Original paper
Rational engineering of cytochromes P450 2B6 and 2B11 for enhanced stability: Insights into structural importance of residue 334

https://doi.org/10.1016/j.abb.2009.11.026Get rights and content

Abstract

Rational mutagenesis was used to improve the thermal stability of human cytochrome P450 2B6 and canine P450 2B11. Comparison of the amino acid sequences revealed seven sites that are conserved between the stable 2B1 and 2B4 but different from those found in the less stable 2B6 and 2B11. P334S was the only mutant that showed increased heterologous expression levels and thermal stability in both 2B6 and 2B11. The mechanism of this effect was explored with pressure-perturbation spectroscopy. Compressibility of the heme pocket in variants of all four CYP2B enzymes containing proline at position 334 are characterized by lower compressibility than their more stable serine 334 counterpart. Therefore, the stabilizing effect of P334S is associated with increased conformational flexibility in the region of the heme pocket. Improved stability of P334S 2B6 and 2B11 may facilitate the studies of these enzymes by X-ray crystallography and biophysical techniques.

Introduction

The cytochrome P450 (P450)2 heme-containing monooxygenases are involved in the oxidation of a broad range of endogenous and xenobiotic compounds [1]. Multiple species of cytochrome P450 found in the endoplasmic reticulum of various tissues of vertebrates catalyze insertion of a single oxygen atom into a wide variety of xenobiotic substrates of differing shapes and sizes. Besides the central role in drug clearance, the ability of mammalian cytochromes P450 to convert various inactive precursors to the respective bioactive compounds makes these enzymes of paramount importance for the healthcare and pharmaceutical industries [2], [3], [4].

The P450 2B subfamily exhibits a relatively low degree of catalytic conservation across mammalian species, making these enzymes an outstanding model system for investigating structure–function relationships of P450s [5]. Investigations utilizing members of the cytochrome P450 2B subfamily have yielded a wealth of biochemical and biophysical information about substrate binding, protein–protein interactions, and the catalytic mechanisms of the microsomal monooxygenase. These enzymes have been studied at length using chimeragenesis, site-directed and random mutagenesis, molecular modeling, X-ray crystallography, and solution biophysics [5], [6]. X-ray structures of an engineered rabbit P450 2B4 (N-terminal modified and C-terminal His-tag) in ligand-free (pdb code 1PO5), 4-(4-chlorophenyl)imidazole (4-CPI)-bound (pdb code 1SUO), bifonazole (BIF)-bound (pdb code 2BDM), and 1-biphenyl-4-methyl-1H-imidazole (1-PBI)-bound (pdb codes 3G5N and 3G93) forms show a remarkable amount of structural plasticity with retention of function [5], [7]. Further studies utilizing isothermal titration calorimetry (ITC) have reinforced the ability of P450 2B4 to accommodate a wide variety of ligands of a wide range of sizes [5]. These studies provide insight into factors that must be considered in understanding and predicting the binding and metabolism of drugs by P450 enzymes.

Despite their importance for human and experimental pharmacology, human P450 2B6 and canine P450 2B11 have not been as extensively studied from a structural or biophysical standpoint as rat P450 2B1 or rabbit 2B4. A major contributing factor is the lower stability of the human and canine enzymes [7]. To surmount these difficulties, a variety of approaches have been used including removal of the membrane associated N-terminal domain, directed evolution, and site-directed mutagenesis [7], [8], [9], [10]. Furthermore, rational engineering and directed evolution have been used to locate important non-active site amino acids and alter function of P450s in the 2B subfamily [7], [10], [11], [12], [13]. Measures of protein stability used to examine 2B enzymes include thermal and pressure tolerance [7], [14], [15], [16].

Recently, sequence comparisons of P450 2B1, 2B4, 2B6, and 2B11 led to the identification of Leu-264 as a major determinant of the lower thermal stability of P450 2B6 [7]. The objective of the present study was to improve stability of P450s 2B6 and 2B11 in order to allow further investigation of their structure–function relationships by X-ray crystallography and solution biophysics approaches. Based on sequence comparison with the relatively more stable 2B1 and 2B4, seven residues in 2B6 and 2B11 were subjected to site-directed mutagenesis. The mutants were then characterized using catalytic tolerance to temperature, thermal stability, and pressure-perturbation spectroscopy. In particular, residue 334 (Ser in 2B1 and 2B4 and Pro in 2B6 and 2B11) was found to play a key role in thermal stability and compressibility of the heme pocket.

