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

Numan Oezguen,Santosh Kumar

doi:10.4172/2157-7609.1000110

Abstract

Rational approaches have been extensively used to investigate the role of active site residues in cytochrome P450 (CYP) functions. However, recent studies using random mutagenesis suggest an important role for non-active site residues in CYP functions. Meta-analysis of the random mutants showed that 75% of the functionally important non-active site residues are present in 20% of the entire protein between helices E and H (E-H) and conserved sequence motif (CSM) between 7 and 11. The CSM approach was developed recently to investigate the functional role of non-active site residues in CYP2B4. Furthermore, we identified and analyzed the CSM in multiple CYP families and subfamilies in the E-H region. Results from CSM analysis showed that CSM 7, 8, 10, and 11 are conserved in CYP1, CYP2, and CYP3 families, while CSM 9 is conserved only in CYP2 family. Analysis of different CYP2 subfamilies showed that CYP2B and CYP2C have similar characteristics in the CSM, while the characteristics of CYP2A and CYP2D subfamilies are different. Finally, we analyzed CSM 7, 8, 10, and 11, which are common in all the CYP families/subfamilies analyzed, in fifteen important drug-metabolizing CYPs. The results showed that while CSM 8 is most conserved among these CYPs, CSM 7, 9, and 10 have significant variations. We suggest that CSM8 has a common role in all the CYPs that have been analyzed, while CSM 7, 10, and 11 may have relatively specific role within the subfamily. We further suggest that these CSM play important role in opening and closing of the substrate access/egress channel by modulating the flexible/plastic region of the protein. Thus, site-directed mutagenesis of these CSM can be used to study structure-function and dynamic/plasticity-function relationships and to design CYP biocatalysts.

Highlights

Understanding the molecular basis of diverse functions of mammalian cytochrome P450s (CYPs), which will enable to predict drug metabolism and drug interactions, is critical [1,2,3,4,5]
Analysis of the functional residues of CYPs identified by random mutagenesis We analyzed CYP mutants that were obtained by a random mutagenesis/directed evolution approach with CYP1A2, CYP2A6, CYP2Bs, and CYP3A4 (Table 1) [25,26,27,28,29,30,31,32,33,34,35,36,37,38]
Directed evolution identified four CYP2A6 residues (287, 297, 300, and 305) and one CYP2B1 residue (295) in the Ihelix that have been described as the backbone of the protein and that belong to substrate recognition site 4 (SRS4)

Summary

Introduction

Understanding the molecular basis of diverse functions of mammalian cytochrome P450s (CYPs), which will enable to predict drug metabolism and drug interactions, is critical [1,2,3,4,5]. We further identified and analyzed CSM 7–11 in important drug- and xenobiotic-metabolizing CYP2A, CYP2B, CYP2C, and CYP2D subfamilies (Figure 1, Table 3). CSM 8 showed 100% amino acid conservation, while CSM 10 had five of the six conserved residues when compared between CYP2B and CYP2C subfamilies.

Results

Conclusion