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 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 >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°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 One of the most intriguing recent discoveries about mammalian cytochromes P450 (CYP)3 is the remarkable conformational plasticity exhibited by a number of the enzymes (1-4). This plasticity allows a single P450 to adapt its ligand binding site to a wide variety of compounds of different size, shape, and chemistry. The rabbit CYP2B4 provides some of the most striking examples of an adaptable active site as inferred from x-ray crystal structures of ligand-free, 4-(4-chlorophenyl)imidazole (4-CPI)-bound, 1-(4-chlorophenyl)imidazole-bound, and bifonazole (BIF)-bound x-ray crystal structures as well as solution thermodynamics derived from isothermal titration calorimetry (5-10). In particular, comparisons of three structures identified five plastic regions in CYP2B4 (8), which account for almost one-third of the protein and contribute to broad substrate specificity by allowing different conformations in response to ligands. These results suggest an important role of non-active site regions/residues in ligand-induced conformational transitions and differential substrate binding and catalysis. However, predicting the role of non-active site regions/residues is currently very difficult.Recently, directed evolution of CYP2B1 has been carried out to locate important non-active site amino acid residues. , and Ser 334 were found to contribute to enzyme catalysis and/or stabil...

Analysis of Cytochrome P450 Conserved Sequence Motifs between Helices E and H: Prediction of Critical Motifs and Residues in Enzyme Functions

“…CSM 7–11 of CYP2 family has been reproduced from our previous work [22] for comparison. These comparisons yielded the following results: 1) CSM 9 is not present in the CYP1 and CYP3 families, suggesting a specific role for CSM 9 in the CYP2 family; 2) CSM 7 and 8 show a relatively higher rank (≥1.8) than CSM 10 and 11(1.4) in the CYP1 family; 3) all four CSM show a high rank (≥1.8) in the CYP3 family, which is similar to the rank in CYP2 family.…”

“…We used a similar approach to that described in our recent work for the CYP2 family [22]. Briefly, we selected 19 CYP1 and 26 CYP3 sequences from species, such as human, mouse, and rat, and generated a multiple sequence alignment with ClustalW [23].…”

“…Briefly, we selected 19 CYP1 and 26 CYP3 sequences from species, such as human, mouse, and rat, and generated a multiple sequence alignment with ClustalW [23]. Similarly, 12 CYP2A, 10 CYP2B, 33 CYP2C, and 25 CYP2D sequences were utilized from various species, as presented earlier [22], and generated a multiple sequence alignment with Clusta1W. These multiple sequence alignments were further analyzed using PCPMer, in which each of the 20 natural amino acids was represented by a 5-dimensional vector [24].…”

“…Recently, we utilized an alternate approach, analysis of conserved sequence motifs (CSM), which resulted in the identification of twenty CSM in the CYP2 family [22]. We investigated CSM 8 (E-F loop) because it is present between plastic/variable regions 3 (177–188) and 4 (203–298) (14) and may regulate ligand-induced flexibility.…”

Computer-based substrate specifity prediction for cytochrome P450

“…В выровненных последовательностях CYP2 найдено 20 мотивов, замены в которых удалось связать с литературными свидетельствами об изменении ферментативных характеристик [18].…”