 1994)
From the
A full-length 1966-base pair clone of the human class IV alcohol
dehydrogenase (-ADH) was isolated from a human stomach cDNA
library. The 373-amino acid
-ADH encoded by this cDNA was
expressed in Escherichia coli. The specific activity of the
recombinant enzyme for ethanol oxidation at pH 7.5 and 25 °C,
calculated from active-site titration of NADH binding, was 92 ±
9 units/mg. Kinetic analysis of the catalytic efficiency (k
/K
) of
recombinant
-ADH for oxidation of primary alcohols indicated
broad substrate specificity. Recombinant human
-ADH exhibited
high catalytic efficiency for oxidation of all-trans-retinol
to all-trans-retinal. This pathway is important in the
synthesis of the transcriptional regulator all-trans-retinoic
acid. Secondary alcohols and 3
-hydroxysteroids were inactive with
-ADH or were oxidized with very low efficiency. The K
of
-ADH for ethanol was 25
mM, and the K
for primary
straight chain alcohols decreased substantially as chain length
increased. There are important amino acid differences in the
alcohol-binding site between the human class IV (
) and human
class I (
) alcohol dehydrogenases that appear to explain the high
catalytic efficiency for all-trans-retinol, the high k
for ethanol, and the low catalytic efficiency
for secondary alcohols of
-ADH relative to
-ADH.
For example, modeling the binding of all-trans-retinol in the
human
-ADH structure suggested that coordination of
retinol to the active-site zinc is hindered by a loop from residues 114
to 120 that is at the entrance to the alcohol-binding site. The
deletion of Gly-117 in human
-ADH and a substitution of Leu for
the bulky Tyr-110 appear to facilitate retinol access to the
active-site zinc.
Humans produce as many as nine different subunits in the family
of dimeric NAD- and zinc-dependent alcohol
dehydrogenases (ADH(
); EC 1.1.1.1). All of these enzymes
exhibit broad substrate specificity. These polypeptides are encoded by
six different genes, two of which are polymorphic. The relationships
between active-site structure and substrate/inhibitor specificity of
the active dimers formed from eight of these subunits (
,
,
,
,
,
,
, and
) have been well
characterized(1, 2) . Those containing
-,
-,
-, and
-subunits have relatively high catalytic efficiency
for ethanol oxidation and even higher catalytic efficiency for
oxidation of long chain primary alcohols. The isozymes have been
grouped into class I (
,
, and
), class II (
), and
class III (
), depending, in part, upon substrate
specificity(1, 2) .
Recently, a new isozyme called
-ADH (or µ-ADH) was isolated from human stomach
tissue(3, 4, 5) .
-ADH exhibits a high K
for ethanol (29 mM) and a
remarkably high k
(1500 min
)
for ethanol oxidation(4, 5) . This enzyme may be
responsible, in part, for ``first pass'' alcohol metabolism
prior to distribution of ethanol into the systemic
circulation(6) . Isozymes with similar electrophoretic
mobilities are found in the human esophagus(7) , rat (8) and mouse (9) stomach, and rat retina(10) .
-ADH has been classified as class IV. The amino acid sequence of
the rat stomach enzyme (8) and a partial amino acid sequence of
the human stomach enzyme (5) have been published. A complete
coding nucleotide sequence for the human stomach enzyme assembled from
partial cDNA clones(11, 12) , a partial genomic
clone(11) , and fragments of the cDNA sequences amplified by
PCR from a cDNA library (12, 13) were reported
recently. Analysis of the deduced amino acid sequence of human stomach
ADH indicates that it has 59-69% sequence identity to the other
four human ADH classes (11) and that class IV ADHs likely
diverged after the avian/amphibian split (8) .
In this
paper, we describe the isolation of the complete cDNA encoding human
stomach -ADH, the expression of an active recombinant enzyme in a
heterologous system, and the characterization of the catalytic
properties and substrate specificity of recombinant human
-ADH.
The cDNA is positively identified by the expression of an active
recombinant isozyme and by the perfect match of the deduced amino acid
sequence with that of
-ADH peptides. Comparison of the amino
acids in the substrate-binding site of
-ADH and the human liver
-isozyme suggests explanations for the high catalytic
efficiency of
-ADH with all-trans-retinol.
