1. Rational engineering of cytochromes P450 2B6 and 2B11 for enhanced stability: Insights into structural importance of residue 334.
- Author
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Talakad JC, Wilderman PR, Davydov DR, Kumar S, and Halpert JR
- Subjects
- Amino Acid Sequence, Amino Acid Substitution, Animals, Aryl Hydrocarbon Hydroxylases isolation & purification, Aryl Hydrocarbon Hydroxylases metabolism, Biocatalysis, Crystallography, X-Ray, Cytochrome P-450 Enzyme System genetics, Cytochrome P-450 Enzyme System isolation & purification, Cytochrome P450 Family 2, Cytochromes metabolism, Dogs, Enzyme Stability, Heme metabolism, Humans, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Pressure, Protein Isoforms, Rabbits, Rats, Spectrum Analysis, Steroid Hydroxylases isolation & purification, Steroid Hydroxylases metabolism, Structure-Activity Relationship, Temperature, Aryl Hydrocarbon Hydroxylases chemistry, Aryl Hydrocarbon Hydroxylases genetics, Cytochrome P-450 Enzyme System chemistry, Cytochrome P-450 Enzyme System metabolism, Protein Engineering, Steroid Hydroxylases chemistry, Steroid Hydroxylases genetics
- 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., (2009 Elsevier Inc. All rights reserved.)
- Published
- 2010
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