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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.

SUBMITTER: Gonzalez-Covarrubias V

PROVIDER: S-EPMC2442771 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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

Gonzalez-Covarrubias Vanessa V Ghosh Debashis D Lakhman Sukhwinder S SS Pendyala Lakshmi L Blanco Javier G JG

Drug metabolism and disposition: the biological fate of chemicals 20070307 6

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. ...[more]

PMID: 17344335

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Project description:MicroRNAs (miRNAs) are small RNAs responsible for the post-transcriptional regulation of a variety of human genes. To date, their involvement in the regulation of CBR1 is unknown. This study reports for the first time the identification of microRNA-574-5p (hsa-miR-574-5p) and microRNA-921 (hsa-miR-921) as two miRNAs capable of interacting with the 3'-untranslated region (3'-UTR) of the CBR1 gene and downregulating CBR1 expression. Furthermore, we demonstrate that a common single-nucleotide polymorphism (SNP) in the CBR1 3'-UTR (rs9024, CBR1 1096G>A) differentially impacts the regulation of CBR1 by hsa-miR-574-5p and hsa-miR-921 dependent on genotype. First, four candidate miRNAs were selected based on bioinformatic analyses, and were tested in Chinese hamster ovary (CHO) cells transfected with CBR1 3'-UTR constructs harboring either the G or A allele for rs9024. We found that hsa-miR-574-5p and hsa-miR-921 significantly decreased luciferase activity in CHO cells transfected with the CBR1 3'-UTR construct carrying the major rs9024 G allele by 35% and 46%, respectively. The influence of these miRNAs was different in cells transfected with a CBR1 3'-UTR construct containing the minor rs9024 A allele in that only hsa-miR-574-5p had a demonstrable effect (i.e., 52% decrease in lucifersase activity). To further determine the functional effects of miRNA-mediated regulation of polymorphic CBR1, we assessed CBR1 protein expression and CBR1 enzymatic activity for the prototypical substrate menadione in human lymphoblastoid cell lines with distinct rs9024 genotypes. We found that hsa-miR-574-5p and hsa-miR-921 significantly decreased CBR1 protein (48% and 40%, respectively) and CBR1 menadione activity (54% and 18%, respectively) in lymphoblastoid cells homozygous for the major rs9024 G allele. In contrast, only hsa-miR-574-5p decreased CBR1 protein and CBR1 activity in cells homozygous for the minor rs9024 A allele, and did so by 49% and 56%, respectively. These results suggest that regulation of human CBR1 expression by hsa-miR-574-5p and hsa-miR-921 depends upon rs9024 genotype status.

Project description:The NADPH-dependent (S)-carbonyl reductaseII from Candida parapsilosis catalyzes acetophenone to chiral phenylethanol in a very low yield of 3.2%. Site-directed mutagenesis was used to design two mutants Ala220Asp and Glu228Ser, inside or adjacent to the substrate-binding pocket. Both mutations caused a significant enantioselectivity shift toward (R)-phenylethanol in the reduction of acetophenone. The variant E228S produced (R)-phenylethanol with an optical purity above 99%, in 80.2% yield. The E228S mutation resulted in a 4.6-fold decrease in the K M value, but nearly 5-fold and 21-fold increases in the k cat and k cat/K M values with respect to the wild type. For NADPH regeneration, Bacillus sp. YX-1 glucose dehydrogenase was introduced into the (R)-phenylethanol pathway. A coexpression system containing E228S and glucose dehydrogenase was constructed. The system was optimized by altering the coding gene order on the plasmid and using the Shine-Dalgarno sequence and the aligned spacing sequence as a linker between them. The presence of glucose dehydrogenase increased the NADPH concentration slightly and decreased NADP(+) pool 2- to 4-fold; the NADPH/NADP(+) ratio was improved 2- to 5-fold. The recombinant Escherichia coli/pET-MS-SD-AS-G, with E228S located upstream and glucose dehydrogenase downstream, showed excellent performance, giving (R)-phenylethanol of an optical purity of 99.5 % in 92.2% yield in 12 h in the absence of an external cofactor. When 0.06 mM NADP(+) was added at the beginning of the reaction, the reaction duration was reduced to 1 h. Optimization of the coexpression system stimulated an over 30-fold increase in the yield of (R)-phenylethanol, and simultaneously reduced the reaction time 48-fold compared with the wild-type enzyme. This report describes possible mechanisms for alteration of the enantiopreferences of carbonyl reductases by site mutation, and cofactor rebalancing pathways for efficient chiral alcohols production.

Dataset Information

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

Publications

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

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