A complete cDNA encoding human class IV ADH was isolated from
a ``5`-stretch'' human stomach cDNA library
(CLONTECH)(14) . The coding region of -ADH was amplified
by PCR with the primers CTTTTTCGGATCCATGGGCACTGCTGGAAAAG (sense) and
CCACTTGAATTCTCAAAACGTCAGGACCGT (antisense). The primers contained
recognition sequences (underlined) for the restriction endonucleases BamHI and EcoRI, respectively. After cleavage, the
amplification product was subcloned into the expression vector pGEX-2T
(Pharmacia Biotech Inc.). The final construct of the
-ADH cDNA
fused with the glutathione S-transferase cDNA contained codons
for 3 extra amino acids (Gly, Ser, and the starting Met) on the N
terminus of
-ADH. The fusion protein (glutathione S-transferase and
-ADH) was expressed and purified using
glutathione-agarose affinity chromatography(14, 15) .
After cleavage of the fusion product with thrombin,
-ADH was
separated from glutathione transferase by elution from a Mono Q column
(Pharmacia Biotech Inc.) with a linear salt gradient. Fractions
containing active
-ADH were combined and concentrated. Purity of
the enzyme was evaluated by SDS-PAGE and staining for protein with
Coomassie Blue(14) . Total protein concentration was determined
by a dye binding assay (Bio-Rad) using bovine serum albumin as a
standard(16) .
Kinetic constants for alcohols, except
retinol, were determined by monitoring the production of NADH at 340 nm
( = 6.22 mM
cm
). The kinetic constants for
all-trans-retinol were obtained by monitoring the production
of all-trans-retinal at 400 nm (
= 29.5
mM
cm
)(17, 18) . Reaction
mixtures contained 2.4 mM NAD
in 0.1 M sodium phosphate, pH 7.4, at 25 °C. The concentration of
active sites was determined by observing fluorescence (excitation
wavelength at 328 nm) at 425 nm while titrating enzyme (1-2
mg/ml) with NADH in the presence of 99 mM isobutyramide in 10
mM sodium phosphate at pH 7.4. The concentration of
-ADH
active sites was evaluated from the intersection point of the linear
regression of the fluorescence titration above and below NADH
saturation(19) . The V
and K
values for alcohol substrates (at 2.4
mM NAD
) were calculated from a fit of the
kinetic data to the Michaelis-Menten equation (V = V
A/(K
+ A),
where A is the concentration of the varied substrate). The k
(min
) was obtained by
dividing V
by the concentration of active sites
assuming a subunit M
of 40,000. The K
values for NAD
(A) and
ethanol (B) were extrapolated to saturation of the second substrate by
a fit of the kinetic data to a sequential Bi Bi mechanism (V = V
AB/(K
B
+ K
A + K
K
+
AB))(20) . The inhibition constant for 4-methylpyrazole was
determined in 0.1 M sodium phosphate buffer containing 2.4
mM NAD
, pH 7.4, at 25 °C by varying both
ethanol and 4-methylpyrazole concentrations. The K
of 4-methylpyrazole was calculated from a fit of the kinetic
data to the equation for competitive inhibition (V = V
B/(K
(1 +
I/K
) + B), where B and I are ethanol and
4-methylpyrazole concentrations, respectively)(20) .
Aldehyde-reducing activity was measured in 0.1 M sodium
phosphate buffer, pH 7.4, at 25 °C employing 0.2 mM NADH
with acetaldehyde (0.25-60 mM) and 2 mM NADH
with
4-trans-(N,N-dimethylamino)cinnamaldehyde
(0.5-60 mM). Acetaldehyde reduction was measured by the
change of NADH absorbance at 340 nm (
= 6.22
mM
cm
). The reduction of
4-trans-(N,N-dimethylamino) cinnamaldehyde
was followed at 398 nm (
= 42 mM
cm
).
The protein sequences of -ADH,
rat class IV ADH, and
-ADH were aligned by the
progressive alignment method of Feng and Doolittle(21) . The
alignment uses the algorithm of Needleman and Wunsch (22) and
the minimum mutation matrix of Dayhoff et al.(23) for
scoring. Gaps are introduced by comparing the most closely related pair
of sequences and are retained by the ``once a gap always a gap
rule.''
