Analysis of the substrate-binding site of human carbonyl reductases CBR1 and CBR3 by site-directed mutagenesis

El-Hawari, Yasser; Favia, Angelo D.; Pilka, E.S.; Kisiela, Michael; Oppermann, U.; Martin, H.; Maser, Edmund

doi:10.1016/j.cbi.2008.11.004

Cited by 31 publications

(26 citation statements)

References 32 publications

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“…Previous studies have shown that DAUN is a better human CBR1 substrate than DOX (Oppermann 2007;Bains et al, 2009). This is interesting because both human CBR1 and CBR3 enzymes are identical in length (277 amino acids) and share 72% amino acid sequence identity and 85% similarity (El-Hawari et al, 2009). Despite the high amino acid sequence identity, the human CBR1 and CBR3 enzymes distinguish between two substrates that differ only at the carbon-14 position (DOX is hydroxylated, but DAUN is not).…”

Section: Discussionmentioning

confidence: 99%

Naturally Occurring Variants of Human CBR3 Alter Anthracycline In Vitro Metabolism

Bains

Karkling²,

Lubieniecka³

et al. 2009

J Pharmacol Exp Ther

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Doxorubicin (DOX) and daunorubicin (DAUN) are anthracycline anticancer agents; however, considerable interpatient variability exists in their pharmacokinetics. This interpatient variability is attributed in part to altered metabolism by nonsynonymous single-nucleotide polymorphisms (ns-SNPs) in genes encoding the carbonyl reductases. This study examines the effect of seven naturally occurring ns-SNPs in the CBR3 gene on in vitro metabolism of anthracyclines to doxorubicinol and daunorubicinol. Kinetic assays measure metabolite levels by high-performance liquid chromatography separation with fluorescence detection by use of purified, histidine-tagged, human CBR3 wild type and variant enzymes. The V224M, C4Y, and V93I variants resulted in significantly reduced maximal reaction velocity (V max ) for both anthracyclines compared with the wildtype enzyme, whereas the M235L variant had significantly reduced V max for DOX only. Significant increases in substrate affinity were found for the V244M variant with DAUN, as well as the C4Y and V93I variants with DOX. The catalytic efficiency values for the V244M, C4Y, and V93I variants were significantly lower than the wild type for DAUN and DOX. Furthermore, DOX was observed to be a better substrate than DAUN for the wild-type enzyme and its variants. HapMap analysis indicated that a haplotype carrying the C4Y and V244M mutations may occur in some individuals in the 11 ethnic populations studied in the HapMap project. Our preparation of the double mutant indicated a significant reduction in activity compared with the wild-type enzyme and single-mutant preparations. These findings suggest that commonly occurring ns-SNPs in human CBR3 significantly alter the in vitro metabolism of DOX and DAUN.

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Section: Discussionmentioning

confidence: 99%

Naturally Occurring Variants of Human CBR3 Alter Anthracycline In Vitro Metabolism

Bains

Karkling²,

Lubieniecka³

et al. 2009

J Pharmacol Exp Ther

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“…However, we found that the substitution of the amino acid residue 230 alone of human CBR1 for the corresponding residue of human CBR3 has no apparent impact on carbonyl reductase activities of CBR3 (Miura et al 2009a). Very recent study (El-Hawari et al 2009) exhibited that, based on the strategy separated from our present study, mutation of human CBR3 to residues 230 and 236-244 of human CBR1 synergically induces high catalytic efficiencies for carbonyl reductase activities toward both isatin and 9, 10-phenanthrene-quinone. Results of a series of our investigations (the present study; Miura et al 2009a) regarding carbonyl reductase activities of mutated enzymes are supported by their study.…”

Section: Discussionmentioning

confidence: 87%

Importance of the substrate-binding loop region of human monomeric carbonyl reductases in catalysis and coenzyme binding

2009

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“…His-tagged human carbonyl reductase 1 (CBR1) was expressed in Escherichia coli and purified as published previously [25].…”

Section: Preparation Of Recombinant Carbonyl Reductasementioning

confidence: 99%

Curcumin is a tight-binding inhibitor of the most efficient human daunorubicin reductase – Carbonyl reductase 1

Hintzpeter¹,

Hornung²,

Ebert³

et al. 2015

Chemico-Biological Interactions

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Analysis of the substrate-binding site of human carbonyl reductases CBR1 and CBR3 by site-directed mutagenesis

Cited by 31 publications

References 32 publications

Naturally Occurring Variants of Human CBR3 Alter Anthracycline In Vitro Metabolism

Naturally Occurring Variants of Human CBR3 Alter Anthracycline In Vitro Metabolism

Importance of the substrate-binding loop region of human monomeric carbonyl reductases in catalysis and coenzyme binding

Curcumin is a tight-binding inhibitor of the most efficient human daunorubicin reductase – Carbonyl reductase 1

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