Structural basis for the high all-trans-retinaldehyde reductase activity of the tumor marker AKR1B10.
Journal: 2008/January - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 1091-6490
Abstract:
AKR1B10 is a human aldo-keto reductase (AKR) found to be elevated in several cancer types and in precancerous lesions. In vitro, AKR1B10 exhibits a much higher retinaldehyde reductase activity than any other human AKR, including AKR1B1 (aldose reductase). We here demonstrate that AKR1B10 also acts as a retinaldehyde reductase in vivo. This activity may be relevant in controlling the first step of retinoic acid synthesis. Up-regulation of AKR1B10, resulting in retinoic acid depletion, may lead to cellular proliferation. Both in vitro and in vivo activities of AKR1B10 were inhibited by tolrestat, an AKR1B1 inhibitor developed for diabetes treatment. The crystal structure of the ternary complex AKR1B10-NADP(+)-tolrestat was determined at 1.25-A resolution. Molecular dynamics models of AKR1B10 and AKR1B1 with retinaldehyde isomers and site-directed mutagenesis show that subtle differences at the entrance of the retinoid-binding site, especially at position 125, are determinant for the all-trans-retinaldehyde specificity of AKR1B10. Substitutions in the retinaldehyde cyclohexene ring also influence the specificity. These structural features should facilitate the design of specific inhibitors, with potential use in cancer and diabetes treatments.
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Proc Natl Acad Sci U S A 104(52): 20764-20769

Structural basis for the high <em>all-trans</em>-retinaldehyde reductase activity of the tumor marker AKR1B10

Results

AKR1B10 Acts as a Retinaldehyde Reductase in a Cellular Environment.

After the recent demonstration of a relevant retinaldehyde reductase activity in vitro (7), we here investigated the involvement of AKR1B10 in retinoid metabolism in vivo. COS-1 cells were transfected with a suitable plasmid containing AKR1B10 cDNA [supporting information (SI) Fig. 5]. all-trans-Retinaldehyde and all-trans-retinol were measured in COS-1 cells transfected with empty vector or with AKR1B10 vector, after incubation with all-trans-retinaldehyde (Fig. 1A). Production of retinyl esters or retinoic acid was undetectable under the conditions used. Control cells had a marked ability to reduce retinaldehyde to retinol because they converted ≈25% of intracellular retinaldehyde. However, cells transiently expressing AKR1B10 showed a 2-fold-higher rate of reduction, reaching ≈55% of conversion. In contrast, incubation with retinol did not produce a significant increase in intracellular retinaldehyde content. Thus, although AKR1B10 can use both retinol and retinaldehyde in vitro, it appears to function only as a retinaldehyde reductase in vivo.

An external file that holds a picture, illustration, etc.
Object name is zpq0510787180001.jpg

Retinaldehyde reductase activity of AKR1B10 and effect of tolrestat in vitro and in vivo. (A) Retinoid metabolism in COS-1 cells transiently expressing AKR1B10. Cellular retinoid content was measured by HPLC after incubating cells for 30 min with 10 μM retinaldehyde or 10 μM retinol. (B) Determination of tolrestat IC50 for retinaldehyde reductase activity of AKR1B10 using 0.5 μM retinaldehyde as a substrate. (C) Tolrestat inhibition of cellular AKR1B10 activity. COS-1 cells transfected with pCMV-HA-AKR1B10 were incubated with 10 μM all-trans-retinaldehyde and different concentrations of tolrestat. Data are expressed as the percentage of conversion of the retinoid taken up by cells (reduced retinaldehyde or oxidized retinol). Conversion for COS-1 cells transfected with empty vector (pCMV-HA) is shown as a control. Results are expressed as the mean ± SEM of at least three determinations.

Tolrestat Inhibition of Retinaldehyde Reductase Activity of AKR1B10 in Vitro and in Vivo.

In a previous work we tested the effect of tolrestat on the retinaldehyde reductase activity of AKR1B1 and AKR1B10, using Tween 80 as a retinoid solvent (3). Similar Ki values were obtained for the two enzymes, but they were 10-fold higher than those reported for AKR1B1 using glyceraldehyde as a substrate (11). Here we reevaluated the effect of the inhibitor on the retinaldehyde reductase activity of AKR1B10 measured by HPLC in the absence of detergent. With the new methodology, we determined an IC50 of ≈10 nM (Fig. 1B), a value similar to that published for the tolrestat inhibition of the glyceraldehyde reductase activity of AKR1B1 (12).

Tolrestat also affected the retinaldehyde reductase activity of COS-1 cells expressing AKR1B10 (Fig. 1C). When cells were incubated with 1 μM tolrestat, the activity contributed by the enzyme was reduced by ≈50%, whereas it was completely suppressed by 10 μM tolrestat. Thus, tolrestat was revealed as a potent inhibitor of AKR1B10 both in vitro and in vivo.

Crystal Structure of AKR1B10 Complexed with NADP and Tolrestat.

The crystal structure of AKR1B10, complexed with the cofactor NADP and the inhibitor tolrestat, was solved and refined at 1.25-Å resolution (Fig. 2A and B and SI Table 3). The structure of AKR1B10 showed the (α/β)8 barrel topology, characteristic of the AKR superfamily. The quality and the resolution of the final electron density map allowed the accurate determination of the conformation of all residues from Met-1 to Tyr-316 (henceforth residue numbering will correspond to that of AKR1B10 with the Swiss-Prot entry {"type":"entrez-protein","attrs":{"text":"O60218","term_id":"322510010","term_text":"O60218"}}O60218) and all non-hydrogen atoms of the cofactor and the inhibitor (SI Fig. 6). The highest mobility, as derived from the thermal B factors, corresponds to a region from Lys-125 to Ala-131 included in the loop A, which contributes to the upper lid of the active-site pocket (Fig. 2B). The NADP cofactor binds at the carboxyl edge of the β-strands of the barrel in an extended conformation that is perpendicular to the axis defined by the strands and with the adenine and nicotinamide moieties located at the periphery and at the center of the barrel, respectively. In turn, tolrestat binds mostly parallel to that axis with its carboxyl group at distances of 2.63 and 3.27 Å from the essential Tyr-49 hydroxyl group and from the cofactor C4 atom, respectively. This geometry is expected to be very close to that of a substrate bound in a catalytically productive manner.

An external file that holds a picture, illustration, etc.
Object name is zpq0510787180002.jpg

Crystal structure of AKR1B10 complexed with NADP and tolrestat. (A) View from the top of the (α/β)8 barrel. The catalytic site is located in the center of the barrel. (B) View of the (α/β)8 barrel after rotating 90°. Cofactor approaches the catalytic site from one side of the barrel, and tolrestat enters the barrel from the upper face. (C) LIGPLOT (32) describing interactions of the tolrestat molecule in the AKR1B10–NADP–tolrestat complex. (D) LIGPLOT of tolrestat in the AKR1B1–NADP–tolrestat complex.

The analysis of interactions between AKR1B10 and the inhibitor shows that residues Tyr-49, His-111, and Trp-112 establish hydrogen bonds with tolrestat atoms O2 and O3 of the carboxyl group and, together with the cofactor-positive charge, define an anion-binding pocket (Fig. 2C). Trp-21, Val-48, Trp-80, Trp-112, Phe-116, Phe-123, Trp-220, Cys-299, Val-301, Gln-303, and the nicotinamide moiety of the cofactor define a strongly hydrophobic pocket, the so-called specificity pocket in AKR1B1, where tolrestat is found. For comparison, a similar analysis was performed for AKR1B1 (13) [Protein Data Bank (PDB) entry 2FZD] in Fig. 2D. For residues interacting with tolrestat, only positions 301 and 303 differ between AKR1B10 and AKR1B1 (Fig. 2D), which might be consistent with the inhibitor having similar effects on AKR1B10 and AKR1B1.

Computer Modeling of Retinaldehyde Binding.

Models of the ternary complexes of AKR1B10 and AKR1B1 with NADP and all-trans-retinaldehyde were built based on the corresponding crystal structures with tolrestat (Fig. 3). In the two enzymes, the retinaldehyde molecule replaced the inhibitor, fitting nicely into the binding pocket. The resulting complexes were submitted to molecular dynamics simulation (SI Text). The average structures, during a time window of 250 ps after equilibration of the complex, were taken for analysis. In both cases, rotation of the cyclohexene ring to accommodate in the binding pocket was observed just after ≈350 ps. During the rest of the simulation the two C1-methyl groups pointed toward the interior of the enzyme. In the anion-binding pocket, the carbonyl group was placed at catalytic distances (≈2.8 Å) from the hydroxyl group of Tyr-49 and the cofactor C4 atom (SI Fig. 7 A and B). Besides this, the larger size of the retinaldehyde molecule with respect to tolrestat resulted in new hydrophobic interactions that were mainly located in the more external part of the binding site. Residues implicated in retinaldehyde binding are strictly conserved in the inner part of the pocket (conformed by Trp-21, Tyr-49, Trp-80, His-111, Phe-116, Phe-123, Trp-220, and Cys-299), resulting in equivalent interactions for the two enzymes. In contrast, all residues (except Pro-124) differ between AKR1B1 and AKR1B10 in the more external part of the channel: Leu125Lys and Val131Ala (loop A), Leu301Val, Ser303Gln, and Cys304Ser (loop C). In particular, whereas Leu-125 results in tight packing in AKR1B1 (Fig. 3C and D), the side chain of Lys-125 is substantially displaced with respect to the conformation seen in the crystal structure of AKR1B10 with tolrestat (Fig. 3A and B).

An external file that holds a picture, illustration, etc.
Object name is zpq0510787180003.jpg

Models of all-trans-retinaldehyde docked into the AKR1B10 and AKR1B1 structures. (A) Tolrestat-binding pocket in the AKR1B10–NADP–tolrestat crystal. (B) all-trans-retinaldehyde-binding pocket of AKR1B10 predicted by our model. (C) Tolrestat-binding pocket in the AKR1B1–NADP–tolrestat crystal (PDB entry 2FZD). (D) all-trans-retinaldehyde-binding pocket of AKR1B1 predicted by our model. The molecular surface is colored according to the local electrostatic potential as calculated with the program PYMOL (www.pymol.org). Residues around the substrate define a highly hydrophobic and well adjusted pocket, protecting the retinaldehyde molecule from the polar solvent.

Models for the complexes of AKR1B10 and AKR1B1 with 9-cis-retinaldehyde were similarly obtained (Fig. 4). In AKR1B10, all residues adopted essentially the same conformation found in the all-trans isomer complex, with the exception of loop A residues (SI Fig. 8). The rmsd between the backbone atoms of the all-trans and 9-cis complexes is much higher for loop A (2.4 Å) than for the rest of the protein (0.6 Å). The most noticeable change in the binding-pocket residues with respect to the all-trans complex corresponds to Lys-125, which in the 9-cis complex retained the conformation observed for tolrestat binding. The different conformation of Lys-125 side chain in the complexes with all-trans and 9-cis substrates is apparent from the representation of the evolution of the N-C-C-C dihedral angle during the simulation (SI Figs. 9 and 10). Thus, when comparing the four complexes obtained with the two enzymes and the two retinaldehyde isomers, the N atom of Lys-125 in the AKR1B10–NADPall-trans-retinaldehyde complex has to move 4 Å away to allow for the complex formation. The location of residue 125 at the entrance of the binding pocket may have variable effects on substrate binding and/or product release.

An external file that holds a picture, illustration, etc.
Object name is zpq0510787180004.jpg

Retinaldehyde isomers docked into the AKR1B10 and AKR1B1 structures. (A) Superimposition of critical residues for 9-cis-retinaldehyde (blue) and all-trans-retinaldehyde (orange) binding predicted by the AKR1B10 model. (B) Superimposition for 9-cis- and all-trans-retinaldehyde in the AKR1B1 model.

Site-Directed Mutagenesis.

To investigate the participation of residues predicted by the modeling in the remarkable kinetic differences between AKR1B10 and AKR1B1 with retinoids, AKR1B10 mutants K125L, V301L, and K125L/V301L were prepared. In these substitutions the AKR1B10 residues were changed to the corresponding ones in AKR1B1.

The kinetics of AKR1B1, AKR1B10, and the AKR1B10 mutants were analyzed toward d,l-glyceraldehyde and the all-trans- and 9-cis-retinaldehyde isomers (Table 1). Comparison of AKR1B1 and AKR1B10 revealed distinct kinetic features. AKR1B1 exhibited a 100-fold-lower Km value with glyceraldehyde, but a similar kcat. In contrast, Km values for all-trans-retinaldehyde were similar for the two enzymes, but kcat was 100-fold higher in AKR1B10. Remarkably, the two AKRs showed identical kinetic results with 9-cis-retinaldehyde, with low Km and kcat values.

Table 1.