The substitutions occurring in human -ADH were
model-built into the human
-structure using the
molecular graphics program QUANTA (Molecular Simulations, Inc.). The
deletion at position 117 was modeled manually by altering local main
chain dihedrals to reconnect the main chain atoms. All residues in this
loop remain in allowed regions of a Ramachandran plot. Following
substitution of all amino acid side chains in the dimer, the model
structure was subjected to 100 cycles of energy minimization using
X-PLOR 3.1 with the x-ray energy term omitted(24) . Docking of
the retinol molecule into the human
-structure was
performed using the program AUTODOCK (25) .
To clone the human stomach -ADH cDNA, a PCR
amplification strategy employing inosine residues in primers was
devised to reduce the degeneracy. After two rounds of amplification
using nested sets of primers, an 873-bp PCR product was obtained and
found to encode human stomach
-ADH(14) . A human stomach
cDNA library was screened with this PCR product, and a 1966-bp cDNA for
human stomach
-ADH was isolated (Fig. 1). The cDNA encodes
the complete
-ADH subunit of 373 amino acids with a M
of 39,902. The 5`-end of this cDNA starts with
the ATG codon immediately followed by the coding region. The
3`-untranslated region is 825 bp long and contains a single
polyadenylation signal at nucleotide 1877.
Figure 1:
Nucleotide sequence and deduced peptide
sequence of -ADH. Amino acid residues that were previously
determined in our laboratory by peptide sequencing (5) are in boldface. Peptide sequences that were used for the design of
degenerate oligonucleotide primers are underlined. The
polyadenylation signal AATAAA is underlined. The termination
codon TGA is indicated (***). The starting Met is present at position
19 of the 1966-bp clone. The 18 bp of the 5`-noncoding sequence found
in the clone (not shown) are apparently a cloning artifact because they
do not appear in the 5`-genomic sequence reported by Satre et
al.(11) . The cDNA sequence is numbered from the
initiating ATG codon as position 19. The amino acid sequence is
numbered from the Gly following the initiating Met codon in accord with
numbering of all other human ADH isozymes.
The -ADH cDNA was
expressed in Escherichia coli, and the recombinant enzyme was
purified to homogeneity. Approximately 1-3 mg of purified
-ADH was obtained per liter of bacterial culture. The fusion
protein (glutathione S-transferase and
-ADH) was
purified by affinity chromatography on a glutathione-agarose column.
After thrombin cleavage,
-ADH was purified by ion-exchange
chromatography on a Mono Q column. The recombinant enzyme was
homogeneous, with an apparent subunit M
of
40,000 on SDS-PAGE(14) . The mobility of recombinant
-ADH was slightly more cathodic than that of native human
-ADH on starch gel electrophoresis(14) .
To
characterize the functional properties of the recombinant enzyme, the
steady-state kinetics for alcohol oxidation and aldehyde reduction were
examined. The specific activity of purified recombinant -ADH
based on a dye-binding protein assay (16) was 47 ± 10
units/mg for three separate preparations. However, calculation of
specific activity by active-site titration that directly measures the
concentration of NADH-binding sites (19) resulted in a specific
activity of 92 ± 9 units/mg. The kinetic constants of
recombinant
-ADH for oxidation of a variety of alcohols are shown
in Table 1. One important property of human
-ADH was the
high k
for ethanol oxidation (1840
min
at pH 7.4). The catalytic efficiency (k
/K
) of
-ADH for primary alcohols measured with 2.4 mM NAD
increased >2 orders of magnitude as the
chain length of the alcohols increased from two to six carbons in
length, but the k
of human
-ADH was
relatively constant for primary alcohols having two to six carbons (Table 1). The K
values ranged from
28 mM for ethanol to 0.13 mM for hexanol. The k
/K
of recombinant
-ADH for all-trans-retinol was 2600 min
mM
. The rates of oxidation of
cyclohexanol and the steroid alcohols etiocholan-3
-ol-17-one and
epiandrosterone, at a maximum solubility of 200-250 mM,
were <2% of that of ethanol. The apparent k
/K
of
-ADH
for acetaldehyde with 0.2 mM NADH was 2030 ± 160
min
mM
.
