A functional genetic polymorphism on human carbonyl reductase 1 (CBR1 V88I) impacts on catalytic activity and NADPH binding affinity

Human carbonyl reductase 1 (CBR1) metabolizes endogenous and xenobiotic substrates such as the fever mediator, prostaglandin E2 (PGE(2)), 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 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 versus V-max CBR1 I88, 121 +/- 12 nmol/min (.) mg, p < 0.05) and PGE(2) (V-max CBR1 V88, 53 +/- 7 versus V-max CBR1 I88, 35 +/- 4 nmol/min (.) mg, p < 0.01). Concomitantly, CBR1 V88 produced higher levels of the cardiotoxic metabolite daunorubicinol compared with 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 mu M versus IC50 CBR1 I88, 15.0 +/- 0.1 mu M, 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 mu M versus K-d CBR1 I88, 3.8 +/- 0.5 mu M). These studies characterize the first functional genetic determinant of CBR1 activity toward relevant physiological and pharmacological substrates.