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

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

Molecular structure of compound JF0064, 2,2’,3,3’,5,5’,6,6’-octafluoro-4,4’-biphenyldiol.

<p>Molecular structure of compound JF0064, 2,2’,3,3’,5,5’,6,6’-octafluoro-4,4’-biphenyldiol.</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

Expression and purification of recombinant human AKR1B15.

<p>(<i>A</i>) SDS-PAGE analysis of protein expression, showing that AKR1B15 was predominantly associated with the insoluble fraction of BL21(DE3) cell lysates. Treatment with 1% (w/v) sarkosyl (Sk) provided a much higher amount of AKR1B15 in the soluble fraction. In the case of BL21(DE3) pBB540 pBB542 cells, a protein band which is highlighted with a red oval was identified as human AKR1B15 by Peptide Mass Fingerprinting. Lanes: C, control for the soluble fraction not induced by IPTG; S, soluble fraction; and I, insoluble fraction. <i>(B)</i> SDS-PAGE analysis of protein purification, showing fractions eluted from the nickel affinity column chromatography using 100 mM imidazole. Lanes: 1, Protein eluted from the soluble fraction of BL21(DE3) + Sk; and 2, Protein eluted from the soluble fraction of BL21 (DE3) pBB540 pBB542. <i>(C)</i> Elution profile from the Superdex 75 10/300 GL column chromatography. AKR1B15 purified from soluble fraction of BL21(DE3) + Sk and from soluble fraction of BL21(DE3) pBB540 pBB542 are shown in grey and black lines, respectively. Major peaks eluting at 7.9 and 11.4 mL correspond to aggregated (132 kDa) and monomer (37 kDa) protein, respectively.</p