-ADH also
exhibited high catalytic efficiency (2900 ± 300 min
mM
with 2 mM NADH) toward
the chromophoric aldehyde
4-trans-(N,N-dimethylamino)cinnamaldehyde,
which has been used for analysis of human ADHs by stopped-flow kinetics (26) . The K
of
-ADH for
NAD
(0.21 ± 0.01 mM) was calculated
from a fit of the kinetic data to the sequential Bi Bi mechanism and
extrapolated to saturating ethanol concentration. The K
for NAD
was calculated
from the same fit of the data to be 0.76 ± 0.10 mM. The K
of
-ADH for NADH with 150 mM acetaldehyde was 219 ± 6 µM. The K
of 4-methylpyrazole calculated from a
fit of the data for competitive inhibition with ethanol as the varied
substrate at pH 10 was 11 ± 1 µM, and the K
at pH 7.4 was 350 ± 20
µM.
Computerized docking simulations were performed
with all-trans-retinol using the program AUTODOCK (25) and the x-ray structure of -ADH (27) or the model of
-ADH built by substituting the
appropriate residues in the
-structure (Fig. 2). The simulations show that retinol can bind in a
productive conformation in both the
-ADH (Fig. 3A) and
-ADH (Fig. 3B)
models. In each model, the alcohol oxygen is directly coordinated to
the active-site zinc (within 2.6 Å), and the hydrogen on C-1 is
in a proper orientation for hydride transfer to C-4 of the
NAD
nicotinamide group (
3.3 Å). The first
four dihedral angles of the all-trans-retinol system in the
-ADH model are in the conjugated planar conformations usually
observed for the alcohol (Fig. 3). However, for
all-trans-retinol to bind productively to
,
rotations about angles 3-5 must be made as shown in Fig. 3C.
Figure 2:
Alignment of -ADH with human class
I
-ADH and rat stomach class IV ADH (R). The
deletion of amino acid 117 in human and rat stomach ADHs is indicated
(*). Amino acid numbers correspond to human class I
-ADH. Key amino acid differences mentioned under
``Results'' and ``Discussion'' are in boldface.
Figure 3:
Results of retinol docking simulations
using the program AUTODOCK(25) . A,
all-trans-retinol docked into the substrate-binding pocket of
human -ADH. The interaction between the docked retinol
molecule and the catalytic zinc atom is illustrated with a dashedline (distance of 2.2 Å). The dashedline between the
ionone ring methyl group and the
carbonyl oxygen of residue 117 represents the closest contact (3.2
Å) between the docked conformation of retinol and the
-ADH substrate-binding pocket. B,
all-trans-retinol docked into the model-built
substrate-binding pocket of human
-ADH. The dashedline illustrates the interaction between the hydroxyl
oxygen of retinol and the catalytic zinc atom (distance of 2.5
Å). C, dihedral angles of all-trans-retinol
docked into the
-ADH and
-ADH active sites.
Our initial attempts to amplify a fragment of human stomach
-ADH by PCR with degenerate oligonucleotide primers, designed
according to the amino acid sequence of the purified
enzyme(5) , proved unsuccessful. This suggested that the amount
of
-ADH mRNA in whole stomach is low. After the degeneracy of
primers was reduced by incorporation of inosine residues(14) ,
an 873-bp PCR fragment was obtained and used to clone a 1966-bp cDNA of
-ADH from a human stomach cDNA library. The deduced amino acid
sequence of the cDNA shown in Fig. 1is identical to the partial
amino acid sequence of
-ADH (5) . The cDNA coding
sequence is also identical to the cDNA and genomic sequences of
-ADH published recently(11, 12, 13) .
The kinetic and molecular characterization of -ADH has been
hampered by the limited quantities of suitable human stomach or
esophageal tissue available for enzyme
purification(4, 5) . Therefore, we cloned the
-ADH coding sequence into the pGEX-2T expression vector and
expressed the enzyme as a fusion protein with glutathione S-transferase in E. coli. After purification of the
fusion protein by affinity chromatography, cleavage with thrombin, and
further purification of
-ADH by ion-exchange chromatography, a
homogeneous protein preparation was obtained. The expressed protein
contained 3 N-terminal amino acids (Gly, Ser, and the starting Met)
that remained from the fusion protein after thrombin cleavage. While
the subunit size of the expressed protein on SDS-PAGE was the same as
that of the native enzyme (40 kDa), the mobility of recombinant
-ADH on starch gel electrophoresis was more cathodic than that of
the native enzyme. This difference in mobility could be explained by
the presence of a free amino group in the recombinant enzyme and a
blocked N terminus in the native enzyme. The human
-subunit (class
I) and other ADH isozyme subunits start with N-acetylated
residues (28) . The specific activity of purified recombinant
-ADH based on an active-site titration that directly measures the
concentration of NADH-binding sites (19) was about twice that
measured by a dye-binding method that estimates total protein
concentration. Hence, we conclude that about half of the purified
recombinant enzyme is catalytically active.
Among human isozymes,
the amino acid sequence of human -ADH is most similar to that of
human class I ADH(11, 12) . Fig. 2shows the
comparison of
-ADH and
-ADH (29) sequences by the progressive alignment
method(21) . The three-dimensional structures for several
enzyme-substrate complexes have been published for human
- and
-isozymes(27, 30) .
All zinc ligands, key coenzyme-binding residues (e.g. Arg-47
and Arg-369), components of the proton relay system (e.g. Thr-48 and His-51), and important residues in the alcohol-binding
site (e.g. Thr-48, Phe-93, Phe-140, and Met-306) are conserved
in
-ADH (Fig. 2). In spite of the similarities in overall
structure of the class I and IV human ADHs, there are remarkable
differences in the catalytic properties of
-ADH (Table 1)
compared with the class I enzymes. One important property of human
-ADH is the high k
for ethanol oxidation
(1840 min
at pH 7.4). This value is 68 times that of
, 200 times that of
, and
21 times that of
(31, 32) . As
suggested by Farrés et al.(12) ,
the substitution of Asn-363 in human
-ADH for His-363 in
-ADH may affect coenzyme affinity and k
(27) ; coenzyme dissociation is
rate-limiting for
-ADH(26) .
Purified
-ADH is similar to other mammalian ADHs in that it exhibits the
highest catalytic efficiency with long chain alcohols, as shown in Table 1. The increase in catalytic efficiency of
-ADH with
increasing chain length of primary alcohols is predominantly due to the
decrease in K
because the k
of human
-ADH remained relatively
constant for primary alcohols with two to six carbons
(
1000-2100 min
in Table 1). The
decrease in primary alcohol K
with
increasing chain length may be due to an increase in the number of
possible van der Waals interactions between the simple aliphatic
primary alcohols and hydrophobic amino acids lining the deep
substrate-binding site.
The kinetic properties of -ADH with
more complex alcohols such as all-trans-retinol, steroid
alcohols, and secondary alcohols are different from those of the class
I ADHs. Vitamin A (all-trans-retinol) is oxidized to retinal
and retinoic acid by alcohol and aldehyde dehydrogenases in a variety
of tissues. The activity of these dehydrogenases is important for the
synthesis of all-trans-retinoic acid, which regulates gene
expression through binding to retinoic acid receptors(33) . It
has been proposed that competitive inhibition of ADH-catalyzed retinol
oxidation by ethanol could depress retinoic acid synthesis and alter
gene expression, thereby causing the developmental abnormalities seen
in fetal alcohol syndrome(34) . We found that
-ADH
purified from human stomach exhibited the highest k
/K
for
all-trans-retinol compared with eight other human ADH
isozymes(18) . The k
/K
of recombinant
-ADH for all-trans-retinol was 2600 min
mM
(Table 1). The most likely
sequence difference between
-ADH and class I ADHs
(
-ADH in Fig. 2) that could account for this
difference in retinol oxidation activity is the deletion of an amino
acid in a loop comprising residues 114-120 that is located on the
surface of the catalytic domain. This loop clearly influences access to
the alcohol-binding site as shown in the x-ray structure of human
- and
-ADHs (Fig. 3A)(29) . The length and sequence of this
loop are quite variable among the enzyme classes and different species.
A docking simulation between all-trans-retinol and either
human - or
-ADH explains why
-ADH exhibits
higher catalytic efficiency for retinol oxidation than the class I
ADHs. The productive docking solution where the retinol oxygen is
coordinated to the active-site zinc and C-1 of retinol is in a proper
orientation for hydride transfer to the nicotinamide of NAD
is shown for the
-ADH structure in Fig. 3A and for the
-ADH model in Fig. 3B. The substitution of the smaller Leu in
-ADH for Tyr at position 110 of
-ADH enables the
change in conformation necessary to model the loop from residues 114 to
120 in
-ADH shown in Fig. 3B. The conformation of
bound retinol in the two enzymes is the same for the first two dihedral
angles of the conjugated side chain of retinol as shown in Fig. 3C. To avoid creating unfavorable van der Waals
contacts with amino acid 117 of the loop in the substrate-binding
pocket of the
-structure, the remaining three dihedral
angles of retinol must be rotated out of plane, thus breaking the
conjugated double bond system of this chromophore. The energetic
penalty to create this unfavorable retinol conformation may account for
the lower k
/K
of
the class I
-isozyme compared with that of class IV
-ADH (Table 1)(18) .
