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Updated: Jul 19, 2026

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
Published on: November 7, 2012
Engineering of cytochrome P450 3A4 for enhanced peroxide-mediated substrate oxidation using directed evolution and
Santosh Kumar1, Hong Liu, James R Halpert
1Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555-1031, USA. sakumar@utmb.edu
Abstract:
CYP3A4 has been subjected to random and site-directed mutagenesis to enhance peroxide-supported metabolism of several substrates. Initially, a high-throughput screening method using whole cell suspensions was developed for H2O2-supported oxidation of 7-benzyloxyquinoline. Random mutagenesis by error-prone polymerase chain reaction and activity screening yielded several CYP3A4 mutants with enhanced activity. L216W and F228I showed a 3-fold decrease in Km, HOOH and a 2.5-fold increase in kcat/Km, HOOH compared with CYP3A4. Subsequently, T309V and T309A were created based on the observation that T309V in CYP2D6 has enhanced cumene hydroperoxide (CuOOH)-supported activity. T309V and T309A showed a > 6- and 5-fold higher kcat/Km, CuOOH than CYP3A4, respectively. Interestingly, L216W and F228I also exhibited, respectively, a > 4- and a > 3-fold higher kcat/Km, CuOOH than CYP3A4. Therefore, several multiple mutants were constructed from rationally designed and randomly isolated mutants; among them, F228I/T309A showed an 11-fold higher kcat/Km, CuOOH than CYP3A4. Addition of cytochrome b5, which is known to stimulate peroxide-supported activity, enhanced the kcat/Km, CuOOH of CYP3A4 by 4- to 7-fold. When the mutants were tested with other substrates, T309V and T433S showed enhanced kcat/Km, CuOOH with 7-benzyloxy-4-(trifluoromethyl)coumarin and testosterone, respectively, compared with CYP3A4. In addition, in the presence of cytochrome b5, T433S has the potential to produce milligram quantities of 6beta-hydroxytestosterone through peroxide-supported oxidation. In conclusion, a combination of random and site-directed mutagenesis approaches yielded CYP3A4 enzymes with enhanced peroxide-supported metabolism of several substrates.
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