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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

Quenching of AKR1B15 and AKR1B10 fluorescence upon binding of NADPH.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).
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pone.0134506.g002: Quenching of AKR1B15 and AKR1B10 fluorescence upon binding of NADPH.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).

Mentions: We attempted the expression and purification of recombinant AKR1B15 using different E. coli strains and procedures. In all cases, the protein appeared to be mostly present in the insoluble fraction of cell lysates. Previously, Salabei et al. [6] had described a successful procedure based on the use of the anionic detergent sarkosyl (sodium N-lauroylsarcosinate). In our hands and in the presence of sarkosyl, the amount of soluble protein and the final yield were acceptable (Fig 1A and 1B). However, AKR1B15 was mainly associated with higher molecular weight aggregates (Fig 1C), which were found to be enzymatically inactive. Only a small fraction of AKR1B15 was found to be in an active monomeric form. A minimal concentration of sarkosyl (0.001%, w/v) was necessary to avoid protein precipitation. Sarkosyl had been described as an enzyme inhibitor [46], and was confirmed that it inhibited AKR1B15 following a non-competitive model (data not shown). As an alternative procedure for expression, an E. coli BL21(DE3) strain co-expressing three chaperone systems (DnaK-DnaJ-GrpE, ClpB and GroEL-GroES) [22] was used. Under these conditions, AKR1B15 was still found to be mainly associated with the insoluble fraction, but the amount of soluble protein recovered increased significantly (Fig 1A). Peptide mass fingerprint analysis of the soluble protein fraction separated by SDS-PAGE identified the isolated band as being human AKR1B15 (data not shown). The enzyme was purified with a final yield of 1 mg protein per liter of culture, when using minimal medium. Analysis by gel filtration chromatography showed a molecular weight of 37 kDa for the purified protein, suggesting that AKR1B15 was obtained as a monomer (Fig 1C). By analyzing the purified protein on SDS-PAGE (Fig 1B, lane 2), the major band corresponded to the 37-kDa enzyme, with some minor contaminating protein bands. Fluorescence analysis of cofactor binding allowed us to determine the KD value for NADPH (113 ± 9 nM, Fig 2). The determined KD value was in the same range with that of AKR1B10 (KD = 92 ± 35 nM).


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)

Quenching of AKR1B15 and AKR1B10 fluorescence upon binding of NADPH.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).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134506.g002: Quenching of AKR1B15 and AKR1B10 fluorescence upon binding of NADPH.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).
Mentions: We attempted the expression and purification of recombinant AKR1B15 using different E. coli strains and procedures. In all cases, the protein appeared to be mostly present in the insoluble fraction of cell lysates. Previously, Salabei et al. [6] had described a successful procedure based on the use of the anionic detergent sarkosyl (sodium N-lauroylsarcosinate). In our hands and in the presence of sarkosyl, the amount of soluble protein and the final yield were acceptable (Fig 1A and 1B). However, AKR1B15 was mainly associated with higher molecular weight aggregates (Fig 1C), which were found to be enzymatically inactive. Only a small fraction of AKR1B15 was found to be in an active monomeric form. A minimal concentration of sarkosyl (0.001%, w/v) was necessary to avoid protein precipitation. Sarkosyl had been described as an enzyme inhibitor [46], and was confirmed that it inhibited AKR1B15 following a non-competitive model (data not shown). As an alternative procedure for expression, an E. coli BL21(DE3) strain co-expressing three chaperone systems (DnaK-DnaJ-GrpE, ClpB and GroEL-GroES) [22] was used. Under these conditions, AKR1B15 was still found to be mainly associated with the insoluble fraction, but the amount of soluble protein recovered increased significantly (Fig 1A). Peptide mass fingerprint analysis of the soluble protein fraction separated by SDS-PAGE identified the isolated band as being human AKR1B15 (data not shown). The enzyme was purified with a final yield of 1 mg protein per liter of culture, when using minimal medium. Analysis by gel filtration chromatography showed a molecular weight of 37 kDa for the purified protein, suggesting that AKR1B15 was obtained as a monomer (Fig 1C). By analyzing the purified protein on SDS-PAGE (Fig 1B, lane 2), the major band corresponded to the 37-kDa enzyme, with some minor contaminating protein bands. Fluorescence analysis of cofactor binding allowed us to determine the KD value for NADPH (113 ± 9 nM, Fig 2). The determined KD value was in the same range with that of AKR1B10 (KD = 92 ± 35 nM).

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