Kinetic analysis for human AKRs and mutant enzymes using the BSA/HPLC method

d,l-Glyceraldehyde
all-trans-Retinaldehyde
9-cis-Retinaldehyde
Km, mMkcat, min−1kcat/Km, mM·min−1Km, μMkcat, min−1kcat/Km, mM·min−1Km, μMkcat, min−1kcat/Km, mM·min−1
AKR1B10.05 ± 0.0131 ± 1660 ± 801.1 ± 0.1*0.35 ± 0.01*320 ± 30*0.4 ± 0.10.7 ± 0.11,500 ± 170
AKR1B106 ± 135 ± 16 ± 10.6 ± 0.1*27 ± 1*45,000 ± 7,600*0.7 ± 0.10.9 ± 0.11,300 ± 160
AKR1B10 K125L5.2 ± 0.835.6 ± 0.17 ± 10.15 ± 0.022 ± 0.0613,100 ± 2,0000.7 ± 0.10.9 ± 0.11,300 ± 190
AKR1B10 V301L6.0 ± 0.741 ± 17 ± 10.6 ± 0.17.7 ± 0.312,700 ± 1,4000.30 ± 0.030.60 ± 0.011,800 ± 140
AKR1B10 K125L/V301L7.3 ± 0.735.4 ± 0.94.9 ± 0.51 ± 0.33.5 ± 0.013,500 ± 320NDNDND

ND, not determined.

*Data taken from ref. 7.

The AKR1B10 mutants, built to mimic the retinoid-binding site of AKR1B1, changed the kinetic constants in the direction predicted by our models. Thus, with glyceraldehyde, a small substrate that occupies a binding position far from the substituted residues, the kinetic constants were identical to those of wild-type AKR1B10. Interestingly, activities with all-trans-retinaldehyde revealed a 13.5-fold decrease in the kcat value for the K125L mutant and a 3.5-fold decrease for the V301L mutant. Although the Km value did not change for the V301L enzyme, it showed a 4-fold-lower value for the K125L mutant. Both mutations failed to modify the kinetic properties of AKR1B10 toward the 9-cis isomer. The double mutation did not further decrease the kcat value for all-trans-retinaldehyde, but yielded a lower kcat/Km than the single mutations.

In addition, the mutants exhibited IC50 values with tolrestat identical to that for wild-type AKR1B10 (data not shown), demonstrating that the mutated residues, important for retinoid kinetics, are not relevant for tolrestat binding.

Kinetics with 4-Hydroxyretinaldehyde.

The structural models of the complexes AKR–retinaldehyde indicate that position 125 and others from the external part of the substrate-binding pocket interact with the cyclohexene ring. If these interactions contribute to the distinct specificity of the AKRs with retinoids, a structural change in the ring is likely to affect the kinetics. To test this hypothesis, (S)-all-trans-4-hydroxyretinaldehyde was synthesized and the kinetics were performed. Unfortunately, the BSA/HPLC method was not feasible, essentially because BSA did not efficiently solubilize the hydroxyl-retinoid and because of a low recovery in the extraction with organic solvents. Therefore, the Tween 80/spectrophotometer methodology was used, although we had shown that this method does not yield reliable Km values because of apparent competitive inhibition by Tween 80. Nevertheless, the kcat results were quite consistent with those obtained with the BSA/HPLC method (7). Comparison of the kcat values for the all-trans- and (S)-all-trans-4-OH-isomers (Table 2) strongly supports the participation of the cyclohexene ring in the specificity for retinoids. Thus, the OH substitution dramatically increased the kcat value of the low-activity enzymes AKR1B1 and AKR1B10 K125L whereas its value did not change for the highly active AKR1B10.

Table 2.

Catalytic constants with all-trans-4-OH-retinaldehyde in Tween 80

kcat, min
all-trans-Retinaldehyde(S)-all-trans-4-Hydroxyretinaldehyde
AKR1B10.37 ± 0.025.80 ± 0.16
AKR1B1017.8 ± 1.017.3 ± 1.5
AKR1B10 K125L4.40 ± 0.0218.5 ± 2.6

Activities were performed in 0.1 M sodium phosphate (pH 7.5)/0.2 mM NADPH/0.02% Tween 80 at 25°C (7).

AKR1B10 Acts as a Retinaldehyde Reductase in a Cellular Environment.

After the recent demonstration of a relevant retinaldehyde reductase activity in vitro (7), we here investigated the involvement of AKR1B10 in retinoid metabolism in vivo. COS-1 cells were transfected with a suitable plasmid containing AKR1B10 cDNA [supporting information (SI) Fig. 5]. all-trans-Retinaldehyde and all-trans-retinol were measured in COS-1 cells transfected with empty vector or with AKR1B10 vector, after incubation with all-trans-retinaldehyde (Fig. 1A). Production of retinyl esters or retinoic acid was undetectable under the conditions used. Control cells had a marked ability to reduce retinaldehyde to retinol because they converted ≈25% of intracellular retinaldehyde. However, cells transiently expressing AKR1B10 showed a 2-fold-higher rate of reduction, reaching ≈55% of conversion. In contrast, incubation with retinol did not produce a significant increase in intracellular retinaldehyde content. Thus, although AKR1B10 can use both retinol and retinaldehyde in vitro, it appears to function only as a retinaldehyde reductase in vivo.

An external file that holds a picture, illustration, etc.
Object name is zpq0510787180001.jpg

Retinaldehyde reductase activity of AKR1B10 and effect of tolrestat in vitro and in vivo. (A) Retinoid metabolism in COS-1 cells transiently expressing AKR1B10. Cellular retinoid content was measured by HPLC after incubating cells for 30 min with 10 μM retinaldehyde or 10 μM retinol. (B) Determination of tolrestat IC50 for retinaldehyde reductase activity of AKR1B10 using 0.5 μM retinaldehyde as a substrate. (C) Tolrestat inhibition of cellular AKR1B10 activity. COS-1 cells transfected with pCMV-HA-AKR1B10 were incubated with 10 μM all-trans-retinaldehyde and different concentrations of tolrestat. Data are expressed as the percentage of conversion of the retinoid taken up by cells (reduced retinaldehyde or oxidized retinol). Conversion for COS-1 cells transfected with empty vector (pCMV-HA) is shown as a control. Results are expressed as the mean ± SEM of at least three determinations.

Tolrestat Inhibition of Retinaldehyde Reductase Activity of AKR1B10 in Vitro and in Vivo.

In a previous work we tested the effect of tolrestat on the retinaldehyde reductase activity of AKR1B1 and AKR1B10, using Tween 80 as a retinoid solvent (3). Similar Ki values were obtained for the two enzymes, but they were 10-fold higher than those reported for AKR1B1 using glyceraldehyde as a substrate (11). Here we reevaluated the effect of the inhibitor on the retinaldehyde reductase activity of AKR1B10 measured by HPLC in the absence of detergent. With the new methodology, we determined an IC50 of ≈10 nM (Fig. 1B), a value similar to that published for the tolrestat inhibition of the glyceraldehyde reductase activity of AKR1B1 (12).

