Biological Role of Aldo–Keto Reductases in Retinoic Acid Biosynthesis and Signaling

Several aldo–keto reductase (AKR) enzymes from subfamilies 1B and 1C show retinaldehyde reductase activity, having low Km and kcat values. Only AKR1B10 and 1B12, with all-trans-retinaldehyde, and AKR1C3, with 9-cis-retinaldehyde, display high catalytic efficiency. Major structural determinants for retinaldehyde isomer specificity are located in the external loops (A and C for AKR1B10, and B for AKR1C3), as assessed by site-directed mutagenesis and molecular dynamics. Cellular models have shown that AKR1B and 1C enzymes are well suited to work in vivo as retinaldehyde reductases and to regulate retinoic acid (RA) biosynthesis at hormone pre-receptor level. An additional physiological role for the retinaldehyde reductase activity of these enzymes, consistent with their tissue localization, is their participation in β-carotene absorption. Retinaldehyde metabolism may be subjected to subcellular compartmentalization, based on enzyme localization. While retinaldehyde oxidation to RA takes place in the cytosol, reduction to retinol could take place in the cytosol by AKRs or in the membranes of endoplasmic reticulum by microsomal retinaldehyde reductases. Upregulation of some AKR1 enzymes in different cancer types may be linked to their induction by oxidative stress and to their participation in different signaling pathways related to cell proliferation. AKR1B10 and AKR1C3, through their retinaldehyde reductase activity, trigger a decrease in the RA biosynthesis flow, resulting in RA deprivation and consequently lower differentiation, with an increased cancer risk in target tissues. Rational design of selective AKR inhibitors could lead to development of novel drugs for cancer treatment as well as reduction of chemotherapeutic drug resistance

Identification of a novel polyfluorinated compound as a lead to inhibit human enzymes aldose reductase and AKR1B10 : structure determination of both ternary complexes and implications for drug design

Aldo-keto reductases (AKRs) are mostly monomeric enzymes which fold into a highly conserved ([alpha]/[beta])8 barrel, while their substrate specificity and inhibitor selectivity are determined by interaction with residues located in three highly variable external loops. The closely related human enzymes aldose reductase (AR or AKR1B1) and AKR1B10 are of biomedical interest because of their involvement in secondary diabetic complications (AR) and in cancer, e.g. hepatocellular carcinoma and smoking-related lung cancer (AKR1B10). After characterization of the IC50 values of both AKRs with a series of polyhalogenated compounds, 2,2',3,3',5,5',6,6'-octafluoro-4,4'-biphenyldiol (JF0064) was identified as a lead inhibitor of both enzymes with a new scaffold (a 1,1'-biphenyl-4,4'-diol). An ultrahigh-resolution X-ray structure of the AR-­NADP+-JF0064 complex has been determined at 0.85 Å resolution, allowing it to be observed that JF0064 interacts with the catalytic residue Tyr48 through a negatively charged hydroxyl group (i.e. the acidic phenol). The non-competitive inhibition pattern observed for JF0064 with both enzymes suggests that this acidic hydroxyl group is also present in the case of AKR1B10. Moreover, the combination of surface lysine methylation and the introduction of K125R and V301L mutations enabled the determination of the X-ray crystallo­graphic structure of the corresponding AKR1B10-NADP+-JF0064 complex. Comparison of the two structures has unveiled some important hints for subsequent structure-based drug-design efforts

Substrate Specificity, Inhibitor Selectivity and Structure-Function Relationships of Aldo-Keto Reductase 1B15: A Novel Human Retinaldehyde Reductase

Human aldo-keto reductase 1B15 (AKR1B15) is a newly discovered enzyme which shares 92% amino acid sequence identity with AKR1B10. While AKR1B10 is a well characterized enzyme with high retinaldehyde reductase activity, involved in the development of several cancer types, the enzymatic activity and physiological role of AKR1B15 are still poorly known. Here, the purified recombinant enzyme has been subjected to substrate specificity characterization, kinetic analysis and inhibitor screening, combined with structural modeling. AKR1B15 is active towards a variety of carbonyl substrates, including retinoids, with lower kcat and Km values than AKR1B10. In contrast to AKR1B10, which strongly prefers all-trans-retinaldehyde, AKR1B15 exhibits superior catalytic efficiency with 9-cis-retinaldehyde, the best substrate found for this enzyme. With ketone and dicarbonyl substrates, AKR1B15 also shows higher catalytic activity than AKR1B10. Several typical AKR inhibitors do not significantly affect AKR1B15 activity. Amino acid substitutions clustered in loops A and C result in a smaller, more hydrophobic and more rigid active site in AKR1B15 compared with the AKR1B10 pocket, consistent with distinct substrate specificity and narrower inhibitor selectivity for AKR1B15

Substrate Specificity, Inhibitor Selectivity and Structure-Function Relationships of Aldo-Keto Reductase 1B15 : a Novel Human Retinaldehyde Reductase