A distinctive feature of
human -ADH is that it does not catalyze oxidation of the
3
-hydroxysteroids etiocholan-3
-ol-17-one and epiandrosterone.
There are two regions of the substrate-binding site (amino acid 48 and
the loop at positions 114-120) that have been shown to affect
3
-hydroxysteroid binding. Both the horse E- and S-isozymes have Ser at position 48. A deletion in the flexible
loop at position 115 (
Asp-115) in the S-isozyme increases
the catalytic efficiency for 3
-hydroxysteroids as compared with
the E-isozyme(35, 36) . For human class I
isozymes that do not have a deletion in the loop at positions
114-120, only the
-isozyme with Ser-48 oxidizes
3
-hydroxysteroids. The
-,
-, and
-isozymes with Thr
at position 48 are inactive with 3
-hydroxysteroids(37) .
Thus, it appears that the deletion of Gly-117 in the loop of
-ADH
is not sufficient to yield 3
-hydroxysteroid-oxidizing activity.
One of the most surprising properties of -ADH is its inability
to oxidize secondary alcohols efficiently. No activity was detected
with cyclohexanol. S(+)-2-Butanol was oxidized at a
measurable rate, but it had an extremely high K
(120 mM) and a 2200-fold lower k
/K
than that of
the primary alcohol homolog 1-butanol. It has been suggested that the
specificity of ADHs toward secondary alcohols is affected by amino
acids at positions 48 and 93(38) . However, human
-ADH
has the same amino acids, Thr-48 and Phe-93, as
-ADH;
therefore, other amino acid substitutions must be responsible for the
inactivity of
-ADH with secondary alcohols. The deletion of
Gly-117 in
-ADH, which opens up access to the alcohol-binding
site, may contribute to the inactivity of
-ADH with secondary
alcohols. For example, Hurley and Vessell (39) recently
demonstrated that the substitution of Leu-116 in
-ADH
with Ala substantially decreases the catalytic efficiency for secondary
alcohols. They suggested that enlarging the entrance to the
alcohol-binding site may yield fewer productive versus unproductive encounters between enzyme and secondary alcohols.
Human -ADH has high (87%) sequence identity to the rat stomach
enzyme. Parés et al.(8) and
Farrés et al.(12) have
discussed amino acid substitutions in human class IV ADH relative to
the rat class I isozymes that could account for its high k
for ethanol oxidation and altered substrate
specificity. Some of the exchanges in the rat stomach enzyme relative
to rat class I ADH are not seen in human
-ADH. For example, the
Val-294 that is important in both coenzyme and substrate binding in
-ADH remains Val in human
-ADH, but is Ala in
rat stomach ADH (Fig. 2). The Arg-47 in
-ADH
that participates in coenzyme binding remains Arg in human
-ADH,
but is Gly in rat stomach ADH (Fig. 2).
Farrés et al.(12) concluded
that the substitution of Arg-47 in the human enzyme by Gly in the rat
stomach enzyme may result in the higher K
for ethanol and the higher K
for 4-methylpyrazole. The unusually high k
and K
of human
-ADH for
NAD
may be due to the His-271 in human
-ADH versus Arg in the class I isozymes (Fig. 2).
These
kinetic studies demonstrate that the -ADH isozyme exhibits unique
alcohol-oxidizing specificity relative to other classes of human ADHs.
When the amino acid sequence of
-ADH is compared with those of
the class I isozymes, the deletion of Gly-117 and the exchange of the
smaller Leu-110 in
-ADH for Tyr in
-ADH may
contribute to differences in substrate specificity. The docking
simulations with
- and
-ADHs clearly indicate
the importance of these residues in the conformational constraints on
all-trans-retinol that are necessary to form a catalytically
competent complex.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U09623[GenBank].