Tolrestat also affected the retinaldehyde reductase activity of COS-1 cells expressing AKR1B10 (Fig. 1C). When cells were incubated with 1 μM tolrestat, the activity contributed by the enzyme was reduced by ≈50%, whereas it was completely suppressed by 10 μM tolrestat. Thus, tolrestat was revealed as a potent inhibitor of AKR1B10 both in vitro and in vivo.

Crystal Structure of AKR1B10 Complexed with NADP and Tolrestat.

The crystal structure of AKR1B10, complexed with the cofactor NADP and the inhibitor tolrestat, was solved and refined at 1.25-Å resolution (Fig. 2A and B and SI Table 3). The structure of AKR1B10 showed the (α/β)8 barrel topology, characteristic of the AKR superfamily. The quality and the resolution of the final electron density map allowed the accurate determination of the conformation of all residues from Met-1 to Tyr-316 (henceforth residue numbering will correspond to that of AKR1B10 with the Swiss-Prot entry {"type":"entrez-protein","attrs":{"text":"O60218","term_id":"322510010","term_text":"O60218"}}O60218) and all non-hydrogen atoms of the cofactor and the inhibitor (SI Fig. 6). The highest mobility, as derived from the thermal B factors, corresponds to a region from Lys-125 to Ala-131 included in the loop A, which contributes to the upper lid of the active-site pocket (Fig. 2B). The NADP cofactor binds at the carboxyl edge of the β-strands of the barrel in an extended conformation that is perpendicular to the axis defined by the strands and with the adenine and nicotinamide moieties located at the periphery and at the center of the barrel, respectively. In turn, tolrestat binds mostly parallel to that axis with its carboxyl group at distances of 2.63 and 3.27 Å from the essential Tyr-49 hydroxyl group and from the cofactor C4 atom, respectively. This geometry is expected to be very close to that of a substrate bound in a catalytically productive manner.

An external file that holds a picture, illustration, etc.
Object name is zpq0510787180002.jpg

Crystal structure of AKR1B10 complexed with NADP and tolrestat. (A) View from the top of the (α/β)8 barrel. The catalytic site is located in the center of the barrel. (B) View of the (α/β)8 barrel after rotating 90°. Cofactor approaches the catalytic site from one side of the barrel, and tolrestat enters the barrel from the upper face. (C) LIGPLOT (32) describing interactions of the tolrestat molecule in the AKR1B10–NADP–tolrestat complex. (D) LIGPLOT of tolrestat in the AKR1B1–NADP–tolrestat complex.

The analysis of interactions between AKR1B10 and the inhibitor shows that residues Tyr-49, His-111, and Trp-112 establish hydrogen bonds with tolrestat atoms O2 and O3 of the carboxyl group and, together with the cofactor-positive charge, define an anion-binding pocket (Fig. 2C). Trp-21, Val-48, Trp-80, Trp-112, Phe-116, Phe-123, Trp-220, Cys-299, Val-301, Gln-303, and the nicotinamide moiety of the cofactor define a strongly hydrophobic pocket, the so-called specificity pocket in AKR1B1, where tolrestat is found. For comparison, a similar analysis was performed for AKR1B1 (13) [Protein Data Bank (PDB) entry 2FZD] in Fig. 2D. For residues interacting with tolrestat, only positions 301 and 303 differ between AKR1B10 and AKR1B1 (Fig. 2D), which might be consistent with the inhibitor having similar effects on AKR1B10 and AKR1B1.

Computer Modeling of Retinaldehyde Binding.

Models of the ternary complexes of AKR1B10 and AKR1B1 with NADP and all-trans-retinaldehyde were built based on the corresponding crystal structures with tolrestat (Fig. 3). In the two enzymes, the retinaldehyde molecule replaced the inhibitor, fitting nicely into the binding pocket. The resulting complexes were submitted to molecular dynamics simulation (SI Text). The average structures, during a time window of 250 ps after equilibration of the complex, were taken for analysis. In both cases, rotation of the cyclohexene ring to accommodate in the binding pocket was observed just after ≈350 ps. During the rest of the simulation the two C1-methyl groups pointed toward the interior of the enzyme. In the anion-binding pocket, the carbonyl group was placed at catalytic distances (≈2.8 Å) from the hydroxyl group of Tyr-49 and the cofactor C4 atom (SI Fig. 7 A and B). Besides this, the larger size of the retinaldehyde molecule with respect to tolrestat resulted in new hydrophobic interactions that were mainly located in the more external part of the binding site. Residues implicated in retinaldehyde binding are strictly conserved in the inner part of the pocket (conformed by Trp-21, Tyr-49, Trp-80, His-111, Phe-116, Phe-123, Trp-220, and Cys-299), resulting in equivalent interactions for the two enzymes. In contrast, all residues (except Pro-124) differ between AKR1B1 and AKR1B10 in the more external part of the channel: Leu125Lys and Val131Ala (loop A), Leu301Val, Ser303Gln, and Cys304Ser (loop C). In particular, whereas Leu-125 results in tight packing in AKR1B1 (Fig. 3C and D), the side chain of Lys-125 is substantially displaced with respect to the conformation seen in the crystal structure of AKR1B10 with tolrestat (Fig. 3A and B).

An external file that holds a picture, illustration, etc.
Object name is zpq0510787180003.jpg

Models of all-trans-retinaldehyde docked into the AKR1B10 and AKR1B1 structures. (A) Tolrestat-binding pocket in the AKR1B10–NADP–tolrestat crystal. (B) all-trans-retinaldehyde-binding pocket of AKR1B10 predicted by our model. (C) Tolrestat-binding pocket in the AKR1B1–NADP–tolrestat crystal (PDB entry 2FZD). (D) all-trans-retinaldehyde-binding pocket of AKR1B1 predicted by our model. The molecular surface is colored according to the local electrostatic potential as calculated with the program PYMOL (www.pymol.org). Residues around the substrate define a highly hydrophobic and well adjusted pocket, protecting the retinaldehyde molecule from the polar solvent.

Models for the complexes of AKR1B10 and AKR1B1 with 9-cis-retinaldehyde were similarly obtained (Fig. 4). In AKR1B10, all residues adopted essentially the same conformation found in the all-trans isomer complex, with the exception of loop A residues (SI Fig. 8). The rmsd between the backbone atoms of the all-trans and 9-cis complexes is much higher for loop A (2.4 Å) than for the rest of the protein (0.6 Å). The most noticeable change in the binding-pocket residues with respect to the all-trans complex corresponds to Lys-125, which in the 9-cis complex retained the conformation observed for tolrestat binding. The different conformation of Lys-125 side chain in the complexes with all-trans and 9-cis substrates is apparent from the representation of the evolution of the N-C-C-C dihedral angle during the simulation (SI Figs. 9 and 10). Thus, when comparing the four complexes obtained with the two enzymes and the two retinaldehyde isomers, the N atom of Lys-125 in the AKR1B10–NADPall-trans-retinaldehyde complex has to move 4 Å away to allow for the complex formation. The location of residue 125 at the entrance of the binding pocket may have variable effects on substrate binding and/or product release.