Section snippets

Materials

7-Hydroxy-4-trifluoromethylcoumarin (7-HFC), 7-methoxy-4-(trifluoromethyl)coumarin (7-MFC), and 7-ethoxy-4-(trifluoromethyl) coumarin (7-EFC) were purchased from Invitrogen (Carlsbad, CA). Sodium hydrosulfite, β-mercaptoethanol, phenylmethylsulphonyl fluoride, and NADPH were obtained from Sigma–Aldrich (St. Louis, MO). Recombinant NADPH-cytochrome P450 reductase (CPR) and cytochrome b5 (b5) from rat liver were prepared as described previously [17]. Oligonucleotide primers for PCR were obtained

Identification of amino acids of interest

Among the P450 2B subfamily, which includes the rat 2B1, rabbit 2B4, human 2B6, and dog 2B11 enzymes, 2B1 and 2B4 were found to be more stable than 2B11 and 2B6. The temperature-induced inactivation of the protein is caused by both P450  P420 formation and the heme loss processes [7]. A multiple sequence alignment of the relatively more stable P450s 2B1 and 2B4 with the less stable 2B6 and 2B11 identified seven non-active site sequence positions, where the residues are identical or similar

Discussion

The realization that an increasing number of drugs are metabolized by human P450 2B6 and that canine P450 2B11 has unique ability to metabolize the anti-cancer prodrugs cyclophosphamide and ifosphamide with high efficiency and to detoxify certain polychlorinated biphenyls has prompted a major effort to understand the structural basis of enzyme action [34], [35]. The recent discovery of the lower inherent stability exhibited by P450s 2B6 and 2B11 compared with the better characterized 2B1 and

Acknowledgments

The authors thank Nadezhda Davydova, Poonam Manwani, and Rebecca Dickerson for their assistance in high pressure experiments. This work was supported, in whole or in part, by National Institutes of Health Grants ES003619 and Center Grant ES006676. P.R.W. is supported by the Training Grant in Heme and Blood Proteins (T32-DK07233).

References (38)

  • E.F. Johnson et al.

    Biochem. Biophys. Res. Commun.

    (2005)
  • Y. Zhao et al.

    Biochim. Biophys. Acta Gen. Subj.

    (2007)
  • E.E. Scott et al.

    Arch. Biochem. Biophys.

    (2001)
  • S. Kumar et al.

    J. Biol. Chem.

    (2005)
  • C.E. Hernandez et al.

    Arch. Biochem. Biophys.

    (2006)
  • L. Sun et al.

    Arch. Biochem. Biophys.

    (2007)
  • D.R. Davydov et al.

    Biochem. Biophys. Res. Commun.

    (1992)
  • D.R. Davydov et al.

    Biochem. Biophys. Res. Commun.

    (2000)
  • G.R. Harlow et al.

    J. Biol. Chem.

    (1997)
  • D.R. Davydov et al.

    Arch. Biochem. Biophys.

    (1995)
  • C. Di Primo et al.

    FEBS Lett.

    (1992)
  • A.J. Dunford et al.

    J. Biol. Chem.

    (2007)
  • E. Anzenbacherova et al.

    Biochem. Biophys. Res. Commun.

    (2005)
  • V.G.H. Eijsink et al.

    J. Biotechnol.

    (2004)
  • G. Hui Bon Hoa et al.

    Biochim. Biophys. Acta

    (2002)
  • A. Al Omari et al.

    J. Pharm. Pract.

    (2007)
  • F.P. Guengerich

    Chem. Res. Toxicol.

    (2001)
  • M. Ingelman-Sundberg

    Naunyn-Schmiedeberg’s Arch. Pharmacol.

    (2004)
  • T.L. Domanski et al.

    Curr. Drug Metab.

    (2001)
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