Received for publication January 10, 2007.
Revised March 1, 2007.
Accepted for publication March 6, 2007.

A functional genetic polymorphism on human carbonyl reductase 1 (CBR1) impacts on catalytic activity and NADPH binding affinity

Abstract

Human carbonyl reductase 1 (CBR1) metabolizes endogenous and xenobiotic substrates such as the fever mediator prostaglandin E2 (PGE₂), and the anticancer anthracycline drug daunorubicin. We screened 33 CBR1 full-length cDNA samples from white and black liver donors and performed database analyses to identify genetic determinants of CBR1 activity. We pinpointed a single nucleotide polymorphism (SNP) on CBR1 (CBR1 V88I) that encodes for a valine-to-isoleucine substitution for further characterization. We detected the CBR1 V88I polymorphism in DNA samples from individuals with African ancestry (p = 0.986, q = 0.014). Kinetic studies revealed that the CBR1 V88 and CBR1 I88 isoforms have different maximal velocities for daunorubicin (V_max CBR1 V88: 181 ± 13 vs. V_max CBR1 I88: 121 ± 12 nmol/min.mg, p < 0.05), and PGE₂ (V_max CBR1 V88: 53 ± 7 vs. V_max CBR1 I88: 35 ± 4 nmol/min.mg, p < 0.01). Concomitantly, CBR1 V88 produced higher levels of the cardiotoxic metabolite daunorubicinol as compared to CBR1 I88 (1.7 fold, p<0.0001). Inhibition studies demonstrated that CBR1 V88 and CBR1 I88 are distinctively inhibited by the flavonoid rutin (IC₅₀ CBR1 V88: 54.0 ± 0.4 µM vs. IC₅₀ CBR1 I88: 15.0 ± 0.1 µM, p<0.001). Furthermore, isothermal titration calorimetry analyses together with molecular modeling studies showed that CBR1 V88I results in CBR1 isoforms with different binding affinities for the cofactor NADPH (K_d CBR1 V88: 6.3 ± 0.6 µM vs. K_d CBR1 I88: 3.8 ± 0.5 µM). These studies characterize the first functional genetic determinant of CBR1 activity towards relevant physiological and pharmacological substrates.

Key words: enzyme inhibitors, enzyme kinetics, enzyme mechanism, genetic polymorphism, human genetics, pharmacogenetics, reductases