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 6-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.