An external file that holds a picture, illustration, etc.
Object name is zpq0510787180004.jpg

Retinaldehyde isomers docked into the AKR1B10 and AKR1B1 structures. (A) Superimposition of critical residues for 9-cis-retinaldehyde (blue) and all-trans-retinaldehyde (orange) binding predicted by the AKR1B10 model. (B) Superimposition for 9-cis- and all-trans-retinaldehyde in the AKR1B1 model.

Site-Directed Mutagenesis.

To investigate the participation of residues predicted by the modeling in the remarkable kinetic differences between AKR1B10 and AKR1B1 with retinoids, AKR1B10 mutants K125L, V301L, and K125L/V301L were prepared. In these substitutions the AKR1B10 residues were changed to the corresponding ones in AKR1B1.

The kinetics of AKR1B1, AKR1B10, and the AKR1B10 mutants were analyzed toward d,l-glyceraldehyde and the all-trans- and 9-cis-retinaldehyde isomers (Table 1). Comparison of AKR1B1 and AKR1B10 revealed distinct kinetic features. AKR1B1 exhibited a 100-fold-lower Km value with glyceraldehyde, but a similar kcat. In contrast, Km values for all-trans-retinaldehyde were similar for the two enzymes, but kcat was 100-fold higher in AKR1B10. Remarkably, the two AKRs showed identical kinetic results with 9-cis-retinaldehyde, with low Km and kcat values.

Table 1.

Kinetic analysis for human AKRs and mutant enzymes using the BSA/HPLC method

d,l-Glyceraldehyde
all-trans-Retinaldehyde
9-cis-Retinaldehyde
Km, mMkcat, min−1kcat/Km, mM·min−1Km, μMkcat, min−1kcat/Km, mM·min−1Km, μMkcat, min−1kcat/Km, mM·min−1
AKR1B10.05 ± 0.0131 ± 1660 ± 801.1 ± 0.1*0.35 ± 0.01*320 ± 30*0.4 ± 0.10.7 ± 0.11,500 ± 170
AKR1B106 ± 135 ± 16 ± 10.6 ± 0.1*27 ± 1*45,000 ± 7,600*0.7 ± 0.10.9 ± 0.11,300 ± 160
AKR1B10 K125L5.2 ± 0.835.6 ± 0.17 ± 10.15 ± 0.022 ± 0.0613,100 ± 2,0000.7 ± 0.10.9 ± 0.11,300 ± 190
AKR1B10 V301L6.0 ± 0.741 ± 17 ± 10.6 ± 0.17.7 ± 0.312,700 ± 1,4000.30 ± 0.030.60 ± 0.011,800 ± 140
AKR1B10 K125L/V301L7.3 ± 0.735.4 ± 0.94.9 ± 0.51 ± 0.33.5 ± 0.013,500 ± 320NDNDND

ND, not determined.

*Data taken from ref. 7.

The AKR1B10 mutants, built to mimic the retinoid-binding site of AKR1B1, changed the kinetic constants in the direction predicted by our models. Thus, with glyceraldehyde, a small substrate that occupies a binding position far from the substituted residues, the kinetic constants were identical to those of wild-type AKR1B10. Interestingly, activities with all-trans-retinaldehyde revealed a 13.5-fold decrease in the kcat value for the K125L mutant and a 3.5-fold decrease for the V301L mutant. Although the Km value did not change for the V301L enzyme, it showed a 4-fold-lower value for the K125L mutant. Both mutations failed to modify the kinetic properties of AKR1B10 toward the 9-cis isomer. The double mutation did not further decrease the kcat value for all-trans-retinaldehyde, but yielded a lower kcat/Km than the single mutations.

In addition, the mutants exhibited IC50 values with tolrestat identical to that for wild-type AKR1B10 (data not shown), demonstrating that the mutated residues, important for retinoid kinetics, are not relevant for tolrestat binding.

Kinetics with 4-Hydroxyretinaldehyde.

The structural models of the complexes AKR–retinaldehyde indicate that position 125 and others from the external part of the substrate-binding pocket interact with the cyclohexene ring. If these interactions contribute to the distinct specificity of the AKRs with retinoids, a structural change in the ring is likely to affect the kinetics. To test this hypothesis, (S)-all-trans-4-hydroxyretinaldehyde was synthesized and the kinetics were performed. Unfortunately, the BSA/HPLC method was not feasible, essentially because BSA did not efficiently solubilize the hydroxyl-retinoid and because of a low recovery in the extraction with organic solvents. Therefore, the Tween 80/spectrophotometer methodology was used, although we had shown that this method does not yield reliable Km values because of apparent competitive inhibition by Tween 80. Nevertheless, the kcat results were quite consistent with those obtained with the BSA/HPLC method (7). Comparison of the kcat values for the all-trans- and (S)-all-trans-4-OH-isomers (Table 2) strongly supports the participation of the cyclohexene ring in the specificity for retinoids. Thus, the OH substitution dramatically increased the kcat value of the low-activity enzymes AKR1B1 and AKR1B10 K125L whereas its value did not change for the highly active AKR1B10.

Table 2.

Catalytic constants with all-trans-4-OH-retinaldehyde in Tween 80

kcat, min
all-trans-Retinaldehyde(S)-all-trans-4-Hydroxyretinaldehyde
AKR1B10.37 ± 0.025.80 ± 0.16
AKR1B1017.8 ± 1.017.3 ± 1.5
AKR1B10 K125L4.40 ± 0.0218.5 ± 2.6

Activities were performed in 0.1 M sodium phosphate (pH 7.5)/0.2 mM NADPH/0.02% Tween 80 at 25°C (7).

Discussion

Previous reports have shown a high catalytic constant (kcat) for the retinaldehyde reductase activity of AKR1B10 (3, 7), which is unique in human AKRs, making the enzyme comparable to retinaldehyde reductases from the SDR superfamily in terms of catalytic efficiency (kcat/Km). Here we have confirmed this observation and have demonstrated the remarkable specificity of AKR1B10 toward all-trans-retinaldehyde. Thus, activity with 9-cis-retinaldehyde, also a physiological compound, is 35-fold lower (Table 1), similar to the activity of other human AKRs, such as AKR1B1, with any retinaldehyde isomer. Therefore, AKR1B10 is strictly an all-trans-retinaldehyde reductase.

