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Engineering of Human Cytochrome P450 2B6 for Enhanced Expression, Stability, and Functional Studies of the Wild‐Type and Genetic Variants
Author(s) -
Kumar Santosh,
Talakad Jyothi C.,
Sun Ling,
Halpert James R.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.919.5
Subject(s) - mutant , cytochrome p450 , xenobiotic , wild type , biochemistry , drug metabolism , computational biology , enzyme , chemistry , biology , microbiology and biotechnology , gene
Despite the emerging importance of human P450 2B6 in xenobiotic metabolism and increasing number of genetic variants, thorough biochemical and biophysical characterization has been impeded due to low expression and stability. Recently, an N‐terminal modified 2B6 (2B6dH) mutant was engineered termed L264F, which showed enhanced expression and stability and retained functional integrity compared with 2B6dH. To further enhance the expression and stability of 2B6dH construction is underway of V234I, E254A, Y325Q, P334S, I427M, and Q473K, which involves changing the residues from the one found in the less stable 2B6dH to the more stable 2B1dH. 2B6dH mutants that show increased stability will be combined with L264F, and the resulting mutant with the highest expression and stability will be used as a template for directed evolution to model a thermostable 2B6dH. To investigate the functional role of the 2B6 variant, K262R, which shows altered activity with several drugs, the mutant was constructed in 2B6dH and L264F backgrounds. K262R and K262R/L264F were then characterized for stability and for functional properties using model and drug substrates/inhibitors. K262R showed decreased protein stability and altered substrate metabolism and inhibitor binding compared with 2B6dH. Structural analysis to understand how K262R alters enzyme functions is underway. [NIH grant ES03619 and Center grant ES06676]