Identification and Analysis of Conserved Sequence Motifs in Cytochrome P450 Family 2

Abstract

Using a multiple alignment of 175 cytochrome P450 (CYP) family 2 sequences, 20 conserved sequence motifs (CSMs) were identified with the program PCPMer. Functional importance of the CSM in CYP2B enzymes was assessed from available data on site-directed mutants and genetic variants. These analyses sug-gested an important role of the CSM 8, which corresponds to 187 RFDYKD 192 in CYP2B4. Further analysis showed that resi-dues 187, 188, 190, and 192 have a very high rank order of con-servation compared with 189 and 191. Therefore, eight mutants (R187A, R187K, F188A, D189A, Y190A, K191A, D192A, and a negative control K186A) were made in an N-terminal truncated and modified form of CYP2B4 with an internal mutation, which is termed 2B4dH/H226Y. Function was examined with the substrates 7-methoxy-4-(trifluoromethyl)coumarin (7-MFC), 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC), 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC), and testosterone and with the inhibitors 4-(4-chlorophenyl)imidazole (4-CPI) and bifon-azole (BIF). Compared with the template and K186A, the mutants R187A, R187K, F188A, Y190A, and D192A showed >2-fold altered substrate specificity, k cat , K m , and/or k cat /K m for 7-MFC and 7-EFC and 3-to 6-fold decreases in differential inhi-bition (IC 50,BIF /IC 50 , 4-CPI). Subsequently, these mutants dis-played 5–12 °C decreases in thermal stability (T m) and 2– 8 °C decreases in catalytic tolerance to temperature (T 50) compared with the template and K186A. Furthermore, when R187A and D192A were introduced in CYP2B1dH, the P450 expression and thermal stability were decreased. In addition, R187A showed increased activity with 7-EFC and decreased IC 50,BIF / IC 50 , 4-CPI compared with 2B1dH. Analysis of long range res-idue-residue interactions in the CYP2B4 crystal structures indicated strong hydrogen bonds involving Glu 149 –Asn 177 – Arg 187 –Tyr 190 and Asp 192 –Val 194 , which were significantly reduced/abolished by the Arg 187 3 Ala and Asp 192 3 Ala substitutions, respectively.