Santosh Kumar

Despite the emerging importance of human P450 2B6 in xenobiotic metabolism, thorough biochemical and biophysical characterization has been impeded as a result of low expression in Escherichia coli. Comparison with similar N-terminal truncated and C-terminal His-tagged constructs (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 guanidinium 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 Leu²⁶⁴→Phe 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.

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 K_{m, HOOH} and a 2.5-fold increase in k_cat/K_{m, 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 k_cat/K_{m, CuOOH} than CYP3A4, respectively. Interestingly, L216W and F228I also exhibited, respectively, a >4- and a >3-fold higher k_cat/K_{m, 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 k_cat/K_{m, CuOOH} than CYP3A4. Addition of cytochrome b₅, which is known to stimulate peroxide-supported activity, enhanced the k_cat/K_{m, CuOOH} of CYP3A4 by 4- to 7-fold. When the mutants were tested with other substrates, T309V and T433S showed enhanced k_cat/K_{m, 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 6β-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.

The previously laboratory-evolved cytochrome P450 2B1 quadruple mutant V183L/F202L/L209A/S334P (QM), which showed enhanced H₂O₂-mediated substrate oxidation, has now been shown to exhibit a >3.0-fold decrease in K_m,HOOH for 7-ethoxy-4-trifluoromethylcoumarin (7-EFC) O-deethylation compared with the parental enzyme L209A. Subsequently, a streamlined random mutagenesis and a high-throughput screening method were developed using QM to screen and select mutants with enhanced tolerance of catalytic activity to temperature and dimethyl sulfoxide (DMSO). Upon screening >3000 colonies, we identified QM/L295H and QM/K236I/D257N with enhanced catalytic tolerance to temperature and DMSO. QM/L295H exhibited higher activity than QM at a broad range of temperatures (35–55°C) and maintained ∼1.4-fold higher activity than QM at 45°C for 6 h. In addition, QM/L295H showed a significant increase in T_m,app compared with L209A. QM/L295H and QM/K236I/D257N exhibited higher activity than QM at a broad range of DMSO concentrations (2.5–15%). Furthermore, QM/K236I/D257N/L295H was constructed by combining QM/K236I/D257N with L295H using site-directed mutagenesis and exhibited a >2-fold higher activity than QM at nearly the entire range of DMSO concentrations. In conclusion, in addition to engineering mammalian cytochromes P450 for enhanced activity, directed evolution can also be used to optimize catalytic tolerance to temperature and organic solvent.

The role of cytochrome b₅ (b₅) in the α-naphthoflavone (α-NF)-mediated inhibition of H₂O₂-supported 7-benzyloxyquinoline (7-BQ) debenzylation by heterologously expressed and purified cytochrome P450 3A4 (CYP3A4) was studied. Although α-NF showed negligible effect in an NADPH-dependent reconstituted system, inhibition of 7-BQ oxidation was observed in the H₂O₂ system. Analysis of the effect of various constituents of a standard reconstituted system on H₂O₂-supported activity showed that b₅ alone resulted in a 2.5-fold increase in the k_cat value and reversed the inhibitory effect of α-NF. In addition, titration with b₅ suggested that only 65% of the CYP3A4 participated in the interaction with b₅, consistent with cytochrome P450 (P450) heterogeneity. Study of the influence of b₅ on the kinetics of H₂O₂-dependent destruction of the P450 heme moiety suggested two distinct conformers of CYP3A4 with different sensitivity to heme loss. In the absence of b₅, 66% of the wild-type enzyme was bleached in the fast phase, whereas the addition of b₅ decreased the fraction of the fast phase to 16%. Finally, to locate amino acid residues that might influence b₅ action, several active site mutants were tested. Substitution of Ser-119, Ile-301, Ala-305, Ile-369, or Ala-370 with the larger Phe or Trp decreased or even abolished the activation by b₅. Ser-119 is in the B′-C loop, a predicted b₅-P450 interaction site, and Ile-301 and Ala-305 are closest to the heme. In conclusion, the interaction of b₅ with P450 apparently leads to a conformational transition, which results in redistribution of the CYP3A4 pool.