The multiple reports on the overexpression of AKR1B10 in different human cancer types (1, 46, 14) have increased the search for physiological or xenobiotic substrates for the enzyme that could reveal a link between a physiological function and its participation in cancer development. Thus, the reducing activity of AKR1B10 has been found with the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, the antiemetic, type 3 serotonin (5-HT3) receptor antagonist dolasteron, and the antitumor drugs daunorubicin and oracin, suggesting that AKR1B10 may play a role in the chemoresistance of tumors toward carbonyl group-bearing drugs (15). In addition, AKR1B10 is able to activate polycyclic aromatic hydrocarbons (16). However, these substrates exhibit 1,000-fold-lower catalytic efficiencies than all-trans-retinaldehyde, stressing the extreme specificity of the enzyme toward this retinoid and reinforcing the concept that a pathological effect of AKR1B10 overexpression in cancer may be through an alteration of the retinoic acid synthesis pathway.

AKR1B10, expressed in COS-1 cells, efficiently reduced retinaldehyde, providing evidence that the enzyme functions as a retinaldehyde reductase also in vivo. The in vitro and in vivo activity of AKR1B10 was inhibited by tolrestat, a well known AKR1B1 inhibitor designed to prevent diabetic complications by blocking sorbitol accumulation. Tolrestat decreased retinaldehyde reductase activity of AKR1B10 with the same IC50 as that reported for the inhibition of glyceraldehyde reduction by AKR1B1 (IC50 ≈ 10 nM) (12). Moreover, tolrestat inhibited AKR1B10 expressed in COS-1 cells with an effect similar to that found on AKR1B4 (the rat ortholog of AKR1B1, IC50 ≈ 1 μM) (17). Tolrestat is therefore an efficient AKR1B10 inhibitor, and thus the overexpression of AKR1B10 in cancer could be counteracted by tolrestat or similar AKR1B1 inhibitors, suggesting potential applications in cancer control. On the other hand, many AKR1B1 inhibitors have side effects precluding their pharmacological use (10, 18). The inhibition of AKR1B10 by AKR1B1 inhibitors may alter retinoid metabolism and contribute to their nonspecific actions. Hence, the design and selection of AKR1B1 inhibitors should also consider their effect on AKR1B10. The recognition of the structural basis of the AKR1B10 specificity with retinoids, and the interest of designing specific inhibitors of potential anticancer activity, make relevant the crystallization and determination of the x-ray structure of the AKR1B10–NADP–tolrestat complex presented here. Tolrestat is bound in the substrate-binding pocket, establishing essentially the same interactions as in AKR1B1 (13). AKR1B1 and AKR1B10 share 70% sequence identity, and, accordingly, their three-dimensional structures are very similar, with an rmsd value between the C atoms of 0.95 Å. Therefore, it was expected that a comparative examination of their structures might explain the 100-fold-higher catalytic efficiency of AKR1B10 with all-trans-retinaldehyde. Attempts to obtain crystals of AKR1B1 and AKR1B10 with NADP and retinoids were unsuccessful. Very few structures of enzymes with retinoid analogs have been reported so far (19), and not a single oxidoreductase structure has been experimentally obtained. Therefore, models of the two enzymes with all-trans- and 9-cis-retinaldehyde were built based on the crystal structures with NADP and tolrestat. all-trans-Retinaldehyde is a larger molecule than tolrestat, which results in its cyclohexene ring interacting with the external part of the cleft, mainly the loops A and C. Comparison of the models for AKR1B1 and AKR1B10 indicates that the residue at position 125 (Lys in AKR1B10 and Leu in AKR1B1) shows the largest differences. In AKR1B10, binding of the all-trans-retinaldehyde molecule requires that Lys-125 move toward the solvent. Such a displacement is not needed in any of the other models analyzed: AKR1B10 with 9-cis-retinaldehyde and AKR1B1 with all-trans- or 9-cis-retinaldehyde. Therefore, Lys-125 appears to be a key residue for AKR1B10 to attain the highest catalytic efficiency with all-trans-retinaldehyde.

To explore this hypothesis, the AKR1B10 K125L mutant was studied. As expected, the activity with glyceraldehyde was not affected by a mutation located in a distant position from the binding pocket of the small substrate. In contrast, with all-trans-retinaldehyde, the kcat value decreased by 13.5-fold with respect to that of the wild-type enzyme. Consistently, all-trans-retinaldehyde modeling into AKR1B10 K125L did not require any rearrangement in the mutated residue, similar to what had been observed for the other enzyme–substrate pairs having poor activity. Kinetics with 9-cis-retinaldehyde was not affected by the mutation. These results fully support a relationship between residue-125 conformational change and AKR1B10 specificity. The AKR1B10 V301L mutant also exhibited a small decrease in the kcat value for all-trans-retinaldehyde, indicating that other residues of the cyclohexene-binding site may also contribute to AKR1B10 specificity. The effect on kcat was not additive in the AKR1B10 K125L/V301L double mutant, but its kcat/Km value further decreased with respect to the single mutants.

The involvement of the substrate-binding region that interacts with the cyclohexene ring in the specificity for retinoids was further supported by using (S)-all-trans-4-hydroxyretinaldehyde as substrate. The cyclohexene hydroxyl group, well oriented toward the 125 position as observed in the models (SI Fig. 11), has a strong influence on the kinetics (Table 2). When interacting with a hydrophilic residue, such as Lys-125 in the highly active AKR1B10, the kcat value is not altered. However, if a hydrophobic residue is found, such as in the less active AKR1B1 or AKR1B10 K125L, then the kcat value increases. Apparently, to reach high activity a hydrophilic group, either in the substrate cyclohexene moiety or in the enzyme position 125, is required. The two situations, either separately or simultaneously present, provide an effect on the substrate binding or, most probably, on the product release, which is translated into a higher kcat value.

The strong differences in kcat values suggest that the limiting step in the reaction mechanism with retinaldehyde differs between the enzymes studied. The limiting step for the low-activity species seems slower than cofactor dissociation, the common limiting step for AKRs with the best substrates (20, 21). The present results are consistent with the dissociation of the retinol product being limiting in these cases, but this should be further investigated by using transient kinetics analysis.

In conclusion, the structural features that make AKR1B10 highly efficient for all-trans-retinaldehyde are localized in the external part of the substrate-binding site, including positions 125 and 301, where the retinoid cyclohexene ring binds. Substitutions in the cyclohexene ring also influence the specificity. These structural characteristics, in both the enzyme and the ligand, should be taken into account for the rational design of new and more specific inhibitors against AKR1B10 for potential cancer control and against AKR1B1 for diabetes treatment.

