In Vitro Directed Evolution And Characterization Of Cytochrome P450 BM-3 Variants

P450 BM-3 from Bacillus megaterium is a self-sufficient natural fusion protein consisting of a P450 heme monooxygenase and a NADPH-dependent diflavin reductase. The natural products of long-chain saturated and unsaturated fatty acids are the substrates for wild type P450 BM-3. A mutant of cytochrome P450 BM-3(F87V/A74G/L188Q) engineered by rational design can hydroxylate indole into indigo and indirubin. In this paper, in order to further improve its capability in the hydroxylation of indole into indigo as well as in terms of higher regioselectivity to less indirubin production, the triple mutant P450BM-3 (A74G/F87V/L188Q) was subjected to further evolution by error-prone PCR and saturation mutagenesis, three P450 BM-3 variants with higher activity had been found. A systematic research of characteristics of mutant enzymes and the molecular mechanism of the activity change of P450 BM-3 mutants was made.First, a combination method of collecting the mutant libraries by E.coli DH5(a) and expressing P450 BM-3 by E. coli BL21 was made, which made the pre-selection system based on color formation agar plate feasible. A spectroscopic assay based on absorbance of indigo assay and NADPH assay in 96-well plate reader was proposed. By using the double screening procedure consisting of a pre-selection based on color formation agar plate and a quantitative comparison of catalytic activity on a 96-well plate reader, the screening efficiency was improved and the intensity of labor was also lessen.Second, the mutagenesis of the monooxygenase domain of the P450 BM-3 (F87V/A74G/L188Q) mutant was performed by error-prone PCR. Mn²⁺ concentration as a gene mutagenesis in vitro was optimized and 0.05 mmol/L Mn²⁺ was found to be optimal in suitable for acquirement of mutant library with appropriate mutation frequency. On this condition, the mutant libraries were made and iteratively screened, four mutants (I39V, K434R, E435D, and D168N/A225V/K440N) based on the triple mutant of P450 BM-3 with a slightly higher hydroxylation activity toward indole than the parental enzyme were obtained only through one round of error-prone PCRrandom mutagenesis.Third, for the identification of target position with a critical effect on P450 BM-3 activity toward indole, the libraries were constructed by saturation mutagenesis based on potential hot spots identified by error-prone PCR. Using this approach, three P450 BM-3 variants (D168H, D168L, E435T) containing A74G, F87V and L188Q substitutions were found to hydroxylate indole into indigo more efficiently than other mutants.Fourth, comparison was made between three mutants D168H, D168L, E435T and the parental enzyme with respect to primary enzymatic properties and kinetic values. The kinetics analysis indicated that the evolved enzymes exhibited higher affinity for substrate indole than the parental enzyme. Their K_m values were 2.2 mM for the parental enzyme, 1.2 mM for D168H mutant, 0.92 mM for D168L mutant and 0.78 mM for E435T mutant respectively. Correspondingly, the k_cat/K_m of all mutants showed up to 5-fold enhancement over that of the parental enzyme, respectively. This enhancement in catalytic eficiency was due to an increase in k_cat and a decrease in K_m, which illustrates that the mutations did affect the catalytic features of enzyme. Three mutants also exhibited higher hydroxylation activity at pH of 8.2. The thermostability of the mutants D168H, D168L decreased a little compared with the parental enzyme, but the thermostability of the mutant E435T did not change.Fifth, the coupling efficiency and regioselectivity of three mutants was investigated. The coupling efficiency of the mutant D168H decreased almost 2 folds compared to the parental enzyme while the coupling efficiency of the mutant D168L, E435T slightly increased. Therefore, exchange at position 168 aspartic acid substituted by hisitidine could influence the interaction between the monooxygenase domain and an FMN-binding reductase domain. The mutants D168H and E435T exhibited a higher regioselectivity forming indigo compared to the parental enzyme P450 BM-3 (A74G/F87V/L188Q), which further emphasized the importance of amino acid 168 and amino acid 435 in the relationship between structure and function of P450 BM-3.Sixth, based on the 3-D structure modeling of mutant enzymes, the changes in molecular structure of evolved enzymes were probed, and the possible explanationsfor the improvement of activity were preliminarily analyzed.Finally, the epoxidation of styrene with similar structure to indole by P450 BM-3 mutants engineered by error-prone PCR directed evolution were investigated. Experiment indicated that P450 BM-3 mutants can epoxidate styrene into styrene oxidate. The mutant E435D had a higher activity than other mutants and parent enzyme, which also emphasized the importance of amino acid 435 in the relationship between structure and function of P450 BM-3. The mutant E435D was choosed as the model enzyme and the effects of reaction conditions such as substrate concentration, reaction pH, temperature and co-solvent were investigated. The experiments showed that the optimum temperature and pH of this enzyme were about 37°C and 8.2. The yield of styrene oxidate decreased faster when styrene concentration came to 8.7mmol/L. And 1.5% co-solvent DMSO could make for favorably process of catalytic reaction in this reaction based on that substrate concentration 4.4 mM.In conclusion, we used directed evolution to improve the catalytic activity of P450 BM-3 toward indole. The evolved enzyme with higher activity and higher regioselectivity is a suitable parent for further directed evolution to improve catalytic rates and enhance regioselectivity. Moreover, all of the results obtained here may serve as the basis for further elucidation of the mechanism of substrate activation in this enzyme, which provides some hints for further study of the catalytic mechanism of P450B M-3.