Human aldo-keto reductase 1B15 (AKR1B15) is a newly discovered enzyme which shares 92% amino acid sequence identity with AKR1B10. While AKR1B10 is a well characterized enzyme with high retinaldehyde reductase activity, involved in the development of several cancer types, the enzymatic activity and physiological role of AKR1B15 are still poorly known. Here, the purified recombinant enzyme has been subjected to substrate specificity characterization, kinetic analysis and inhibitor screening, combined with structural modeling. AKR1B15 is active towards a variety of carbonyl substrates, including retinoids, with lower kcat and Km values than AKR1B10. In contrast to AKR1B10, which strongly prefers alltrans-retinaldehyde, AKR1B15 exhibits superior catalytic efficiency with 9-cisretinaldehyde, the best substrate found for this enzyme. With ketone and dicarbonyl substrates, AKR1B15 also shows higher catalytic activity than AKR1B10. Several typical AKR inhibitors do not significantly affect AKR1B15 activity. Amino acid substitutions clustered in loops A and C result in a smaller, more hydrophobic and more rigid active site in AKR1B15 compared with the AKR1B10 pocket, consistent with distinct substrate specificity and narrower inhibitor selectivity for AKR1B15

Comparison of the cofactor-binding site between the AKR1B15 model (<i>A</i>) and the AKR1B10 crystal structure (<i>B</i>).

<p>Interactions of Met265 and His269 with NADP⁺ in AKR1B15 are similar to those of Val265 and Arg269 in AKR1B10 (black dotted lines). His269 forms a π-stacking interaction with the adenine ring of the cofactor. The substitution of Lys22 by Arg in AKR1B15 prevents its interaction with the pyrophosphate bridge of NADP⁺. The salt bridge between Asp217 and Lys263 (red dotted line), acting as a safety belt in the coenzyme binding, and the π-stacking interaction of Tyr210 with the cofactor nicotinamide ring are conserved between the two AKRs. Carbon atoms of the cofactor are shown in green, whereas those of the enzyme are colored grey. Figures have been drawn using PyMOL.</p

Molecular docking of substrates or inhibitors to the active-site pocket of AKR1B15.

<p>(<i>A</i>) Residues implicated in binding all-<i>trans</i>- and 9-<i>cis</i>-retinaldehyde are displayed in light and dark grey sticks; while the substrates are shown in light and dark blue, respectively. The residues found in the most external part of all-<i>trans</i>-retinaldehyde binding channel in AKR1B10 are highlighted in yellow. The energy minimized <i>apo</i>-conformation is displayed in magenta cartoon. (<i>B</i>) and (<i>C</i>) Side view of the surface contour of the active-site pocket, depicted in grey and orange for AKR1B15 and AKR1B10, respectively, to show the inhibitor “specificity pocket”. A thick grey curved line indicates the “specificity pocket” in AKR1B10. As it is shown, this pocket may not be opened in AKR1B15, likely due to the presence of bulky Phe residues. (<i>D</i>) The inhibitor JF0064 (PDB ID 4ICC) bound to AKR1B15 is displayed as sticks with C atoms in magenta, while residues interacting with the inhibitor are shown as sticks with C atoms in grey. (<i>E</i>) Steric hindrance preventing tolrestat (in blue) and sorbinil (in orange) from binding to the active site of AKR1B15. For this analysis, the AKR1B15 structure model was superimposed with the AKR1B10 crystallographic structures with tolrestat (PDB ID 1ZUA) and sorbinil (PDB ID 4GA8). NADP⁺ is colored in orange. Figures have been drawn using PyMOL.</p

Quenching of AKR1B15 and AKR1B10 fluorescence upon binding of NADPH.

<p>Change of the protein fluorescence intensity (in percentage) upon addition of cofactor is shown. All proteins were used at a concentration of 0.5 μM in 20 mM sodium phosphate, pH 7.0, at 25°C. Graph symbols: AKR1B15 (diamonds), AKR1B10 (open squares).</p

Inhibitory effect of different compounds on enzymatic activity.

<p>The enzymatic activity assay with inhibitors was performed by using d,l-glyceraldehyde as a substrate. JF0064: 2,2’,3,3’,5,5’,6,6’-octafluoro-4,4’-biphenyldiol</p><p>^aData from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134506#pone.0134506.ref070" target="_blank">70</a>].</p><p>^bData from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134506#pone.0134506.ref052" target="_blank">52</a>].</p><p>^cData from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134506#pone.0134506.ref071" target="_blank">71</a>].</p><p>^dData from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134506#pone.0134506.ref066" target="_blank">66</a>]</p><p>Inhibitory effect of different compounds on enzymatic activity.</p

Comparison of the cofactor-binding site between the AKR1B15 model (<i>A</i>) and the AKR1B10 crystal structure (<i>B</i>).

<p>Interactions of Met265 and His269 with NADP⁺ in AKR1B15 are similar to those of Val265 and Arg269 in AKR1B10 (black dotted lines). His269 forms a π-stacking interaction with the adenine ring of the cofactor. The substitution of Lys22 by Arg in AKR1B15 prevents its interaction with the pyrophosphate bridge of NADP⁺. The salt bridge between Asp217 and Lys263 (red dotted line), acting as a safety belt in the coenzyme binding, and the π-stacking interaction of Tyr210 with the cofactor nicotinamide ring are conserved between the two AKRs. Carbon atoms of the cofactor are shown in green, whereas those of the enzyme are colored grey. Figures have been drawn using PyMOL.</p

Kinetic constants with retinaldehyde isomers.

<p>Enzymatic activity was measured by using the HPLC-based method.</p><p>^aData from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134506#pone.0134506.ref023" target="_blank">23</a>]</p><p>Kinetic constants with retinaldehyde isomers.</p