Materials and Methods

Site-Directed Mutagenesis.

K125L, V301L, and K125L/V301L mutants were obtained by standard procedures performed as described in SI Text.

Expression and Purification of AKR1B10.

Wild-type AKR1B10 and mutants were purified as described (7). Details of purification are presented in SI Text.

Enzyme Kinetics.

Standard activities were measured before each kinetic experiment by using d,l-glyceraldehyde as a substrate (3). d,l-Glyceraldehyde kinetics were performed as described (3) but in the presence of 90 mM potassium phosphate (pH 7.4)/40 mM KCl (reaction buffer) at 37°C. Activity with retinoids in the presence of BSA was performed in reaction buffer at 37°C in siliconized glass tubes (22), and the products were analyzed by HPLC. Activity in the presence of Tween 80 was determined as described (7). The kinetic constants were expressed as the mean ± SEM of at least three independent determinations. (S)-all-trans-4-Hydroxyretinaldehyde was synthesized as reported (23).

HPLC Analysis.

After extraction, retinoids were separated by chromatography on a Spherisorb S3W column (4.6 × 100 mm; Waters) in hexane:methyl-tert-butyl ether (96:4, vol/vol) mobile phase, at a flow rate of 2 ml/min using Waters Alliance 2695 HPLC. Elution was monitored at 350 nm with a Waters 2996 photodiode array detector (7).

Cell Culture and Transfection.

Cells were grown in DMEM supplemented with 10% (vol/vol) FBS (GIBCO BRL). Transfection was done with Lipofectamine Plus (Invitrogen) as detailed in SI Text.

Crystallization and Structure Determination.

Crystals were grown at 20°C by vapor diffusion using the hanging-drop method. One microliter of protein solution (18 mg/ml) was mixed with 1 μl of precipitant solution containing polyethylene glycol 6000 and 100 mM sodium cacodylate (pH 9.0). The hanging drop was equilibrated with 0.8 ml of precipitant solution. Crystals belonged to the hexagonal space group P61 (a = b = 89.1 Å, c = 78.4 Å, α = β = 90.0°, γ = 120.0°), with one molecule in the asymmetric unit. Data were collected at 100 K on an ADSC Q4R CCD detector in the beamline ID14-4 at the European Synchrotron Radiation Facility. Diffraction data were integrated and scaled by using DENZO and SCALEPACK (24). The structure was solved by molecular replacement with MOLREP (25), using as a searching model the PDB coordinates with entry 1FRB (26). Subsequent refinement was carried out by using REFMAC (27) and manual model building in O (28) (SI Table 3). Building of the AKR1B10–NADP–retinaldehyde complexes was performed with the graphic program O and regularized with the idealization option in REFMAC. AUTODOCK 3.05 software (29) was used to build the initial structure of the AKR1B1–NADP–retinaldehyde complexes, using the crystal structure of AKR1B1 (13) (PDB entry 2FZD).

Molecular Dynamics Simulations.

Molecular dynamics simulations were performed to allow retinaldehyde substrates to accommodate in the binding cavity as described in SI Text. Analysis of the trajectories was carried out by using standard tools of AMBER (30) and with VMD (31).

Coordinates.

Atomic coordinates and structure factors of the AKR1B10–NADP–tolrestat complex have been deposited in the PDB (ID code 1ZUA).

Site-Directed Mutagenesis.

K125L, V301L, and K125L/V301L mutants were obtained by standard procedures performed as described in SI Text.

Expression and Purification of AKR1B10.

Wild-type AKR1B10 and mutants were purified as described (7). Details of purification are presented in SI Text.

Enzyme Kinetics.

Standard activities were measured before each kinetic experiment by using d,l-glyceraldehyde as a substrate (3). d,l-Glyceraldehyde kinetics were performed as described (3) but in the presence of 90 mM potassium phosphate (pH 7.4)/40 mM KCl (reaction buffer) at 37°C. Activity with retinoids in the presence of BSA was performed in reaction buffer at 37°C in siliconized glass tubes (22), and the products were analyzed by HPLC. Activity in the presence of Tween 80 was determined as described (7). The kinetic constants were expressed as the mean ± SEM of at least three independent determinations. (S)-all-trans-4-Hydroxyretinaldehyde was synthesized as reported (23).

HPLC Analysis.

After extraction, retinoids were separated by chromatography on a Spherisorb S3W column (4.6 × 100 mm; Waters) in hexane:methyl-tert-butyl ether (96:4, vol/vol) mobile phase, at a flow rate of 2 ml/min using Waters Alliance 2695 HPLC. Elution was monitored at 350 nm with a Waters 2996 photodiode array detector (7).

Cell Culture and Transfection.

Cells were grown in DMEM supplemented with 10% (vol/vol) FBS (GIBCO BRL). Transfection was done with Lipofectamine Plus (Invitrogen) as detailed in SI Text.

Crystallization and Structure Determination.

Crystals were grown at 20°C by vapor diffusion using the hanging-drop method. One microliter of protein solution (18 mg/ml) was mixed with 1 μl of precipitant solution containing polyethylene glycol 6000 and 100 mM sodium cacodylate (pH 9.0). The hanging drop was equilibrated with 0.8 ml of precipitant solution. Crystals belonged to the hexagonal space group P61 (a = b = 89.1 Å, c = 78.4 Å, α = β = 90.0°, γ = 120.0°), with one molecule in the asymmetric unit. Data were collected at 100 K on an ADSC Q4R CCD detector in the beamline ID14-4 at the European Synchrotron Radiation Facility. Diffraction data were integrated and scaled by using DENZO and SCALEPACK (24). The structure was solved by molecular replacement with MOLREP (25), using as a searching model the PDB coordinates with entry 1FRB (26). Subsequent refinement was carried out by using REFMAC (27) and manual model building in O (28) (SI Table 3). Building of the AKR1B10–NADP–retinaldehyde complexes was performed with the graphic program O and regularized with the idealization option in REFMAC. AUTODOCK 3.05 software (29) was used to build the initial structure of the AKR1B1–NADP–retinaldehyde complexes, using the crystal structure of AKR1B1 (13) (PDB entry 2FZD).

Molecular Dynamics Simulations.

Molecular dynamics simulations were performed to allow retinaldehyde substrates to accommodate in the binding cavity as described in SI Text. Analysis of the trajectories was carried out by using standard tools of AMBER (30) and with VMD (31).

Coordinates.

Atomic coordinates and structure factors of the AKR1B10–NADP–tolrestat complex have been deposited in the PDB (ID code 1ZUA).

