Mutual Influence of Human Cytochrome P450 Enzymes and UDP-Glucuronosyltransferases on Their Respective Activities in Recombinant Fission Yeast

Abstract

Cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs) are the most important human drug metabolizing enzymes, but their mutual interactions are poorly understood. In this study, we recombinantly co-expressed of each one of the 19 human members of the UGT families 1 and 2 with either CYP2C9, CYP2D6, or CYP4Z1 in fission yeast. Using these strains, we monitored a total of 72 interactions: 57 cases where we tested the influence of UGT co-expression on CYP activity and 15 cases of the opposite approach. In the majority of cases (88%), UGT co-expression had a statistically significant (p < 0.05) effect on P450 activity (58% positive and 30% negative). Strong changes were observed in nine cases, including one case with an activity increase by a factor of 23 (CYP2C9 activity in the presence of UGT2A3) but also four cases with a complete loss of activity. When monitoring the effect of CYP co-expression on the activity of five UGTs, activity changes were generally not so pronounced and, if observed, always detrimental. UGT2B7 activity was not influenced by CYP co-expression, while the other UGTs were affected to varying degrees. These data suggest the notion that mutual influence of CYPs and UGTs on each other's activity is a widespread phenomenon.

Keywords: CYP2C9; CYP2D6; CYP4Z1; Cytochrome P450; UDP-glucuronosyltransferase; UGT2B7.

Figure 1

Cloning Strategy. (a) Parental haploid strain JMN12 (mating type h-) was transformed with the integrative vector pCAD1 and with 19 derivatives of pCAD1 that contain one of the human UGT genes to yield 20 new strains (SAN3 to SAN22; top, marked blue). Parental haploid strain JMN11 (mating type h+) was transformed with pCAD1-CPR to yield the new strain SAN2 (top, marked green). Mating of these strains yielded 19 new diploid strains that co-express CPR, with one of the UGTs plus a control that only expresses CPR (SAN100 to SAN119; top right). These 20 new diploid strains were then individually transformed with three different autosomal replicating plasmids (pREP1-CYP2C9, pREP1-CYP2D6, and pREP1-CYP4Z1, respectively; marked red) to yield three new series of diploid strains that co-express CPR together with a CYP and a UGT (including controls); these strains are SAN200 to SAN219 (CYP2C9 series), SAN300 to SAN319 (CYP2D6 series), and SAN500 to SAN519 (CYP4Z1 series), respectively. (b) Parental haploid strain JMN11 was transformed with pCAD1 yielding SAN1. Mating of this strain with SAN3, SAN6, SAN11, SAN15, SAN17, and SAN22 yielded the new strains SAN120 (no insert), SAN123 (UGT1A4), SAN128 (UGT1A9), SAN132 (UGT2A3), SAN134 (UGT2B7), and SAN139 (UGT2B28) used as diploid controls for UGT activity assays.

Figure 2

Enzymatic activities of enzyme bags prepared from diploid strains co-expressing CYP2C9, CPR, and different UGTs as indicated. Activities towards the substrate Luciferin-H are shown in comparison to the control strain SAN200 (which co-expresses CYP2C9 and CPR without a UGT). Activities that are not different from the control are shown in grey, while those that are statistically significantly higher or lower than the control are shown in black or white, respectively. * p < 0.05, *** p < 0.005, **** p < 0.001.

Figure 3

Enzymatic activities of enzyme bags prepared from diploid strains co-expressing CYP2D6, CPR, and different UGTs as indicated. Activities towards the substrate Luciferin-MEEGE are shown in comparison to the control strain SAN300 (which co-expresses CYP2D6 and CPR without a UGT). Activities that are not different from the control are shown in grey, while those that are statistically significantly higher or lower than the control are shown in black or white, respectively. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.

Figure 4

Enzymatic activities of enzyme bags prepared from diploid strains co-expressing CYP4Z1, CPR, and different UGTs as indicated. Activities towards the substrate Luciferin-4FBE are shown in comparison to the control strain SAN500 (which co-expresses CYP4Z1 and CPR without a UGT). Activities that are not different from the control are shown in grey, while those that are statistically significantly higher or lower than the control are shown in black or white, respectively. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.

Figure 5

Enzymatic activities of enzyme bags prepared from diploid strains co-expressing five different UGTs with CPR and four different CYPs as indicated. Activities towards UGT-Glo substrate A are shown in comparison to the respective control strains (which only express one of the UGTs). Activities that are not different from the control are shown in grey, while those that are statistically significantly lower than the control are shown in white. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.