Structure of Cytochrome P450 2C9*2 in Complex with Losartan: Insights into the Effect of Genetic Polymorphism

Insights into the Genetic Variations of Human Cytochrome P450 2C9: Structural Analysis, Characterization and Comparison

“…Importantly, the orientation of losartan in the active site of CYP2C9*8 is inconsistent with the demonstrated metabolism as the site of hydroxylation of losartan is located further away from the heme iron. Such an altered pose has been observed with several crystal structures of P450 complexes, including the losartan complexes of CYP2C9 described previously [ 25 , 28 ]. The crystallization is performed in the absence of NADPH or cytochrome P450 reductase, and the substrate may likely reorient to a catalytically favorable pose in the presence of the redox partner during the catalytic cycle.…”

“…The CYP2C9*3 variant illustrated significant reduction in binding affinity to losartan compared with WT and to the *2 variant. The average apparent dissociation constant ( K D ) value for losartan binding to CYP2C9*3 was 24.5 ± 2.7 µM, which was more than three-fold weaker than the WT enzyme with a previously published K D value of 6.8 ± 0.7 µM for losartan binding [ 28 ]. On the other hand, the average K D for losartan binding to CYP2C9*8 was 8.95 ± 0.97 µM, which was comparable to that yielded previously by the *2 variant (9.0 ± 0.5 µM).…”

“…It is in this access channel region that the third molecule of losartan is bound in the WT complex but not in the *3 or the *8 structures. In addition, the structural overlay of the CYP2C9*8 with the recently solved CYP2C9*2 (R144C)-losartan complex revealed changes in the overall conformation (RMSD ~0.71 Å) that was largely reflected in the F-G cassette, which comprises the F, F’, G’ and G helices ( Figure 6 A) [ 28 ]. The differences in the conformation between the *8 and the *2 complexes were similar to those observed between the WT and the *2 complexes of losartan; however, the losartan in the access channel was not observed in both (*8 and *2) structures.…”

“…The differences in the conformation between the *8 and the *2 complexes were similar to those observed between the WT and the *2 complexes of losartan; however, the losartan in the access channel was not observed in both (*8 and *2) structures. Furthermore, the losartan binding site at the periphery was unclear in the *2 complex but not in the other losartan complexes solved thus far [ 25 , 28 ]. Figure 6 B shows the distinct orientation of losartan in the active site and differences in the secondary structural elements at position R144C between the two structures that were consistent with the WT complex.…”

“…Enzymatic assays were performed using 50 pmol reconstituted CYP2C9 (WT, *3 and *8) incubated with 50 pmol human cytochrome P450 reductase on ice for 5 min [ 28 , 43 ]. An amount of 10 µM losartan was added, and the reaction was further incubated on ice for 5 min.…”