Supplementary Material

Supporting Information:
*Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain;
Institut de Biologia Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Institut de Recerca Biomèdica, and
Centre de Recerca en Química Teòrica, Parc Científic de Barcelona, Josep Samitier 1–5, E-08028 Barcelona, Spain;
Institució Catalana de Recerca i Estudis Avançats, Passeig Lluís Companys 23, E-08010 Barcelona, Spain; and
Departamento de Química Orgánica, Universidade de Vigo, E-36200 Vigo, Spain
**To whom correspondence should be addressed. E-mail: se.bau@serap.reivax
Edited by Wayne A. Hendrickson, Columbia University, New York, NY, and approved November 6, 2007

Author contributions: O.G., C.R., J.F., I.F., and X.P. designed research; O.G., F.X.R., A.A., and I.F. performed research; M.D., R.A., A.R.d.L., and C.R. contributed new reagents/analytic tools; O.G., F.X.R., A.A., C.R., J.F., I.F., and X.P. analyzed data; and O.G., A.R.d.L., C.R., J.F., I.F., and X.P. wrote the paper.

Present address: European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
Edited by Wayne A. Hendrickson, Columbia University, New York, NY, and approved November 6, 2007
Received 2007 Jun 16

Abstract

AKR1B10 is a human aldo-keto reductase (AKR) found to be elevated in several cancer types and in precancerous lesions. In vitro, AKR1B10 exhibits a much higher retinaldehyde reductase activity than any other human AKR, including AKR1B1 (aldose reductase). We here demonstrate that AKR1B10 also acts as a retinaldehyde reductase in vivo. This activity may be relevant in controlling the first step of retinoic acid synthesis. Up-regulation of AKR1B10, resulting in retinoic acid depletion, may lead to cellular proliferation. Both in vitro and in vivo activities of AKR1B10 were inhibited by tolrestat, an AKR1B1 inhibitor developed for diabetes treatment. The crystal structure of the ternary complex AKR1B10–NADP–tolrestat was determined at 1.25-Å resolution. Molecular dynamics models of AKR1B10 and AKR1B1 with retinaldehyde isomers and site-directed mutagenesis show that subtle differences at the entrance of the retinoid-binding site, especially at position 125, are determinant for the all-trans-retinaldehyde specificity of AKR1B10. Substitutions in the retinaldehyde cyclohexene ring also influence the specificity. These structural features should facilitate the design of specific inhibitors, with potential use in cancer and diabetes treatments.

Keywords: aldo-keto reductases, aldose reductase, protein structure, retinoic acid, tolrestat
Abstract

AKR1B10 (human small intestine aldose reductase or aldose reductase-like) is a recently identified NADP-dependent aldo-keto reductase (AKR) (1, 2). Similar to other members of the enzymatic family, AKR1B10 is a monomer of Mr 36,000 and can reduce a variety of aldehydes and ketones, such as glyceraldehyde, methylglyoxal, diacetyl, and aromatic aldehydes (1, 3). However, its physiological function remains still unclear. Two recent findings have made the study of this enzyme especially relevant: its detection in several human cancer types and its high activity with retinaldehyde.

AKR1B10 was reported to be increased in several studies on human hepatocellular carcinoma (1, 4). Recently it was proved that the expression of the enzyme was induced in patients with non-small cell lung carcinoma (NSCLC), which is mainly linked to tobacco consumption (5). Accordingly, AKR1B10 has been proposed as a new diagnostic marker for smoking-related NSCLC. Interestingly, expression of AKR1B10 was found to be induced already in squamous metaplasia, a precancerous lesion in squamous cell carcinoma, suggesting that the enzyme could be involved in the onset of carcinogenesis and stressing its potential use as a therapeutic target (5). Consistent with this report is the several-fold induction of AKR1B10 in oral cancer cells by cigarette smoke condensate (6).

We have demonstrated that AKR1B10 and other AKRs catalyze retinol–retinaldehyde conversion (3, 7), the essential first step in the retinoic acid synthesis pathway. Retinoic acid participates in a large number of biological processes, ranging from fetal development to cell proliferation and differentiation, controlling the expression of multiple genes (8). Two other enzyme types are well known to be involved in this initial redox step, alcohol dehydrogenases from the medium-chain dehydrogenases/reductases and retinol dehydrogenases from the short-chain dehydrogenases/reductases (SDR) (8). Thus, highly distinct protein structures have converged to the same function. The multiplicity of members of the three enzyme superfamilies that can contribute to retinol–retinaldehyde interconversion stresses the importance of this critical step in retinoic acid synthesis (7). An alteration of this step, e.g., by changing enzymatic levels, may result in disturbances in gene control and cell proliferation. Thus, the up-regulation of retinaldehyde reductase activity of AKR1B10 found in cancer could be linked to the depletion of retinoic acid levels and subsequent loss of cell differentiation and cancer development (9).

In addition to AKR1B10, other human AKRs—AKR1B1 (aldose reductase) (3, 7), AKR1C3, and AKR1C4 (F.X.R. and X.P., unpublished data)—are also active with retinaldehyde, but with very low kcat values. AKR1B10 is an exception; it exhibits low Km and high kcat values, resulting in a catalytic efficiency comparable to that of the best retinaldehyde reductases from the SDR superfamily (7). In the present work we have further studied the functional properties of AKR1B10, in vivo and in vitro, and have obtained its crystal structure to discover the specific features that provide its high activity with retinoids. We have compared the functional and structural properties of AKR1B10 with those of AKR1B1 as a model of an AKR with low retinaldehyde reductase activity. Thus, we have demonstrated the inhibition of AKR1B10 with tolrestat, a powerful inhibitor of AKR1B1, designed to treat secondary complications of diabetes (10). The crystal structures show that AKR1B10 and AKR1B1 similarly bind tolrestat, but differences are observed in the corresponding models of complexes with retinaldehyde. This could be related to the distinct kinetics of the two enzymes with retinoids, which is supported by results from site-directed mutagenesis.

ND, not determined.

Activities were performed in 0.1 M sodium phosphate (pH 7.5)/0.2 mM NADPH/0.02% Tween 80 at 25°C (7).

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ACKNOWLEDGMENTS.

We thank Professor T. Geoffrey Flynn (Department of Biochemistry, Queen's University, Kingston, Ontario, Canada) for his support. We acknowledge the computer support, technical expertise, and assistance provided by the Barcelona Supercomputing Center—Centro Nacional de Supercomputación. This work was supported by grants from Dirección General de Investigación (BMC2003-09606, BFU2005-02621, BFU2005-08686-C02-01, and FIS2005-00655) and Generalitat de Catalunya (2005SGR-00036 and 2005SGR-00112).

ACKNOWLEDGMENTS.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 1ZUA).

This article contains supporting information online at www.pnas.org/cgi/content/full/0705659105/DC1.

Footnotes

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