title: A functional genetic polymorphism on human carbonyl reductase 1 (CBR1 V88I) 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 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 PGE2 (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 microM versus IC50 CBR1 I88, 15.0 +/- 0.1 microM, 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 microM versus K(d) CBR1 I88, 3.8 +/- 0.5 microM). These studies characterize the first functional genetic determinant of CBR1 activity toward relevant physiological and pharmacological substrates.
authors.affiliation: Department of Pharmaceutical Sciences, The State University of New York at Buffalo, Buffalo, NY 14260-1200, USA.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Gonzalez-Covarrubias
authors.forename: Debashis
authors.initials: D
authors.lastname: Ghosh
authors.forename: Sukhwinder S
authors.initials: SS
authors.lastname: Lakhman
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Pendyala
authors.forename: Javier G
authors.initials: JG
authors.lastname: Blanco
mesh_terms: D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D003630:Daunorubicin / Q000031:analogs & derivatives / Q000378:metabolism; D005838:Genotype; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009249:NADP / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D012431:Rutin / Q000494:pharmacology; D017422:Sequence Analysis, DNA
chemical_list: D018076:DNA, Complementary; D009249:NADP; D012431:Rutin; D000429:Alcohol Oxidoreductases; C000847:daunorubicinol; D003630:Daunorubicin