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

Giménez-Dejoz J, Kolář MH, Ruiz FX, Crespo I, Cousido-Siah A, Podjarny A, Barski OA, Fanfrlík J, Parés X, Farrés J, Porté S - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Faculty of Biosciences, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Model of AKR1B15 structure.(A) Side view of the (α/β)8 barrel. In red, the divergent residues between AKR1B15 and AKR1B10. (B) and (C) Active-site pockets of AKR1B15 and AKR1B10, respectively. The AKR1B15-specific residues are displayed in magenta. NADP+ cofactor is colored in orange. The surface contour of pockets is shown in grey and orange for AKR1B15 and AKR1B10, respectively. (D) The local conformational changes in holo forms of AKR1B15 (red line) and AKR1B10 (blue line) derived from computer simulations, as indicated by root mean square fluctuations (RMSF) of backbone atoms. The residues of loops A, B and C are highlighted by grey background. The difference in RMSF between the two enzymes is displayed as a black line in the top. (E) AKR1B15 loops A and C indicating potential contacts between different residues are shown as sticks, which may explain the low flexibility of the protein in this region. Figures have been drawn using PyMOL.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4519324&req=5

pone.0134506.g004: Model of AKR1B15 structure.(A) Side view of the (α/β)8 barrel. In red, the divergent residues between AKR1B15 and AKR1B10. (B) and (C) Active-site pockets of AKR1B15 and AKR1B10, respectively. The AKR1B15-specific residues are displayed in magenta. NADP+ cofactor is colored in orange. The surface contour of pockets is shown in grey and orange for AKR1B15 and AKR1B10, respectively. (D) The local conformational changes in holo forms of AKR1B15 (red line) and AKR1B10 (blue line) derived from computer simulations, as indicated by root mean square fluctuations (RMSF) of backbone atoms. The residues of loops A, B and C are highlighted by grey background. The difference in RMSF between the two enzymes is displayed as a black line in the top. (E) AKR1B15 loops A and C indicating potential contacts between different residues are shown as sticks, which may explain the low flexibility of the protein in this region. Figures have been drawn using PyMOL.

Mentions: With the aim to compare the substrate-binding site of AKR1B15 with that of AKR1B10, and upon unsuccessful attempts of protein crystallization, a 3D homology model of AKR1B15 was built (Fig 4A). The SCWRL server was chosen, as it is designed specifically for predicting side-chain conformations, provided a fixed backbone usually obtained from an experimental structure. In practical terms, this is the case of AKR1B15, given its 92% sequence identity with AKR1B10. Model quality was checked by PROCHECK analyses, indicating that most residues are in the preferred regions (94.8%), whereas residues in allowed regions and outliers are 3.5 and 1.6%, respectively. The compatibility of the atomic model (3D) was checked with its own amino acid sequence (1D) using Verify 3D analysis. This analysis shows that there is no region with negative scores, which would otherwise indicate potential problems. In addition, the analysis using the QMEAN server indicates the reliability of the model, with a QMEAN score of 0.67 out of 1, and with all the Z-scores being consistent with the good quality of the structure. Thus it may be concluded that this model is suitable for structural studies.


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

Giménez-Dejoz J, Kolář MH, Ruiz FX, Crespo I, Cousido-Siah A, Podjarny A, Barski OA, Fanfrlík J, Parés X, Farrés J, Porté S - PLoS ONE (2015)

Model of AKR1B15 structure.(A) Side view of the (α/β)8 barrel. In red, the divergent residues between AKR1B15 and AKR1B10. (B) and (C) Active-site pockets of AKR1B15 and AKR1B10, respectively. The AKR1B15-specific residues are displayed in magenta. NADP+ cofactor is colored in orange. The surface contour of pockets is shown in grey and orange for AKR1B15 and AKR1B10, respectively. (D) The local conformational changes in holo forms of AKR1B15 (red line) and AKR1B10 (blue line) derived from computer simulations, as indicated by root mean square fluctuations (RMSF) of backbone atoms. The residues of loops A, B and C are highlighted by grey background. The difference in RMSF between the two enzymes is displayed as a black line in the top. (E) AKR1B15 loops A and C indicating potential contacts between different residues are shown as sticks, which may explain the low flexibility of the protein in this region. Figures have been drawn using PyMOL.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4519324&req=5

pone.0134506.g004: Model of AKR1B15 structure.(A) Side view of the (α/β)8 barrel. In red, the divergent residues between AKR1B15 and AKR1B10. (B) and (C) Active-site pockets of AKR1B15 and AKR1B10, respectively. The AKR1B15-specific residues are displayed in magenta. NADP+ cofactor is colored in orange. The surface contour of pockets is shown in grey and orange for AKR1B15 and AKR1B10, respectively. (D) The local conformational changes in holo forms of AKR1B15 (red line) and AKR1B10 (blue line) derived from computer simulations, as indicated by root mean square fluctuations (RMSF) of backbone atoms. The residues of loops A, B and C are highlighted by grey background. The difference in RMSF between the two enzymes is displayed as a black line in the top. (E) AKR1B15 loops A and C indicating potential contacts between different residues are shown as sticks, which may explain the low flexibility of the protein in this region. Figures have been drawn using PyMOL.
Mentions: With the aim to compare the substrate-binding site of AKR1B15 with that of AKR1B10, and upon unsuccessful attempts of protein crystallization, a 3D homology model of AKR1B15 was built (Fig 4A). The SCWRL server was chosen, as it is designed specifically for predicting side-chain conformations, provided a fixed backbone usually obtained from an experimental structure. In practical terms, this is the case of AKR1B15, given its 92% sequence identity with AKR1B10. Model quality was checked by PROCHECK analyses, indicating that most residues are in the preferred regions (94.8%), whereas residues in allowed regions and outliers are 3.5 and 1.6%, respectively. The compatibility of the atomic model (3D) was checked with its own amino acid sequence (1D) using Verify 3D analysis. This analysis shows that there is no region with negative scores, which would otherwise indicate potential problems. In addition, the analysis using the QMEAN server indicates the reliability of the model, with a QMEAN score of 0.67 out of 1, and with all the Z-scores being consistent with the good quality of the structure. Thus it may be concluded that this model is suitable for structural studies.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Faculty of Biosciences, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus