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

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 suggested an important role of the CSM 8, which corresponds to(187)RFDYKD(192) 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 > or =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 (IC(50,BIF)/IC(50,4-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 IC(50,BIF)/IC(50,4-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)-->Alasubstitutions, respectively.

FIGURE 1.

Structure of ligand-free CYP2B4 (1PO5) and CYP2B4-CPI (1SUO). The segment in green shows all the CSM and in red is the CSM 8, which was studied experimentally.

FIGURE 2.

Structures of 7-MFC, 7-EFC, 7-BFC, 4-CPI, and BIF.

FIGURE 3.

A, thermal inactivation of CYP2B4dH mutants (1 μm) monitored by a decrease in the amount of total heme protein as a function of temperature as described under “Experimental Procedures.” Determination of the total concentration of the heme protein was done by non-linear least square approximation of the spectra by a linear combination of spectral standards of CYP2B4 low spin, high spin, and P420 states. The data were fit to a sigmoidal curve to obtain T_m. B, catalytic tolerance to temperature monitored by a decrease in the enzyme activity with 7-EFC as a function of temperature as described under “Experimental Procedures.” The data were fit to a sigmoidal curve to obtain T₅₀.

FIGURE 4.

Crystal structure of CYP2B4-4-CPI (1SUO) representing the hydrogen bond pattern (dashed yellow line) in residue-residue interactions. Panel A shows the interaction among Glu¹⁴⁹-Asn¹⁷⁷-Arg¹⁸⁷-Tyr¹⁹⁰, and panels B and C are models of R187K and R187A. Panel D shows the interaction between F188A-F195A and panel E is a model of F188A. Similarly, panel F shows the hydrogen bonds between Asp¹⁹² and Val¹⁹⁴ in H226Y, and panel G shows the same region of the D192A mutant. Except for H226Y all the mutant models were energy-minimized using AMBER.