Identification and analysis of conserved sequence motifs in cytochrome P450 family 2 -: Functional and structural role of a motif 187RFDYKD192 in CYP2B enzymes

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 suggested an important role of the CSM 8, which corresponds to (RFDYKD192)-R-187 in CYP2B4. Further analysis showed that residues 187, 188, 190, and 192 have a very high rank order of conservation 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 bifonazole (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 inhibition (IC50, BIF/IC50,4-CPI). Subsequently, these mutants displayed 5-12 degrees C decreases in thermal stability (T-m) and 2-8 degrees 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 IC50,BIF/IC50,4-CPI compared with 2B1dH. Analysis of long range residue-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) --> Ala and Asp(192) --> Ala substitutions, respectively.