Received for publication July 6, 2007.
Revised August 14, 2007.
Accepted for publication August 16, 2007.

Rational Engineering of Human Cytochrome P450 2B6 for Enhanced Expression and Stability: Importance of a Leu²⁶⁴Phe Substitution

Abstract

Despite the emerging importance of human P450 2B6 in xenobiotic metabolism thorough biochemical and biophysical characterization has been impeded due to low expression in E. coli. Comparison with similar N-terminal truncated and C-terminal His-tagged constructs, namely rat P450 2B1dH, rabbit 2B4dH, and dog 2B11dH, revealed that P450 2B6dH showed the lowest thermal stability, catalytic tolerance to temperature, and chemical stability against guanidium chloride-induced denaturation. Eleven P450 2B6dH mutants were rationally engineered based on sequence comparison with the three other P450 2B enzymes and the solvent accessibility of residues in the ligand-free crystal structure of P450 2B4dH. L198M, L264F, and L390P showed ~3-fold higher expression than P450 2B6dH. L264F alone showed enhanced stability against thermal and chemical denaturation compared with P450 2B6dH, and was characterized further functionally. L264F showed similar preferential inhibition by pyridine over imidazole derivatives as P450 2B6dH. The Leu²⁶⁴Phe substitution did not alter the K_s for inhibitors or the substrate benzphetamine, the K_m for 7-ethoxy-4-(trifluoromethyl)coumarin, or the benzphetamine metabolite profiles. The enhanced stability and monodisperse nature of L264F made it suitable for isothermal titration calorimetry studies. Interaction of 1-benzylimidazole with L264F yielded a clear binding isotherm with a distinctly different thermodynamic signature from P450 2B4dH. The inhibitor docked differently in the binding pocket of a P450 2B6 homology model than in 2B4, highlighting the different chemistry of the active site of these two enzymes. Thus, L264F is a good candidate to further explore the unique structure-function relationships of P450 2B6 using X-ray crystallography and solution thermodynamics.

Key words: Thermodynamic and kinetic processes and modeling, Mutagenesis/Chimeric approaches, Cytochrome P450, Enzymology, Structure/function/mechanism

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